Marine biogenic humic substances control iron biogeochemistry across the Southern Ocean

Domain: earth_science
Published: 2025-03-18
Editorial outcome: major_revision

Abstract
Iron, which is an essential element for marine photosynthesis, is sparingly soluble in seawater. In consequence, iron bioavailability controls primary productivity in up to 40% of the world’s ocean, including most of the Southern Ocean. Organic ligands are critical to maintaining iron in solution, but their nature is largely unknown. Here, we use a comprehensive dataset of electro-active humics and iron-binding ligands in contrasting regions across the Southern Ocean to show that humic substances are an important part of the iron binding ligand pool, as has been found elsewhere. However, we demonstrate that humics are mostly produced in situ and composed of exopolymeric substances from phytoplankton and bacteria, in contrast to other regions where terrestrially-derived humics are suggested to play a major role. While phytoplankton humics control the biogeochemistry, bioavailability and cycling of iron in surface waters, humics produced or reprocessed by bacteria affect iron cycling and residence time at the scale of the global ocean. Our findings indicate that autochthonous, freshly released organic matter plays a critical role in controlling primary productivity and ocean-climate feedbacks in iron-limited oceanic regions.

Introduction
The ocean modulates both the global carbon cycle and Earth’s climate. Each year, the ocean absorbs roughly one-fourth of anthropogenic CO2 emissions1,2, de facto mitigating a large proportion of their global warming potential. Around 40% of this carbon uptake occurs in the Southern Ocean3 (SO), where cold waters absorb atmospheric CO2 and transfer it into the ocean interior by convection and mixing. This solubility pump is enhanced by the biological carbon pump, whereby marine phytoplankton fix CO2 into organic carbon biomass, reducing CO2 concentrations in surface waters and amplifying ocean carbon uptake4. Phytoplankton carbon is partly recycled in shallow waters and partly exported to depths where it will remain sequestered for centuries to millennia2,4. Iron (Fe), essential for photosynthesis5, is the dominant micronutrient limiting phytoplankton growth over most of the SO, especially south of the Polar Front6–9. Thus, the scarcity of bioavailable Fe limits the efficiency of the biological carbon pump, thereby curtailing atmospheric CO2 drawdown. The contribution of the SO to global carbon uptake is expected to increase with global warming, mostly due to increased Fe and light availability10.

While it is widely accepted that Fe chemical forms largely explain Fe bioavailability9,11, Fe chemistry alone cannot predict whether this micronutrient will sustain phytoplankton growth9,12,13. In marine ecosystems, more than 99% of the dissolved Fe is bound to organic ligands within the dissolved organic matter (DOM) pool6, one of Earth’s largest organic carbon reservoirs that nonetheless remains poorly characterised. Iron-binding organic ligands (Fe-L) exert a major control on (i) the oceanic residence time of dissolved Fe, (ii) the subsurface ocean Fe inventory, and (iii) Fe bioavailability, i.e., the acquisition of Fe-substrate by microorganisms6,9,12–14. Iron bioavailability is not controlled by seawater chemistry only, but it is also influenced by the biological uptake mechanisms at play, as well as biological competition for Fe acquisition9,11–13. Iron bioavailability varies far more widely in the SO (>200-fold variation)13 than in the global ocean (5-fold variation)12, highlighting that our poor knowledge of the nature of Fe-L is a major impediment to our understanding of the biological carbon pump in the SO.

It has recently been proposed that Fe biogeochemistry across the global ocean is regulated by humic substances (HS). This conclusion was based on measurements of fluorescence and chemical DOM properties14–19, as well as through coupling of Fe-binding stoichiometry from terrestrially-derived standard humics (Suwanee River humics) to electrochemically-detected iron-bound HS (eHS)14,19–23. HS represent a continuum of organic compounds resulting from the degradation of (terrestrial or aquatic) biomass, including the degradation of polysaccharides, proteins, lipids, nucleic acids, and lignin14,17. Within HS, humic and fulvic acids are two operationally-defined fractions, with humic acids (HA) being soluble in natural and alkaline solutions only, and fulvic acids (FA) being soluble at all pH levels17.

The important role of terrestrial humics in the HS pool has been clearly demonstrated in the transpolar drift of the Arctic Ocean20,21 and in coastal systems18, as well as through their long-term persistence in marine systems17,18. However, most studies have also recognised the importance of other sources of humics including atmospheric dust deposition24, microbial reworking of marine DOM25–27 and remineralisation in the ocean’s water column28. The widespread abundance of marine humics14,17 has even been determined in the landlocked Mediterranean Sea22,29 and in coastal systems such as at the East Antarctic Peninsula30. Humics have also been reported in SO14,23, yet their sources and role in Fe biogeochemistry remain largely unexplored despite recent data suggesting that Fe-binding ligands are critical drivers of Fe residence time and bioavailability in surface waters31. Our study addresses this important knowledge gap.

Results
Uniqueness of iron-binding organic ligands across the Southern Ocean
During the Antarctic Circumnavigation Expedition (ACE) in the summer 2016–2017, we conducted a circumpolar assessment of the connections among ice, ocean, climate, and life across contrasting regions of the SO (Fig. 1, Supplementary Figs. 1 and 2, Supplementary Table 1)13. Unprecedented quasi-synoptic measurements (n = 70) of eHS, hydrolysable carbohydrates (Carb), dissolved Fe (DFe), and Fe-L concentrations were carried out on seawater samples collected all around Antarctica along vertical profiles spanning the upper 1000 m of the water column, to assess the role of eHS in Fe biogeochemistry. Carb showed an inverse correlation with depth (Fig. 1; R = −0.21, p < 0.001, n = 239, Supplementary Table 2), suggesting vertical attenuation of carbohydrates and CHONS-containing molecules as a result of microbial DOM processing24–27,32. A strong positive correlation between Fe-L and eHS (Fig. 1b; R = 0.7, n = 70, p = 1.8 × 10−11, Supplementary Table 2, Fig. 2d) suggests that eHS, despite being a subset of the HS pool, might represent a substantial fraction of the Fe-L across the SO. Notably, the correlation also holds (R = 0.8, n = 39, p = 9.2 × 10−10, Supplementary Table 3) when considering surface waters (0–100 m) only. Overall, the intercept was 1.3 nmol L−1 and the slope was 0.045 nmol µg−1 eHS (Supplementary Fig. 4). Thus, on average, 1 mg eHS could be associated with 45 nmol L−1 Fe-L, which is close to the terrestrial SRHA value (32 nmol Fe mg−1 SRHA)14,33,34. Fe-L and eHS values associated with sedimentary input close to the Balleny Islands13,35 were amongst the highest measured and could explain a Fe-binding capacity for eHS close to that of terrestrial humics.

Although this finding supports the postulated role of eHS in Fe biogeochemistry across the global ocean14,19–23,36, no correlation between DFe and eHS was observed for the SO (Fig. 2b) in contrast to all other oceanic regions for which comparable data are available (Fig. 2a, Supplementary Fig. 3, except for the South Pacific Ocean). In the global ocean, typically >99% of the DFe is associated with Fe-L6; therefore, if eHS represents the bulk of Fe-L, one would expect a correlation between DFe and eHS unless (i) eHS are undersaturated with Fe, as might be anticiapated in an Fe-limited region such as the SO, and/or (ii) a strong in situ competition between different metals and cations to bind the same eHS molecule exists6,13,14. Similar eHS concentrations have been reported for several ocean basins and the Mediterranean Sea (Fig. 3a). The Arctic Ocean emerges as a notable exception, where eHS concentrations are elevated, likely due to strong riverine inputs and high concentrations of terrestrial humics in the transpolar drift20,21. The DFe levels associated with eHS are within or smaller than the range observed for saturated Suwannee River FA and HA for a significant portion of the data collected in the North Pacific, North Atlantic and South Pacific Oceans. In the other regions, even lower values were measured (Fig. 3b). Together, competition between other metals and cations to bind eHS, and low DFe resulting in undersaturated eHS will typically lower the measured DFe:eHS values. Surprisingly, similar average DFe:eHS were found across regions globally (18.4 ± 19.1 nmol Fe mg−1 eHS, Fig. 3b); a value that is representative of SRFA and SRHA maximal Fe-binding capacity or stoichiometry14,33. This finding suggests that the role of humics in the global ocean could be assessed using Suwanee River reference material, although this approach might not be suitable for investigating the southernmost waters. For the SO, a median DFe:eHS of 8 nmol Fe mg−1 eHS was observed, suggesting an undersaturation of eHS and/or Fe-L properties or sources different from SRHA and SRFA. Indeed, the relationships between DFe, eHS, and Fe-L indicate a distinct nature of organic matter – Fe interactions in this southernmost ocean (Fig. 2, Supplementary Figs. 3 and 5).

To explore whether, by considering the iron-binding stochiometry from standard terrestrial HA and FA, humics represent the bulk of the Fe-L and could dominate Fe biogeochemistry, we compiled measurements of DFe, in situ Fe-L and eHS from several oceanic regions14,19–22,24 and estimated the humic contribution (as LFA; Fig. 3c, and LHA; Fig. 3d) to in situ iron-binding ligands, as in refs. 19,21,22. We note that this approach inherently assumes that all DFe is bound to eHS with similar Fe binding capacity as for standard humics in a 1:1 Fe:eHS stoichoimetry, which is a necessary simplification given the complex chemistry of aqueous DOM and Fe20,37, the fact that Fe can bind to other ligands on a “first come, first served” basis38, and that other trace elements can compete for the available binding sites. Therefore, the Fe-L attributed to FA and HA should represent an upper estimate of in situ Fe-binding ligands. Only a strong Fe-binding capacity (as for HA) succeeded in representing the bulk of the in situ ligands in the Arctic, North Atlantic and Mediterranean Sea (Fig. 3c, d). In the Arctic, previous direct measurements suggested that 80 ± 51% of Fe was bound to eHS20, which falls within the range of LFA and LHA contributions estimated here. In the South Pacific and SO, such an approach fails to represent the bulk of in situ ligands, except for marginal values in the SO associated with sediment inputs near the Balleny Islands13,35. Overall, estimated Fe-L eHS only accounted for 10–30% of in situ Fe-L, suggesting that the bulk of eHS measured within the upper 1000 m of the SO and South Pacific cannot be described by standard FA and HA substances. This observation, coupled with the absence of a relationship between DFe and eHS, highlights a different behaviour and/or nature of eHS in these southernmost ocean regions.

The origin of Fe organic ligands in the Southern Ocean and their relevance to Fe biogeochemistry
Coexisting nutrients and organic matter sources and degradation processes often weaken the observed relationships between the biomass of phytoplankton and bacteria with Fe-L compared to what is expected based on our understanding of known processes at play30., yet several studies have demonstrated the role of microorganisms in Fe-L production and transformation28,39–41. Here, we take advantage of our unique dataset to explore Carb, Fe-L, and eHS relationships with phytoplankton, bacteria, and depth as a way to diagnose key sources and sinks.

Fe-L and eHS concentrations were negatively correlated with bacterial abundances (R = −0.4, n = 45, p = 0.0045; and R = −0.4, n = 46, p = 0.0064, respectively) (Supplementary Table 2). These relationships suggest that microbial reworking of DOM during remineralisation affects both Fe-L and eHS, as previously observed28,39. This idea is further supported by the observation that the negative correlation was not observed when only surface waters were considered (0–100 m; n = 32, p = 0.38, Supplementary Table 3). In general, bacterial and phytoplankton abundances were positively correlated with Carb (Supplementary Table 3), suggesting that these compounds were predominantly photosynthates and exudates. While only weak correlations between phytoplankton and DFe were observed for the ACE dataset, the efficiency of photosystem II, indicated by the Fv/Fm ratio5, was positively related to both Fe-L (R = 0.5, n = 45, p = 0.0003) and eHS (R = 0.3, n = 47, p = 0.036). Despite the rather low R values, these correlations suggest a ligand-mediated Fe bioavailability to phytoplankton that cannot be predicted from DFe alone, as previously observed for natural phytoplankton assemblages9. The discrepancy between DFe concentrations and ligand-bound Fe bioavailability could be related to the occurrence of a significant proportion of DFe that is less bioavailable because it is associated with colloids. Colloidal Fe plays a critical role in Fe residence time, with consequences for the dynamics of marine ecosystems31; unfortunately, we have no measurements of colloidal Fe and thus cannot explore whether these forms are responsible for the lack of correlation between DFe and the biomass of phytoplankton or bacteria.

We identified both allochthonous and in situ-produced compounds as potentially important for Fe biogeochemistry across the SO; unfortunately, our measurements of only eHS and Fe-L prevented us from differentiating them. Among in situ biologically produced Fe-binding ligands, exopolymeric substances (EPS) are described as key compounds9,12,40–42. Similar to HS, EPS are loosely defined organic macromolecules able to bind several metals and cations41,42. Heterotrophic bacteria and phytoplankton excrete EPS41–43, which contribute to the eHS pool (Table 1) and are known to represent an important fraction of labile DOM25,44. EPS act as weak Fe-binding organic ligands41,43 (Table 1) that make Fe bioavailable to phytoplankton12,40,41. Carbohydrates are often considered key constitutive components given that EPS are generally rich in high molecular weight acidic polysaccharides, many of which contain carboxylic groups45-a good functional site for Fe binding. For these reasons, we further investigated the role of EPS and carbohydrates in SO Fe biogeochemistry.

High-resolution depth profiles of EPS in the ocean do not exist, and the distinction between phytoplankton and bacterial EPS has never been made. Here, we use the chemical characterisation of EPS previously isolated from phytoplankton, bacteria, or a naturally occurring bloom (see “Methods” section) to explore their potential role in Fe biogeochemistry. EPS excreted by phytoplankton and bacteria had similar labile Carb (similar to polysaccharides) but contrasting eHS content (Table 1), suggesting distinct compositions yet similar organic carbon lability. Bacterial EPS had a lower eHS and Fe-L content than phytoplankton EPS, but a similar conditional stability constant with Fe. Together, these observations indicate that the ability of EPS to bind Fe cannot be predicted from their Carb content.

We compiled data on isolated phytoplankton and bacterial EPS to test whether biologically produced EPS could represent the bulk of the in situ Fe-L (Table 1). The maximal contribution to Fe-L was estimated for each ligand type (Fe-LCarb, Fe-LEPS; Fig. 4a) using their respective maximal iron-binding capacity (Table 1), as was done for the LFA and LHA. Further, the complexing capacity of each ligand type was calculated using the in situ excess ligands (eL = Fe-L – DFe) multiplied by their respective conditional binding affinities for Fe (Table 1). In theory, for a ligand to potentially influence the in situ Fe biogeochemistry, its complexing capacity should be equal to or higher than that of the in situ Fe-L (eL × KFe’L; Fig. 4b). We note that such a calculation relies on similar assumptions as those for Fe-LeHS. Hence, to be of significance for Fe biogeochemistry in the SO, an estimated ligand type should have a complexing capacity much greater than that of the in situ Fe-L. Carb should not play a major role in Fe biogeochemistry as their log KFe’L are low5,46 (Table 1). Moreover, their average complexing capacity is 3.8-fold lower than the in situ value, similar to what is observed for FA (Fig. 4b). Overall, Fe-LEPSin was similar to in situ Fe-L, while both Fe-LEPSphyto and Fe-LEPSbact were greater than Fe-L by two orders of magnitude (Fig. 4a). The most plausible explanation for these results is that EPS are not explicitly produced to bind Fe, in contrast to siderophores. Rather, EPS also bind numerous other trace metals24,40–42. Nonetheless, with only 1% saturation with Fe, EPS could represent the bulk of the in situ Fe-L. Given that in the EPS used here, Fe was present at a level largely exceeding zinc (by 2–15 fold) and copper (by 20–50 fold), this 1% Fe saturation is a robust approximation40,41. We conclude that the complexing capacity of EPS from phytoplankton and bacteria was high enough to regulate in situ Fe biogeochemistry and bioavailability (Fig. 4b). As such, our observations shed light on the nature of the marine humics controlling Fe biogeochemistry, showing that autochthonous EPS and biogenic humics can represent the bulk of the eHS and Fe-L across the SO, and most likely also in the South Pacific Ocean.

Because the eHS and Fe-L properties of in situ EPS are much closer to those of phytoplankton than bacterial EPS (Table 1), phytoplankton are likely the dominant source of the in situ EPS in surface waters. The relative contributions of phytoplankton and bacterial cells to total particulate organic carbon (POC) varied greatly across the SO. In the Mertz polynya, for instance, the absolute heterotrophic bacterial abundance was similar to that in other SO regions despite the much greater phytoplankton biomass (Supplementary Fig. 2), suggesting that bacterial EPS might be relatively less important in productive waters, with bacteria instead increasing their share of total EPS in oligotrophic waters.

Methods
Watersampling
Sampling was conducted onboard the R/V Akademik Tryoshnikov from 20th December 2016 to 22nd February 2017 (Fig. 1, Supplementary Table 1). Water for trace metal and ligand measurements was sampled using an autonomous rosette (Model 1018, General Oceanics, USA) on a Dyneema line equipped with acid-washed Teflon-coated 10 L Niskin X bottles, with bottles manipulated in a clean container under a HEPA filter as described in ref. 69. Water from 0 to 1000 m was filtered by gravity through 0.2 µm acid-washed capsule filters (Acropak 200, Pall). Sampling and manipulation related to Fe biogeochemistry followed GEOTRACES guidelines70. Water from the biological parameters was sampled using a 12 L Niskin bottle deployed on a rosette equipped with a Seabird 911 CTD71. Both rosettes were deployed within a 1 h time window to collect comparable data. Macronutrient data from the two deployments showed a similar water column structure72.

Dissolved Fe concentrations
Samples for trace-metal concentrations were collected in LDPE bottles acidified with HNO3 to pH < 1.8 and stored for at least 6 months before analysis. Laboratory work was carried out under trace-metal clean conditions in ISO 5 clean hoods, using ultrapure reagents. Two different analytical methods and sample sets were used, and they showed good accuracy and analytical metrics35,69,73. Briefly, dissolved Fe concentrations for Leg 1 (Cape Town to Hobart, Fig. 1) were determined at ANU by isotope dilution. Samples were pre-concentrated, and the seawater matrix was removed (Nobias PA1 resin) offline using a home-built, automated pre-concentration system, and then eluted in 1 M HNO3. Weighed sample aliquots were spiked with enriched isotopes (57Fe) and internal standards (Sc, In, Yb), then analysed by inductively coupled mass spectrometry (ICP-MS) on either a Neptune or Element XR instrument (both Thermo Scientific).

Dissolved Fe concentrations for Leg 2 (Hobart to Punta Arenas) were measured using samples taken for metal stable isotope composition, which were acidified and amended with Fe double spikes. Metals were extracted using Nobias PA1 resin and purified using AG-MP1 resin, following previously published methods35,69. Fe concentrations and isotope ratios were analysed at the University of South Florida using a Neptune with an Apex-Omega desolvating system, a Jet Ni sampler cone and an X-type Al skimmer cone.

Electrochemistry
Electrochemical measurements were made as per 9 using a 0.52 mm2 hanging mercury drop electrode and stirring during deposition steps using a rotating PTFE rod set to a stirring speed of 3000 rpm. Analyses were conducted in a laminar flow cabinet (600 PCR workstation, AirClean Systems) at ambient temperature.

Fe(III) chemical speciation. The Fe speciation was determined by a CLE-AdCSV technique as in 9,74. Briefly, a seawater aliquot was buffered to pH 8.2 with a 1 M borate buffer to which 0–9 nM Fe was added. After 1 h equilibration at room temperature, 5 µM salicylaldoxime solution (SA, Acros Organics, competitive ligand) was added to all the tubes. Following overnight equilibration, the analysis was conducted using the following parameters: 30 s air purging, 200 s deposition time at 0 V. Voltammograms were analyzed with ECDSoft75, considering the 4th derivative with a tangential baseline, and the software ProMCC76 was used to analyse the resulting Fe-binding ligand titration data and quantify both the total concentration of in situ ligands (Fe-Linsitu) and the conditional stability constant (KFe’L) of the complexes. Parameters were calculated assuming the detection of one ligand class and considering van den Berg fitted values.

Electroactive humic substances (eHS). Electroactive HS was determined using the voltammetric method from 33 with standard addition of SRFA (IHSS, std 1, 0–90 µg L−1)9. This method is based on CSV and makes use of the adsorptive properties of Fe–HS complexes on the mercury drop electrode at natural pH. As such eHS can be described as the fraction of HS binding Fe that is electroactive. A mixed reagent solution (KBrO, EPPS, NH4OH, 750 µl) was added to 100 mL of seawater in order to obtain a pH of 8.2, along with 20 nM of inorganic Fe (ICP standard, Sigma) to saturate the in situ eHS. After 2 h in the dark at room temperature, analysis was done using a 300 s nitrogen purge time (Pan Gas) and 200 s deposition time. Detection limit was 1.9 µg L−1 SRFA equivalent and accuracy was within 3% for a solution of 95.0 µg L−1 SRFA77.

Carbohydrates
Hydrolysable mono- and polysaccharides are considered as labile organic compounds that were quantified by 2,4,6-tripyridyl-s-triazine spectroscopy78. D-Glucose was used as a standard. The instrumental detection and quantification limits were 2.46 µMC and 4.29 µMC, respectively79.

EPS and in situ, DOM selected as marine humics
Most of the EPS selected are relevant to the SO and its low iron levels; all EPS are of marine origin or from marine strains. The bacterial strains included Pseudoalteromonas sp. CAM025 isolated from Antarctic sea ice41, Cobetia marina L 9,80, and Vibrio alginolyticus (Hassler, Beaudoux, unpublished data). Two separate isolations of Cobetia marina L were analysed. The phytoplankton strains included Prymnesiophytes from the Australian National Culture Collection, Phaeocystic antarctica (CS243, Prydz Bay, Antarctica) and Emiliania huxleyi (CS812, Mercury Passage, Tasmania, Australia). The in situ EPS or DOM was isolated from the North Atlantic43 (1000 m depth, 35°39.8’N 74°30.8’W) and from the Sub-Antarctic Zone40 (depth of fluorescence maximum, 25 m, 159°5 E 46°2 S) coincident with a coccolithophorid bloom40 (0.69 µg L−1 Chl a). The respective contributions to these selected organic materials of hydrolysable carbohydrates, eHS and Fe-L were measured in inorganic trace-metal clean seawater9,41.

Data analysis
To estimate the potential contribution of different humics to in situ Fe-binding ligands in the ocean, SRHA (standard 1, IHSS, referred as HA), SRFA (standard 1, IHSS, referred as FA), hydrolysable carbohydrates (Carb) and biologically produced EPS (phytoplankton, bacteria and in situ) were selected. First, the in situ measured eHS was transformed into the selected humics concentration using their specific eHS content (Table 1). Considering the average maximal Fe binding properties (Table 1) for each humics type (HA, FA, Carb, and EPS), the corresponding Fe-binding ligand concentration was calculated assuming that all eHS behave as a specific ligand type (LHA, LFA, LCarb, and LEPS). Their contribution to Fe-binding ligands was then obtained by comparing LHA, LFA, LCarb, and LEPS with in situ Fe-L. Overall, this approach followed calculations made previously19,21,22. For example, to estimate the contribution of EPS to in situ Fe-L, one applies the following formulae:

Equivalent concentration of EPS (mg L−1) = eHS (mg L−1) / eHS content of EPS (mg eHS mg−1 EPS) (1)

L EPS (nM) = equivalent concentration of EPS (mg L−1) × Fe-L content of EPS (nmol Fe mg−1 EPS) (2)

Potential contribution to in situ ligands (%) = L EPS / Fe-L × 100 (3)

To further assess the potential for each selected humic to impact SO Fe biogeochemistry, we calculated the complexation capacity using the product of excess ligands (eL = Fe-Linsitu – DFe) and multiplied it by the conditional stability constant of each ligand type. For a ligand to potentially influence SO Fe biogeochemistry, its average complexing capacity should equal or exceed the in situ complexing capacity given by eL × KFe’L. Data are discussed with respect to different humics types and support investigation into the respective roles of bacterial and phytoplankton EPS.

Statistical relationships amongst different parameters were investigated using ACP and a Pearson correlation table using SigmaPlot ver. 14 with a statistical significance set at the level of 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper presents important new data on the ligands for iron from the Southern Ocean, an under-sampled region, and provides insight into identifying those ligands.\nStrengths:\nThe strength in this paper lies in new data for ligands, humic substances and EPS from the Southern Ocean, an iron-limited region that is under-sampled and poorly understood. This is an important topic that deserves attention. The additional analyses into identifying the sources of the ligands are an important contribution. This study builds on previous work by the lead author into the importance of EPS as a complexing agent for iron in the Southern Ocean, and points to a biogenic source of ligands. The general assumption within the scientific community would be that most of the ligand pool in the upper Southern Ocean is biogenic, and therefore these findings are not necessarily a surprise and thus the narrative is a little off the mark, although that shouldn’t discredit the importance of this new work in providing better insight into ligand sources and identity.\nConcerns:\nThis paper struggles with some key concepts and is weak on getting across its true importance, which is crucial for an article in this journal. It lacks discussion of many other key works on this topic, which for some reason are omitted from the ‘global ocean’ data in figure 3, yet which includes data from less relevant studies. For example, there is no discussion of a study that already reported biologically mediated production was the main source of eHS in the Mediterranean, a nearly landlocked basin and therefore arguably more surprising to not be dominated by terrestrial humics (Dulaquais et al., 2018), or of work in the Arctic as a contrasting polar region (Slagter et al., 2019; Slagter et al., 2017). Instead, the justification for this work appears to be that the findings here are unexpected when considering a single previous study by Whitby et al. (2020), yet the conclusions of that study appear to have been misrepresented in order to fit the narrative here.\nThis paper needs to be written more accurately and transparently to best place these findings in the context of existing research. The paper should be revised to better emphasize that its novelty is in answering questions posed by previous studies to enhance our understanding of this little understood topic, and in studying this important region.\nThe paper would also benefit from more clarity for the non-expert on what the different ligand types are, and how they are related, particularly an explanation of the crossover between EPS and humics, as well as more detail in the methods section and a more honest view of any assumptions and limitations.\nBackground info\nFirstly, relevant for lines 50-55, 196 and elsewhere:\n‘Humic substances’ is a catch-all term which represents a continuum of organic compounds following degradation of (terrestrial or aquatic) biomass, including the degradation of polysaccharides, proteins, lipids, nucleic acids, lignin, cutin, melanin etc. Within humic substances, humic and fulvic acids are merely operationally-defined fractions, with humic acids soluble in natural and alkaline solutions only, and fulvic acids soluble at all pH levels. EPS is not a separate operationally defined fraction of humic substances (as inferred in line 196).\nBy definition, all soluble humic substances are a mixture of both HA-like and FA-like material; i.e. all samples contain a mixture of some ratio of material that either is soluble at all pH, or is not. HA and FA cannot empirically be distinguished from one another using the voltametric method referenced. eHS is itself an operationally defined fraction (electroactive humic-like material), and thus also a mixture of compounds that are either soluble at all pH (FA-like) or are not (HA-like). Any EPS that is detected by the eHS method would fall into either the HA-like or FA-like category purely because of this operational definition, as will many other compounds, not be distinct from it - it cannot be soluble and not be one of these if it contributes to the humic pool. The fact that some natural EPS in a sample behaves electrochemically similarly to the added FA used for the standard addition and is thus caught up in the measurement demonstrates that it shares FA-(or HA)-like properties. One can either argue that humic substances do not include EPS and thus this is an interference, or (some) EPS are HA/FA, but both cannot be true, as the authors appear to suggest.\nFinally, the eHS method cannot distinguish between sources of eHS, and cannot alone inform of the presence terrestrial or marine material, regardless of the standard used for quantification.\nLiterature:\nThere are many other relevant works that the authors may wish to consider for a broader understanding of this topic and in building their narrative, and the recommended expanded global eHS dataset for Figure 3; it is not necessary for all of these to be included in this article (not all have the relevant data for the figure), but I share in case they are of relevance (Dulaquais et al., 2023; Dulaquais et al., 2018; Fourrier et al., 2022; Laglera et al., 2019; Mahieu et al., 2024; Muller, 2018; Slagter et al., 2019; Slagter et al., 2017; Sukekava et al., 2018; Sukekava et al., 2024; Whitby et al., 2020a; Yamashita and Nishioka, 2023; Yamashita et al., 2020).\nComments\nTitle is appropriate\nL17-18 This would benefit with a sentence introducing what humic substances are before discussing their relationship with iron.\nL18 – this statement as written is incorrect, or the citation used does not support the statement.\n*(Note that reference 6 in the bibliography also has a typo: Whitby, H. et al. humic-like 'subtsances')\nThe Whitby et al paper does not propose that terrestrial humics dominate the ligand pool globally. There is a suggestion that the North Atlantic specifically has a higher degree of terrestrial influence, but not that terrestrial humics form the majority of iron-binding ligands in general. There is nothing to suggest terrestrial humics would be expected to dominate the ligand pool in the surface of the Southern Ocean, and I don’t believe the community think this is the case.\nSee figure 4 in Whitby et al which specifically separates iron limited from terrestrially-influenced regions, and the following relevant excerpts from the Whitby et al. study:\n‘Higher concentrations at low salinities demonstrate a terrestrial source, as would be expected. However, elevated concentrations in mesopelagic waters and relatively consistent values at open ocean locations also agree with a marine source and a refractory component [of humic substances]’.\n‘When considering that a large fraction of organic matter in the deep ocean is aged and allochthonous10, our results suggest that the persistence of terrestrial humics in the deep ocean may be pivotal in the global ocean iron cycle. However, there are many sources of both iron and humic material to seawater and it is likely that aged humics in the deep ocean are derived from a combination of terrestrial34, marine37 and even hydrothermal sources16. Regardless of origin…’\n‘Thus unsurprisingly, the humic contribution to iron complexation is dependent on the concentration of other, stronger ligand groups, varying regionally and with depth.’\n‘Deep waters reflect an accumulation of aged humic material of higher aromaticity28,29, linked to the persistence of terrestrial humics32,34, the selective decomposition of organic matter35, and the hypothesized aging process of humification24,28,34.’\nConsidering all of that, I think it necessary to reconsider the justification for this study and the text alluding to this within the abstract and other parts of the paper.\nE.g. the authors may consider something more transparent, along the lines of:\nBased on iron stoichiometry with electroactive humic substances, it has been proposed that humic material composes an important fraction of the iron-binding ligands in the ocean[6]. Humic substances are [definition]….., originating from a variety of sources. Some regions have a strong terrestrial humic influence, such as the Arctic Ocean[refs]. However, there is very limited data on humic ligands in the Southern Ocean, which is characterized by…..\nL20-22 Humics do not account for most of the ligands, but EPS account for most of the humics and most of the ligands? Is this because only a small fraction of EPS is picked up by the humic measurements? Please clarify the wording here.\nL43 – how does this work fit in with the recent study (in Nature) that claims that the ligand pool is less important than previously thought and that most iron in seawater may instead exist in authigenic colloids? (Tagliabue et al., 2023).\nL50-55 states:\n‘It has recently been proposed that Fe biogeochemistry across the global ocean is regulated by humic substances (HS, resulting from the degradation/humification of bacterial, plant and animal matter in soil, sediments and natural waters)6 . However, the study considered only terrestrially derived humics (Suwanee River fulvics and humics, further referred as HA and FA) as the main contributors to electrochemically detected iron-bound HS (eHS), despite other sources like atmospheric dust deposition15 and microbial reworking of marine DOM16-18 having been reported.’\nThe eHS method relies on a standard for quantification of eHS concentration in seawater; typically, Suwannee River Fulvic Acid or Humic Acid isolates are used, which are commercially available from the International Humic Substances Society. While a marine standard (and improved extraction procedure) would be preferable, currently there is not one commercially available at scale and the IHSS standard allows different studies to be compared with one another (as the authors do here). Terrestrial and marine humics share many similar components, and a terrestrial standard does allow quantification of the natural eHS, which in seawater is likely composed of some ratio of terrestrial and marine-derived humic material (‘humic’ encompassing many different things). It does not infer through its use that all of the material is of terrestrial origin. Furthermore, all humics and thus we can only assume all eHS is a variable mixture of FA-like and HA-like material, which cannot be determined using the existing method.\nThe cited study uses these known operational definitions of HA and FA to explain apparent trends regionally and with depth, and to infer the likely source of the material, such as using aromaticity from fluorescence measurements to infer changes to binding capacity, based on what is known for the FA and HA standards. However, I don’t believe the cited paper suggests that the FA and HA like components exclude EPS (or any of the many other components of humic substances). As stated above, EPS is not an accepted operationally defined fraction separate to FA or HA. Neither the paper cited (Whitby et al. 2020) nor this paper can empirically distinguish how much of the eHS is terrestrial, or how much actually is FA-like or HA-like. Just because an FA standard is used does not mean all of the eHS is FA, and vice versa, as the natural eHS peak can be quantified with either standard.\nFurthermore, as noted above the cited study discusses a variety of sources of humics, not only terrestrial, and thus the discussion is again incorrect.\nL59 – add space contrasting regions\nL68-69 please clarify what is meant by ‘how the correlation between DFe and eHS that served as a foundation for this assertion is not seen (Figure 2)’. Figure 2A appears to show data from citation 6, where the data from the Southern Ocean also appear to be in a similar position to the data here in panel B – i.e. low DFe, high eHS concentrations, unless I am missing something? (noting that Figure 2A is challenging to read when using triangles for every basin, even if they are different orientations. I recommend improving the figure by changing the icons). Is the point being made here that there are some values where DFe exceeds the humic binding capacity, suggesting that other ligands are important? If so, this is interesting and needs to be stated more clearly, although if that is the intention, it does not fit with the next two lines of text that suggest that DFe concentrations were low but eHS was similar to concentrations found elsewhere (lines 71-72). Clarity needed for this section.\nL77 – why Liverpool Bay, which is coastal, and the Western Antarctic study, which does not have ligand data, yet many other more relevant studies are omitted? There needs to be justification for the data used in this figure – and similarly, why other studies are ignored. I strongly urge the authors to include a wider range of published humic studies in order to have a transparent and comprehensive comparison that actually is an update on the original paper. See comment under Figure 3 below.\nL99 – It should be noted that the percentage contribution of eHS to FeL here is similar to findings in the South Pacific (Cabanes et al., 2020; Dulaquais et al., 2023).\nL118 – reworking of DOM, and also of POM - see ‘Contribution of electroactive humic substances to the iron-binding ligands released during microbial remineralization of sinking particles’ (Whitby et al., 2020a). It may be relevant elsewhere in this paper too as it includes data from the Southern Ocean.\nL126 – how is the amount of ligand that is allochthonous determined? (and similarly elsewhere, the amount of eHS that is terrestrial?) or is this purely through order of elimination when using measurements of known biogenic components? Clarity needed here.\nL129 – add space before ‘Whereas’ and then either reword this sentence so it is a complete standalone sentence, or join it with the subsequent sentence ‘…phytoplankton, carbohydrates…’ – there is also a typo there and the word should be ‘constitutive’.\nL193 – add is ‘is what determines’\nL205 – 207 reword this section to better convey the true novelty and importance. Remove revisit – see above, there is nothing to revisit, just build on. What is important here is that we have new data for the Southern Ocean, and further evidence that eHS has a strong biogenic component, supporting previous studies e.g. Dulaquais et al (2018). The authors could use this work to better clarify the relationship between EPS and HS.\nOne could argue, if there is a lot of excess ligand here, and a similar amount of humic material here to other regions, it is not then purely the low iron supply that is limiting productivity, and the ligands themselves have little impact on iron geochemistry, no matter what they consist of? The message of why this study is important does not come across as powerfully as it should for a Nature Communications article.\nI recommend the authors try to emphasise why it is important to know that most of the eHS present is of biogenic origin and thus explain the true novelty of this study.\nFigures\nFigure 1 is clear and well explained. However, I think it is a shame not to have the map shown as extended fig 1 within the main text.\nFigure 2A – use different symbols instead of all circles – even in different orientations, they are still challenging to distinguish between (and maybe more so for those with visual disabilities or dyslexia for instance).\nFigure 3 - In general I like this figure, but as currently presented it is misleading as it cherry-picks only some published humic data to support a certain narrative, omitting other data, and with incorrect labels.\nIn panel C, ‘Global Ocean’ should either read ‘North Atlantic Ocean’, as according to the paper, only the samples where both humic and ligand data was available was used for this percentage which was only the N Atlantic, or actually be a compilation of the existing global data that is available. I strongly urge the authors to do the latter.\nFor example, multiple studies have shown eHS to compose around 20%–60% of the iron-binding ligand pool (Dulaquais et al., 2018; Fourquez et al., 2023; Whitby et al., 2020b), while iron-humic complexes can be the dominant form of dissolved iron in the terrestrially-influenced Arctic Ocean (Laglera et al., 2019; Slagter et al., 2019). There is also another study in the Pacific which corroborates the Cabanes study with low eHS relative to DFe (Dulaquais et al., 2023). Combining these would give a more comprehensive global dataset. I therefore encourage the authors to either separate the data by basin, or, to have a Southern Ocean and ‘Global’ or ‘Rest of ocean’ dataset, to be truly transparent.\nFigure 4, Table 1, and section L135 -169 – This section and the linked visual material is challenging to follow, particularly considering line 150 ‘Together, this shows that EPS ability to bind Fe cannot be predicted from their hydrolysable carbohydrate content’. This section would be improved by a simple explanation for the lay audience (not EPS/ligand or DOM experts) explaining what is being compared and how they are related, particularly relating to the different forms of EPS and how they relate to eHS. Out of curiosity, how do EPS compare to other humic measurements, such as fluorescence measurements?\nMethods\nI hoped the methods section would give a more detailed explanation of some concepts that are difficult to follow in the main text, but it is rather sparse and missing some key details.\nGeneral:\nEnsure throughout that units are correctly written, e.g. µg instead of ug.\nThere is confusion with references – for example, on line 477 in methods, quotes reference 60, but there are only 19 references in the methods and 50 in the main text, so which is reference 60?\nThis may be up to the journal, but with the separate bibliography and numbering system restarting in the methods, there are then some duplicate references where a single paper has two numbers depending on the section being read, which can make it confusing for the reader and possible to accidentally read the wrong bibliography and use the wrong reference. Is it possible to have a continuation of the original numbered reference list in the methods so each paper has only one number?\nIt looks like there is the inclusion of some methods for data that is not shown or discussed in the main text; depending on the rules of the journal, I would recommend moving any methods relating to only supplementary figures and tables to the supplementary info so that space can be saved in the methods to better explain some of the key parameters.\nSpecific:\nL393 - missing some punctuation?\nL401 – HNO3\nL420 – remove stop after ‘as per 6’\nL422 – remove comma after ‘done’\nL428 – only 30s purge?\nL430 – add space ‘and the’\nL432 – why was only one ligand class fitted when SA typically gives multiple classes? It would have been interesting to see if two ligand classes were given and to compare the log K values of L1 and L2.\nL434 – why was the Laglera and van den Berg 2009 method used and not the updated method (Sukekava et al., 2018)?\nWhat is the full code for the IHSS standard; was the FA standard saturated with Fe prior to use? How many standard additions were performed? How was the detection limit determined?\nL474 – change ‘than in’ to ‘as in’. Typo in ‘largely’\nL476-479 Wording here is unclear. Used in situ ligand data instead of DFe but for what? They are different things.\nComparing to L gives an estimate of the % composition of L as eHS (ignoring that L is controlled by the artificial ligand selected, the detection window, pH buffered to etc). Comparing to DFe gives the relationship between the eHS and DFe, in turn ignoring that eHS can bind other metals and that DFe can be bound to other ligands. Both require assumptions, both are useful comparisons that give you an insight, but insights into different things and as written this section does not make sense. It’s impossible to check the reference 60 as it doesn’t exist in the bibliography.\nL477 – space between a potential\nL480-482 – again this whole section needs to be written more clearly. ‘we were able to transform measured eHS in an estimated Fe binding ligands concentration’ does not make sense grammatically. Also, please detail how this was done, give references/calculations/assumptions etc.\nL489 – ‘should be equal to, or exceed, the’\nL490 – ‘with respect to different ligands’ and ‘investigation into the respective roles’\nSee attached for bibliography.", "reviewer_2": "The manuscript entitled “Marine Biogenic Humic Substances Control Iron Biogeochemistry across the Southern Ocean” presents a comprehensive investigation into humic substances, with particular emphasis on extracellular polymeric substances (EPS) as a crucial component. Although only 1% of iron appears to be complexed with these substances, the study is original and makes a significant contribution to the field by addressing a critical gap in the current literature. The importance of exopolysaccharides in the pool of iron ligands, as well as their substantial influence on the humic fraction, is well-documented. However, this paper effectively demonstrates, through a large dataset, the specific influence of these substances on iron biogeochemistry.\nWhile the authors have successfully identified and addressed this research gap, some methodological and analytical choices require further consideration. Specifically, the use of correlations with low p-values but weak r-values suggests only a modest influence between the variables, rather than a strong correlation. Although the relationship is not random, the weak correlation raises concerns about the strength of the inferred relationships between the parameters.\nAlthough the data presented are robust and the experimental design is generally well-structured, the reliance on these correlations may not sufficiently support the accuracy of the data analysis. It is recommended that the authors minimize the emphasis on these correlations as currently presented.\nWhile the methodologies employed are appropriate for addressing the research question, it is unfortunate that the authors did not utilize a more recent version of the method for humic substances, such as the updated approach described by Laglera et al. (2007). The use of a more current methodology could have provided additional insights, particularly given the reliance on eHS results. Nevertheless, this does not detract from the overall quality of the study, which remains well-structured and informative.\nIn conclusion, I recommend acceptance with minor revisions.\nGENERAL OVERVIEW\nThe paper provides a good explanation of how ligands and humic substances interact. However, the explanation is not always clearly presented, leading to some confusion in certain sections. I strongly suggest reading papers that describe the kinetics of humic substances with other ligands, as these could help address some of the information gaps.\nRecommendations and Questions\n-Line 28: Change “de facto” to “in practice.”\n- Line 54:The paper should not focus exclusively on SRFA and SRHA as the only methods for measuring humic substances. This sentence appears too restrictive. The current phrasing implies that these are the only approaches, which is not accurate. Consider rewording to allow for alternative methods in the study of humic substances.\n- Line 59: Change “contrastingregions” to “contrasting regions.”\n- Line 61: Despite the significant p-value, the correlation is so small that it is difficult to believe any inverse relationship exists.\n- Line 65: Did you extract the percentage of eHS to represent binding capacity? Since you transformed humic substances into eHS using the binding capacity of SRFA and SRHA, these percentages could help reinforce the strong positive correlation.\n- Line 68: There are many other papers that describe this, such as Laglera et al. (2019). Be sure to reference relevant literature in the discussion, including Laglera et al. (2019), which provides additional insights into this topic.\n- Line 69: It is unfortunate that the method was not updated, as using a more recent methodology would have increased the value of this data. By applying the updated approach, it would have been possible to observe the amount of iron complexed with humic substances and to calculate the percentage of iron bound to humic substances. Despite this limitation, I agree that deep-water data could have had a greater influence on this outcome. Please reference the relevant paper that describes this issue.\n- Line 70: The comparison method in Figure 2b seems unnecessarily complex, despite the values being similar. I suggest changing the triangle symbols to different shapes to improve clarity. Adjusting the graphical representation may help the reader interpret the data more easily, without introducing unnecessary complexity.\n- Line 77: I disagree with the comparison of Liverpool Bay to other oceans. It would be more appropriate to use data from regions with similar environmental conditions or from areas with well-known concentrations of humic substances, such as the Arctic Ocean. The Arctic demonstrates high concentrations of humic substances and has a significant influence on the ligand pool. Liverpool Bay, with its distinct riverine inputs from major rivers such as the Alt, Clwyd, Dee, Ribble, and Mersey, and unique circulation patterns, is less comparable to open ocean environments.\n- Line 87: Change “theFe-binding” to “the Fe-binding.”\n- Line 95:T his could be associated with the presence of other ligands. It is well known that if DFO binds to iron before humic substances, the latter cannot displace it. This explanation should reference Sukekava et al. (2024) and the concept of 'first come, first served.' A discussion on ligand competition, particularly about existing literature, would enhance this explanation.\n- Line 102: The examples provided do not mention the Southern Ocean. I suggest reformulating the sentence to clarify that humic substances have been identified in various regions globally. A clearer description of the global distribution of humic substances would improve the reader's understanding.\n- Line 133: There is a need to reinforce the importance of conducting kinetic studies to address uncertainties about EPS and their classification as weak ligands. This could be an important measurement for future studies.\n- Line 189: The data is particularly interesting, as no riverine input seems capable of explaining the results. Dust and autochthonous humic substances appear to be the only plausible sources.\n- Line 390: I assume all the materials for sampling and analysis were prepared following the GEOTRACES protocol. Should this be explicitly mentioned in the paper?\n- Line 412: Has the zinc concentration data been utilized at any point in the analysis? If not, is it necessary to describe it in the text?\n- Line 433: Why were calculations performed with only one ligand class? Could this be clarified further?\n- Line 477: The reference appears to be incorrect; I could not locate any reference numbered 60. Please verify this.\n- Line 482: I am unclear on how you calculated the different ligand concentrations for humic substances and carbohydrates. Could you provide a more detailed explanation of this section?\n- Extended data Figure 1: While visually appealing, it is not clear how this figure contributes to the overall paper. It may be more effective to display only the sample locations without including the nitrate variability.\n- Extended data Table 1:If all the sampling locations are already included in Figure 1, I would recommend omitting this table, as it may be redundant.\nExtended data Figures 2 and 3, I recommend placing greater emphasis on the distinct behaviors observed across various locations, rather than focusing primarily on correlations between variables. Different regions exhibited unique patterns, particularly in ligand distribution. For instance, the High Nutrient Low Chlorophyll (HNLC) region and the Mertz Polynya displayed distinct groups of carbohydrates and eHS. These differences could be better integrated to highlight the varying chemical influences across different water masses, especially considering the influence of differing oceanic currents, as described in Smith et al. (2022) and Ardiningsih et al. (2020, 2021).", "reviewer_3": "Review of \"Marine biogenic humic substances control iron biogeochemistry across the Southern Ocean\" By Christel Hassler et al., submitted to Nature Communications, 2024\nScope of the manuscript, general assessment and recommendation\n--------------------------------------------------------------\nThe manuscript by Hassler et al. is a contribution to the long-standing and biogeochemically very important question concerning the nature of the organic ligands that have been shown to control iron\nresidence time and bioavailability in the ocean. It does so based on a new data set of measurements of electroactive humic substances, iron binding ligands, and dissolved iron concentrations mostly from the upper 200m depth below the surface in the Southern Ocean. It is argued that the bulk of the electroactive (iron-binding) humics and ligand iron binding in their samples is based on exopolymeric substances produced by bacteria and phytoplankton, as part of the marine humic pool.\nBoth the data set and its interpretation are welcome additions to an important question regarding the iron cycle in the ocean, and in principle merit the publication in a high-profile journal such as Nature Communications.\nBesides several minor points listed below, I have, however, one main point of criticism of the present manuscript, and can therefore not recommend publication before major revision.\nThe manuscripts refers mainly to Whitby et al, 2020, another paper on the role of humics in the global iron cycle, to show the novelty of the results and interpretations here, with respect to previous ideas. In doing so, however, it builds a strawman, and strongly misrepresents what has been said in that paper.\nThis already starts in the abstract that states:\n\"Based on iron stoichiometry with electroactive humics, it has been proposed that humic substances of terrestrial origin may form the majority of iron-binding ligands in the ocean(6). Here, we (...)\ndemonstrate that terrigenous humics cannot account for the bulk of in-situ ligands.\"\nHere (6) is a reference to Whitby et al, 2020. The statement that humic substances of terrestrial origin may form the majority of iron binding ligands in the ocean is never made in that paper. Instead, allochthonous humics (including riverine terrestrial as well as dust-derived) is discussed as just one component of humics, besides marine humics, derived mostly from phytoplankton. It is stated that allochthonous humics have a higher aromaticity as opposed to marine humics, which are more carboxylic-rich and/or aliphatic, consistent with the present manuscript. And moreover, it is also said explicitly that\n\"In our samples from the upper 1000 m of the Northeast Pacific and in all samples from the Southern Ocean, where most humic-like material comes from the recent microbial degradation of sinking autochthonous organic matter (30), the DFe/eHS ratio was below or equivalent to the lowest reported binding capacity of terrestrial humics\".\nSo the finding that most humics in the surface Southern Ocean is not terrestrially-derived has already been made in Whitby et al.\nFurther down, Whitby et al. state\n\"When considering that a large fraction of organic matter in the deep ocean is aged and allochthonous (10), our results suggest that the persistence of terrestrial humics in the deep ocean may be pivotal in the global ocean iron cycle. However, there are many sources of both iron and humic material to seawater and it is likely that aged humics in the deep ocean are derived from a combination of terrestrial(34), marine(37) and even hydrothermal sources (16).\"\nIt is also stated that this statement is largely based on the data from the North Atlantic and that there is a lack of deep samples in the Pacific and Southern Ocean. All in all a much more nuanced statement than the statements ascribed to it in this manuscript.\nThe current manuscript also states, referring to Whitby et al. that\n\"the study considered only terrestrially derived humics (Suwanee River fulvics and humics, further referred as HA and FA) as the main contributors to electrochemically detected iron-bound HS (eHS),\ndespite other sources like atmospheric dust deposition (15) and microbial reworking of marine DOM (16-18) having been reported\".\nBesides forgetting photodegradation of terrestrial humics as another source of aliphatic DOM, this is simply a wrong representation of the statements in Whitby et al., as shown with the\ncitations above. But it maybe explains where the authors of the present manuscript have their misconception from: Indeed the extent of iron complexation from eHS is estimated in Whitby using the published values for iron complexation for HA and Fa as bounds (which, by the way is also what is done in this manuscript. But, Whitby justify this by stating\n\"we account for the full range of published iron binding capacities for terrestrial standards from the Suwannee River, using an envelope that encompasses the maximum (Suwannee River Humic Acid, SRHA) and minimum (Suwannee River Fulvic Acid, SRFA) reported values (13,21,27) as a first approximation (Fig. 2). While marine humics could well incorporate a wider range, the mean iron-binding capacity of our samples (Supplementary Table S1), as well as in the Pacific Ocean (13) and Mediterranean Sea (12), indicate a similar range to terrestrial standards, lending confidence to this assumption\"\nThis misreading of the published reference literature has to be corrected before this manuscript could be published.\nMajor comments\n--------------\nLine 25: \"Thus autochthonous and freshly produced organic matter\": I understand the argument for autochthonous, but not for \"freshly produced\". As far as I know some marine humics can also be quite\nrefractory (CRAM), so I do not see the two as equivalent. What is the argument here?\nLines 64ff: mathematically, I do not agree that a strong positive correlation suggests that eHS represent most of the FeL pool. A positive correlation per se just emphasizes a linear relation between the two variables, but does not exclude a large y-axis intercept of the relation and also not a 1:1 slope. I assume that this statement is probably based on more than the correlation alone, but that should be stated, or else the inference be formulated more cautiously here.\nLine 68-69: It is a misrepresentation that the assertion in Whitby et al, (2020) was founded on correlation. The term, used in Whitby is much more cautious: \"We find broad correspondence between dissolved iron and humic ligands\", immediately followed by a statement that a variability of binding capacity hinders straightforward comparisons. The main argument here is that the calculated iron-binding capacity of eHS is generally larger than the dFe concentration, with clear and systematic differences between surface/deep waters and between Atlantic/Pacific/Southern Oceans. Actually, the statement in Whitby that \"In 49% of the samples shallower than 200m DFe/eHS values near or below the lower binding capacity suggested undersaturation of humics with iron\", which also includes SO data, agrees well with the data shown in Figure 2b of the present manuscript.\nLine 140: what is \"binding complexity\"? I suppose this means complexing capacity, but this remains unclear. Also, how is it calculated and does the quantity have a unit?\nIn lines 187 ff, Hassler estimate a contribution of dust deposition to in-situ eHS, coming to the conclusion that it is a negligible factor. While I agree to the result, the calculation done here is\nsimply wrong: Firstly, the authors convert the deposition to a concentration by arbitrarily setting the depth of the surface layer to one metre. As the mixed layer of the ocean is typically well-mixed on timescales longer than a day, a more appropriate depth range would be the mixed layer depth, further decreasing the contribution. Secondly, a flux has the unit of a mass/m^2/day; the time unit has been completely forgotten here. Dividing a flux by a depth does not give a concentration, but an increase rate of a concentration per day, or whatever time unit the flux is defined in.\nLine 199-200 \"that become relevant at different dFe concentrations\": I would add \"and at different pH values\" to that, see Lodeiro, P., Rey-Castro, C., David, C., Humphreys, M.P., Gledhill, M., 2023. Proton Binding Characteristics of Dissolved Organic Matter Extracted from the North Atlantic. Environ. Sci. Technol. https://doi.org/10.1021/acs.est.3c01810\nLine 204: i would add \"surface\" before SO, as this is what this paper shows. It is probably different in the deep ocean, as discussed both by Whitby et al (2020`), and this manuscript. Of course one could argue that bioavailability is relevant only in the surface ocean, and this is what is talked about here. That's o.k., but maybe one should then explicitly state \"phytoplankton bioavailability\" as bacterial bioavailability might be different.\nFigure 4B: I am missing the exact definition and unit of the quantity shown here. Also there is no explanation what the underlying red bars are. One can assume that it is the measured values, but this not stated explicitly here, and the range shown here is also not explained.\nTable 1: I must admit that I failed to comprehend what is shown here, and the explanation 158-160 didn't help me really. What is the measured quantity, what is calculated from it?\nMinor comments & typos\n----------------------\nLine 59: \"contrastingregions\" -> contrasting regions\nLine 129ff: The sentence starting with \"Whereas\" is incomplete, I suspect some part has been lost in a previous copyediting operation. There is also no whitespace between this and the preceeding\nsentence. There is also no whitespace between this and the preceeding sentence.\nLine 380: \"measure\" -> measured\nLine 474: \"similar .. than\" does not sound right.\nLine 474ff: The sentence starting with \"Because\" is missing a verb, I believe\nLine 477: \"apotential\" -> a potential"}, "author_response": "We are very grateful for reviewers’ comments, which have greatly strengthened our manuscript. Our revised manuscript addresses all the comments and concerns listed.\nTo show how the reviewers’ comments have been addressed in our revised version of the manuscript, we copied their original reviews and added our reply below each comment in italics, with references to line numbers of the revised manuscript. Changes can also easily be assessed in the version with track changes shown."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Most of the reviewer comments have been addressed appropriately and the manuscript is far more inclusive and transparent than the previous version. There are some issues with figures, particularly fig 2, noted below. Some parts of the new manuscript do feel rushed with typos or referencing mistakes which are highlighted below, or text that assumes readers already have a greater understanding of ligands, humics etc than I would expect the general audience of this journal to be expected to have, and so it does still require some minor attention. There is still some reliance on correlations which was noted by multiple reviewers, although the text has been modified to tone down the language and a suitable rebuttal given.\nThere is however still an issue with the way the novelty of this study is presented. For example, in lines 188-190 at the start of the Discussion – it’s great that there is now more data to further understand Fe biogeochemistry in the Southern Ocean, and this data deserves publication for that alone, as well as the expansion to including Carb and EPS, but as was pointed out by two different reviewers, the differing role of eHS in the Southern Ocean compared to elsewhere was already noted in Whitby et al. 2020 (see comments from R1 and R3). Furthermore, the difference in eHS contribution to FeL between the South Pacific, Southern Ocean and elsewhere was also recently highlighted in Whitby et al., 2024. It’s fine to keep the text as it is, but it would be appropriate to reference the earlier studies here to demonstrate they are being built on and this new data augments and concurs with those earlier findings. The novelty is in the expansion of this complicated story with the inclusion of additional complementary measurements, and this still needs to be better highlighted.\nDetailed comments:\nThe abstract is much improved, but it could still be clearer. Essentially, I think you want to say that you find that humic substances are an important part of the ligand pool for Fe in the Southern Ocean as has been found elsewhere, but that it is mostly produced in situ and composed of EPS, in contrast to other regions where terrestrial-derived humics may play a greater role(?). As written this doesn’t really come across.\nFigure 1B – the arrow is hiding the label of eHS, move the label so that it can be clearly seen\nFigure 2:\n- caption – is DDFe a typo? Also, what about FeL in this description.\n- missing references within the caption text (just says ‘refs’ in multiple places).\n- The data for the Southwest Pacific is missing from the Fig 2a, or the description for 2b should clearly state that this figure also includes data for the Southwest Pacific as well as the Southern Ocean, otherwise this is misleading.\n- I cannot see lines on the figures for ‘Calculated DFe saturation of eHS is shown (dotted lines) considering HA (top) and FA (bottom)’ on the pdf although they are weakly visible in the tracked changes document.\n- The caption states ‘Relationship between eHS and L is shown for (c) different ocean basins…’ the axis labels instead show DFe vs L, not eHS. Either axes or caption incorrect. Again, d says ‘for Southern Ocean’ when actually it is Southern Ocean + Southwest Pacific.\n- L517 – typo in ligands\n- For the Mediterranean Sea, only one reference is provided to Dulaquais et al. 2018, however that paper uses FeL data first published by Gerringa et al. 2017. If the Gerringa data is used it should be correctly cited. There is no Northeast Pacific FeL data in ref 14 and no other reference is provided so I’m not sure what data figure 2c and d is using for the Northeast Pacific. Is it necessary to distinguish between North Atlantic and Northwest Atlantic in the figure? In fact, shouldn’t it be Northeast Atlantic, not Northwest, if it’s referring to data from Dulaquais et al 2018? These confusions could be resolved by making it clearer to which references the data on the figure refer to. One solution would be to include the numbered references next to the basin name within the legend on each subplot, because it’s difficult to associate the data on the figure with the correct references in the caption.\nFigure 3 – ‘as in ref 6’ – ref 6 is Boyd et al., 2010? I don’t think this is the correct reference. I cannot see the dotted line that represents the 100% contribution to FeL on the pdf (although I can see it on the tracked changes document). You may wish to just list the ocean basins along the y axis instead of numbers 1 – 6 with numbered references, it would be easier to follow. Also why does the x axis on D not start at 0? Finally, similar issues to Fig 2 for references.\nFigure 4 – I don’t fully understand this figure, the caption could better explain what is being shown, what is meant by ‘role on in situ Fe biogeochemistry’. What is ‘Maximal Fe-L’?\nSpecific comments:\nL35 – thee to the\nL70 – ‘despite recent data suggest’ is missing something, e.g. ‘which suggests’ or ‘suggesting’\nL74 –what is the ‘Uniqueness of iron..’?\nL76 – move comma after life\nL82-88 – give some more information on binding capacities, what it means to be similar to Suwannee river HS etc. The average reader without knowledge of humics will not understand this section.\nL92 – is DFe supposed to be here or a typo\nL89-92 – It could just show that humics are less saturated with DFe, which is not surprising in an Fe-limited ocean.\nL89-100 – overall this section would be challenging for many readers to follow if they are not already experts in ligands and humics. I suggest making this a little more accessible to non-experts.\nL108 – “first served”\nL116 – what is meant by ‘the bulk of eHS… cannot be described by standard FA and HA substances” – are you inferring that these eHS have a different binding capacity due to being marine-derived? Or that other ligands are important in these regions?\nL121-122 – I do not understand this sentence (weaker relations to what?), plus it has multiple typos - “Coexistence of numerous coumpounds [change to compounds] typically results in “weaker” relations that [change to than] expected on known processes28”\nL124 – ‘to specific microorganisms’ – I did not see any data presented here on comparing to individual microorganisms, only to bacteria vs phytoplankton. Reword this for clarity.\nL140 – add here “EPS, which compose part of the eHS pool, …” (or however you wish to associate them) for clarity\nL174 – add space before ‘We’\nL240 – add stop after inputs.\nL268 – the lack of ‘a’ marine humic standard\nL277 – add space between ‘they modulate’\nL288 – should this be particles\nSome comments from previous review round have no response, e.g. R1 asks ‘only 30s purge?’ with no response, and ‘A single ligand class was obtained’ doesn’t really address the question as to why only one ligand class was obtained when usually SA gives 2. Was an attempt made to fit multiple and it was not possible? Why might this be the case as this is unusual. I don’t consider receiving responses to these queries should be an impediment to publication, but if further review iterations occur, it would be good to see the explanation.", "reviewer_2": "After reviewing the revised manuscript, I am pleased to confirm that the authors have significantly improved the paper. Many concerns raised in earlier reviews, including mine, have been addressed thoughtfully, resulting in a more cohesive and compelling narrative.\nThe flow of the manuscript has greatly improved, and even sections that I initially found problematic—such as the statistical analysis—now make more sense within the revised structure of the text. By integrating various components effectively, the authors have summarized their findings in a way that better conveys the significance and novelty of the study.\nThe methodological clarifications and the inclusion of additional references have strengthened the paper's foundation. The authors successfully addressed concerns about previous misinterpretations of data and justified their analytical approaches with clearer explanations. The revised figures and improved statistical presentation enhance the paper's readability and reliability.\nIn conclusion, this paper presents a valuable and well-substantiated contribution to the field, and I recommend it for publication.", "reviewer_3": "Review of the first revised version of \"Marine biogenic humic substances control iron biogeochemistry across the Southern Ocean\" by Hassler et al, submitted to Nature Communications\nIn their revised version, the authors have corrected my main concern with the first version of the paper, which was the misreprepresentation of the conclusions drawn in the study by Whitby et al., 2020. They have also clarified many of their unclear statements and now discuss much better how the different, often operationally defined, subclasses of marine DOM (humic substances, electroactive humics, ligands, EPS) relate to each other. Although the methods used do not allow to directly quantify the role of EPS in binding iron, the authors now quite convincingly show that EPS has the possibility to be an important contributor to Fe-binding ligands in the Southern Ocean and South Pacific. I would therefore now recommend publication of the study in Nature communications, after some minor revisions.\nMinor comments\n--------------\nLine 50: 'Iron bioavailability varies ...'; the authors of course know very well that the bioavailability, as it is defined in citations 12 and 13 is not a property of seawater iron chemistry alone, but is also influenced by the uptake mechanisms of the phytoplankton species involved; maybe it would be helpful for the reader if this was mentioned briefly in this paragraph.\nLine 82 ff: I find the statement made here (especially 'suggest' and 'most') maybe a bit too strong for the fact that a) the intercept 1.3 nmol/L ligand for zero eHS is not negligible, and b) that the calculation has been done using the lab-based maximum Fe binding capacity for Suwanee river humics, i.e. neglecting the competition of other cations for binding.\nBesides, I do not understand to which graph the mentioned intercept and slope in line 85 belong, to Figure 2d? Maybe it would help here whether the nmol/L are actually nmol/L of Fe or of 'Fe-L'?\nAnd, to add to my confusion: If the slope is 0.045 nmol/L / (ug eHS) (which, I assume should be 0.045 nmol/L / (ug eHS / L)), why is then 1 ug eHS equivalent to the inverse 1/0.045 = 22.2? This whole calculation is unclear.\nLine 134 ff, '...Fe bioavailability, more than Fe concentration, is key to phytoplankton photosynthesis and growth': I have some difficulty with this statement. The positive correlations of Fv/Fm with Fe-L and eHS indeed suggest that iron that is bound to ligands and electroactive humics is bioavailable. But then why is there no correlation observed to DFe? Does this suggest that some part of the DFe is NOT bioavailable, and which part would that be, given that ligand concentrations shown in Fig 2d are fairly high, and ligands are mostly unsaturated with Fe? Colloidal DFe? Iron bound to some strong ligands that are not detected?\nLine 223: Given that it is based to a large part on assumed iron-binding capacities for humics, the statement 'Our study identifies EPS as the bulk of Fe-binding marine humics and ligands' is maybe a bit strong.\nLine 227: The citation 56, given as support for the association of hydrothermally derived iron with (e)HS, is rather indirect: It deals with humics in hydrothermally influenced sediments. Is there more direct evidence for the association of humics with Fe in hydrothermal plumes?\nFigure 1B: would it be possible to separate the labels eHS and L in the figure somewhat? And why is 'Fe-L' called 'L' in the figure and caption? In the caption it is mentioned that logK and Carb are not statistically represented. Does that mean that only a low part of their variance is explained by the first two EOFs? Then I would remove them from the plot alltogether, right?\nFigure 2: The dotted lines are not visible in my printout; they are visible in the pdf at some magnification. Maybe make them somewhat more prominent? In the caption, line 512: 'DDFe' -> 'DFe', Maybe ligands should also be mentioned in the bold part of the caption? Obviosly some references were intended to be included but weren't '(refs)'. What does the 'mostly this study' mean?\nLine 558 'following GEOTRACES guidelines': This merits a citation.\nLine 634: How large, approximately, is the error in the calculated complexation capacity, as calculated from the errors in L_insitu, DFe and the conditional stability constants?\nTypos and small comments\n------------------------\nLine 35: thee -> the\nLine 48 and several following: I find it somewhat unfortunate to abbreviate iron-binding ligand concentrations as Fe-L, as this might be mistaken for the concentration of iron bound to ligands. Maybe LFe (possibly with Fe as subscript) is less confusing and more in line with other papers on the subject?\nLine 92: I think 'South Pacific)DFe' should just be 'South Pacific'.\nLine 121: 'that' -> 'than', 'on' -> 'from'\nLine 138: Here ligands are sudden;y abbreviated as 'L', not as 'Fe-L'. Why?\nLine 189: 'humic' -> 'humics'\nLine 517: 'liagnds' -> 'ligands'. There should be a '1,' before 'Arctic'.\nLine 650: '_H. Forrer' -> ' H. Forrer'"}, "author_response": "We are very grateful for this second round of reviewers’ comments, which have improved our manuscript. Our revised manuscript addresses all the comments and concerns raised.\nTo show how the reviewers’ comments have been addressed in our revised version of the manuscript, we copied their original reviews and added our reply below each comment in italics, with references to line numbers of the revised manuscript. Changes can also easily be assessed in the version with track changes shown and corresponding lines indicated in our reply."}]

Decision letter