Microbial strong organic ligand production is tightly coupled to iron in hydrothermal plumes

Hoffman, Colleen L.; Monreal, Patrick J.; Albers, Justine B.; Lough, Alastair J. M.; Santoro, Alyson E.; Mellett, Travis; Buck, Kristen N.; Tagliabue, Alessandro; Lohan, Maeve C.; Resing, Joseph A.; Bundy, Randelle M.

doi:https://doi.org/10.1101/2023.01.05.522639

Preprints

https://doi.org/10.1101/2023.01.05.522639

Preprints

06 Oct 2023

| 06 Oct 2023

Microbial strong organic ligand production is tightly coupled to iron in hydrothermal plumes

Colleen L. Hoffman, Patrick J. Monreal, Justine B. Albers, Alastair J. M. Lough, Alyson E. Santoro, Travis Mellett, Kristen N. Buck, Alessandro Tagliabue, Maeve C. Lohan, Joseph A. Resing, and Randelle M. Bundy

Abstract. Hydrothermal vents have emerged as an important source of iron to seawater, yet only a subset of this iron is soluble and persists long enough to be available for surface biological uptake. The longevity and solubility of iron in seawater is governed by organic iron-binding ligands, and strong organic ligands called siderophores are produced by marine microorganisms to access and retain iron in soluble forms. Siderophores and other microbially-produced ligands comprise part of the ocean’s dissolved iron-binding ligand pool, which is hypothesized to aid in the persistence of soluble iron in hydrothermal environments. To explore this hypothesis, we measured iron, iron-binding ligands, and siderophores from 11 geochemically distinct sites along a 1,700 km section of the Mid-Atlantic Ridge. For the first time, we identified siderophores in hydrothermal plumes at all sites. Proximity to the vent played an important role in dictating siderophore types and diversity. The notable presence of amphiphilic siderophores may point to microbial utilization of siderophores to access particulate iron in hydrothermal plumes, and the exchange of iron between dissolved and particulate phases in these systems. A tight coupling between strong ligands and dissolved iron was observed in the neutrally buoyant plume across six distinct hydrothermal environments, and the presence of dissolved siderophores with siderophore-producing microbial genera suggests that biological production of siderophores exerts a key control on hydrothermal dissolved iron concentrations.

Received: 02 Sep 2023 – Discussion started: 06 Oct 2023

Status: closed

RC1:
'Comment on egusphere-2023-2017', Anonymous Referee #1, 06 Dec 2023
Review of "Microbial strong organic ligand production is tightly coupled to iron in hydrothermal plumes" by Hoffman and coauthors:
The manuscript employs a combination of classical electrochemical methods (CLE-AdCSV) to measure iron ligand concentrations in solution with novel developments in chromatographic recognition and quantification of siderophores to shed further light in our understanding of iron speciation in the ocean, in this case in the plumes of hydrothermal vents. The goal of the chromatographic section is to detect a broader range of these compounds for which a careful description of the level of confidence in the assignment of molecular formulae is described. The manuscript is well-written and accessible to specialists.
However, although the experiment planning and the resulting database are extremely valuable for the scientific community, I have strong reservations about the interpretation of the results, particularly in the electrochemical part and I am going to suggest major changes. Whole sections should be thought through using the arguments hereafter and followed by rewriting of many sections. Accordingly, I would only consider this manuscript as a letter if those changes are implemented.
My major concern with the current version is the assumption that the outcome of CLE-AdCSV represents "organic" ligands' concentration and stability constant. Unfortunately, a wrong assumption, once established, might be challenging to reverse. In waters with high terrestrial dissolved organic matter (DOM) input or significant biological activity (resulting in ligand exudation), this assumption might be close to the actual situation. However, it cannot be universally granted. The technique involves introducing an artificial ligand that equilibrates with the sample (CLE step), extracting a fraction of the iron in solution, and then measuring the concentration bound to the artificial ligand (the electrolabile fraction, the AdCSV step). If there are no sequestering natural ligands, the artificial ligand recovers the entire iron concentration, and electrolabile iron equals total. If all iron is so refractory bound that it does not exchange at all overnight in contact with the artificial ligand, the concentration of electrolabile iron is zero, and all iron is considered complexed with a stability constant that should be infinite (K~∝) but that the analytical window limitation of the technique forces to fall inside this window. Real samples exhibit an intermediate situation, with a partition between iron complexed to the original ligands and the added ligand, dependent on the concentration and stability constant of both ligands.
It is evident from this description that CLE-AdCSV cannot discriminate between organic and inorganic fractions, only between "labile" and "refractory" fractions competing for exchange with the added ligand. Historically, since the oceanographic studies in the 80s, researchers assumed that the partially exchanged fraction should mostly be of organic origin, especially since the technique was initially used for copper, which has no solubility issues. As the field started the study of iron in the 90s, this assumption was carried over and it was assumed that inorganic oxyhydroxides do not contribute to the partially exchangeable fraction, as their concentration is presumed to be very small and their stability very small compared to organic ligands. However, this is hardly compatible with the well known low solubility, formation of aggregates characterized by very different reactivity as a function of aging, estuarine trapping and open ocean scavenging suffered by iron.
It is certain that the manuscript's assumption about the organic complexation of iron in hydrothermal fluids relies on the literature. However, this stems from the assumption that CLE-AdCSV experiments exclusively determine "organic" ligands (Buck et al., 2018; Kleint et al., 2016; Sander and Koschinsky, 2011). This assumption is highly unlikely in hydrothermal waters with surges of Fe(II) at substantial concentrations. Fe(II) quickly oxidizes (hours-days at the local temperature and pH), creating stable colloids that continue to grow until reaching a size that induces precipitation. And no local nanomolar concentration of ligands can prevent this precipitation of micromolar iron. In this scenario, if the sample for CLE-AdCSV contains any inorganic phase smaller than 0.2 µm, that does not undergo complete solubilization overnight by the extracting effect of the artificial ligand, it will be counted as a ligand and labelled as "organic”. This is very unlike in this case. Some ligand concentrations presented here are of the same order of magnitude as those measured in cultures using cell growth media and an order of magnitude higher than iron ocean fertilizations. This is very suspitious.
In my opinion, considering that part of the ligands may be inorganic would explain 1) the high ligand concentrations, 2) the high correlation of ligands and dissolved iron independent of any biological variable, 3) and the high concentration of ligands in the absence of primary producers that could support the ligand production of bacteria. Actually arguing fot the presence of inorganic fractions in the definition of ligands, would drift from the current paradigm and add extra relevance to the manuscript.

Other concerns:
There is a non explicit, but implicit, argument pointing to siderophores as a “major” part of the so-called L1 (my interpretation). This is not based in literature where there is no evidence of such, it seems very unlikely (siderophore production is very energy demanding and bacteria requirements are not that high, and the bibliography used is very one sided to this argument. In my opinion the ecological importance of siderophores is paramount but, since the fraction of iron complexed by siderophores in minor (<2% with our current analytical tools), siderophores should not be considered a major driver of iron cycling. No author has found important concentrations of siderophores to justify this relevance (Bundy, Boiteau, Gledhill, Ahner and so on manuscripts).

The authors do not seem aware of the limitations of the technique and the limitation caused by the existence of an analytical window. In table S3 stability constants (K1 to K3) are spread in almost 5 orders of magnitude. Such accuracy is impossible, it would mean negligible analytical error. The analytical window is approximately 4 orders of magnitude wide (depends on the error, there is related bibliography from Apte, van den Berg, Pizeta, Laglera, Gledhill and Gerringa) and K fall inside the range, cannot fall on the very edges or beyond like in a few cases here. Could this be related to the use of salycilaldoxime as competing ligand (recent Geriinga et al BG manuscript)?

In the methodology section the authors state that “forward” and “reversed” titrations were performed but this is not shown in the results section neither discussed. Is that high K1 coming from reverse and low K3 from forward titrations? Results form both treatments cannot concur if more than one ligand (as it is 100% the case here) are present and a separate discussion and comparison are due. This must be added to a future version

Self citation is excessive and the authors give as settled some particular arguments that are not accepted by the whole scientific community, example the referred prominence of siderophores as source of strong ligands in the ocean. I think that papers should reflect and discuss other visions in the field.

I know that this is quite radical but I would suggest to mend the interpretation of the voltammetric data, commenting on inorganic complexes and discussing forward and reverse and focus on the interesting chromatographic finding. The paper must be put into the context of the ecological relevance of these finding more than in the relevance for iron cycling since the siderophore concentrations found here apparently only binds a very minor fraction of the iron concentration in solution.

As I was reading I took some note that should be of interest to the editor and authors. I attach them, part are a repetition of what I stated above:

48 I find here that self-citation is a bit excessive, there are more people involved in this type of studies
For being a L1 it must be a L2. This is based in results from a particular technique that do not match results from other CLE-AdCSV protocols. I mean that other analytical approaches do not always measure L1 and L2 using a different artificial ligand. Moreover, when log K is outside the analytical window, the limitation of the analytical window brings the value inside. The classification of ligands as a function of log K is not a sensible strategy.

53 again self-citation. There are many more studies about transition of iron from estuarine waters to the sea that so not concur with this vision. The importance of humics (that I assume from the authors’ previous publications that they consider weak ligands) has been well established in many studies (Laglera/van den Berg, Slagter, Yang and Muller studies by CLE-AdCSV and many other studies using fluorescence, coprecipitation). Other studies have found that transport is a function of the molecular weight of the ligand with prevalence of smaller fractions. The process and visions of different research groups are quite more diverse than simplified here.
60-64 please revise grammar
66 see my previous comment about organic ligands and hydrothermal fluids
71 word repetition
72-76 impressive range of sampling sites with different physicochemical conditions. This gives relevance to the manuscrit

Appendix 226……. Methods
245 the concentration of buffer is possibly wrong. It should be millimolar and not micromolar. This concentration would not buffer at all against the bicarbonate natural buffer, let alone against the huge formation of hydroxides inevitably associated to the polarographic analysis of oxygen saturated solutions. If the buffer was settled at such concentration, the analysis was carried out at pH close to 9 (Laglera et al 2016)
10 micromolar SA seems a compromise solution between the concentration suggested by Abualhaija and van den Berg (5 uM) and the concentration traditionally used by Buck and collaborators (25 uM). Since doubts about the use of SA increase (Gerringa et al BG paper) it is not clear that the effect of the Fe(SA)2 complex has been removed and not counted as L3. It would be good to show a linear titration of UV digested seawater in this condition to rule out such effect.
254 here the boric acid is at the same low concentration which makes me think that perhaps the buffer was not correctly implemented and the analysis was carried out at a very basic pH (at the surface of the electrode). For instance Hawkes et al (2013) 5 mM in each aliquot (50 mM in the paper is wrong).
What was the pH of the solution here? NN is supposed to work only at low pH (8 or less) according to the intial Gledhlii/van den Berg papers. This pH is so far away from the pK of the buffer (close to 9) and the buffer concentration so low that its buffering effect would be null. This is usually detected by changes in the peak potential. Can the authors compare peaks for this work with peaks obtained in studies with higher concentrations of buffer?
260 I could not find in Hawkes et al (2013) any reference to χmin = 0.8, χmax = 0.9, and c1high = 0.75. What are these constants and what is the implication of fixing them at this value and not other? I found them in the R script and although I am not expert in R it seems that the authors of the R routine suggested to use 0.9 or 0.8 as maximum value reached during the RV if the shape of the curve was not that of a double michaelis-menten. I know the topic of this manuscript is not to criticize such but it all looks very arbitrary to me and not sure how much change in L and K would bring a change of value here. I do not suggest to recalculate anything but the method used is a bit arbitrary in the assignment of constants.
265 onwards: I congratulate the authors for the effort to apply an internal quality criterion. This is randomly the case and improves greatly the relevance of this work.

266 why the result is called L1? Is there L2 in samples?
310 “siderophore concentrations reported here are estimates of siderophore concentrations in these environments based on ferrioxamine E.” although this is obviously a strong limitation, possibly this is the only way to move forward. In cases like this applied to concentrations obtained by means of other techniques, cconcentrations of other siderohores are reported in DFOE equivalents and not simply as nM. Hopefully, at the time to evaluate total siderophore concentrations, overestimations and underestimation may compensate but it would be interesting to evaluate whether DFOE gives sensitivities around the average for all compounds commercially available. Because if DFOE is particularly more or less sensitive to the detector, the authors would incur in substantial over or under estimations of concentrations for other siderophores. Was this considered at the time to select DFOE? A comment should be added

80 this sentence is 1) not based in a prior understanding of CLE-AdCSV in the case of 2018 Buck; as I referred before, the technique does not discriminate organic or inorganic ligands 2) not based in any experimental evidence in the case of the other two papers that are one a review and the other one a model where CLE-AdCSV ligand have been added.
83 onwards. Although the argument about complexation seems right and coherent, again a concentration of 10-90 nM ligands are 1 to two orders of magnitude higher than observed in very concentrated cultures or fertilization at any growth stage. Since hydrothermal plumes are not watermasses especially abundant in biomass, the biological release of tens of nanomols per litre of “organic” ligands is extremely unlikely. This would be energetically absurd, to release ligands for concentrations that are well over the iron requirement. That some aged/stabilized oxyhydroxides and/or iron sulphides are part of the sample is a more likely explanation.

Log K3 values around 8.8 are difficult to reconcile with what we know about analytical windows and CLE-AdCSV. This is especially true if the authors claim that can resolve ligands of log K 12-13 and 9-10 (separated 3 orders of magnitude) from the same titration. It would be a mathematical artifact
94-96. Again there is only self citations about rivers where there is no consensus about and there are available results from other groups that differ substantially with the processes described here. In any case it is good that the adjective organic dropped in this paragraph.
100-101 Again self citation. Recent evidence shows that a fraction of humics of riverine origin compete with siderophores for dFe (Slagter and Laglera papers in Arctic waters). Moreover, I insist that stabilized/growing inorganic fractions (of no biological origin) could be found in the L1 fraction and in the physicochemical conditions described here, constitute most of L1.
101-103 all these processes are no doubt present, but very unlikely to produce L1 ligands in the order of tens of nM.
107 None of the Cowen references include ligands measurements or even include the word ligand. The Lauderdale paper is a modelling paper and does not constitute empiric evidence. The bibliography does not support the argument
118-129 this section is very speculative and as such should be remarked. please remove significant since this term implies some statistics behind and this is not the case, it is just a speculation. The Hider and Kong reference is a review and only speculates about whether more products are expected. My problem here is that the paragraph is based in repeating a speculation. Other sources of L1 referred to in the bibliography do not deserve even a mention (humics, EPS, etc)
126 this calculation is addressed to increase the relevance of the paper but again is very speculative. A factor of 10 was found for overall ligands but the factor for siderophores following the evidence presented here should be 2.5. If the range in line 118 is increased ten fold, the range is 0.2-4% but it would be fairer to use a range about 0.03 to 0.1 %.
132-133 I agree but a reference would be nice here.
138-139 apart of bringing back again the argument that the technique cannot measure “organic” L1, since the contribution of siderophores to L1 is estimated by authors as 4% tops. This is less than the CLEAdCSV error, that it is very difficult to bring down to ~5%, there are simulations at different error level of copper titrations in the literature. This uncertainty and low contribution would impede any statistically robust contribution of siderophores to the coupling of L1 and dFe. For that, siderophores should be a substantial contribution to L1 and their concentrations be well above the analytical error in the determination of both dFe and L..
142-153 I like this paragraph and its finding, implying somehow more biodiversity in on-axis locations (assuming a wider variety would imply more bacterial species). The problem is that the relevance would be diminished if the last paragraph stands as it is. If the fraction of siderophores found is a minimum fraction of the total, these variabilities of small fractions would be irrelevant. I suggest to reduce the number of previous speculations and leave this paragraph as it is.
160-161. Not so surprising if most of L1 is very refractory/low bioavailable inorganic iron released by the vent and stabilized in the oxic environment. Bacteria would need to solubilize a fraction of such iron and the likely mechanism would be siderophore release.
169-170 this paragraph fits with the explanation that part of what is interpreted here as L1 is inorganic (<0.2 um) refractory iron.
175-182. I assume there were no bacteria counts in particles or free living. Particles in the ocean are hot spots of bacterial activity. It could be that this difference here in siderophore producers it is simply a matter of bacteria density.
202 In my opinion tis argument that concentrations of units to tens of nM of iron cannot be enough to suppress siderophore production. It is clearly a matter either of passive siderophore production (continuous production, and not a response to low iron concentrations) or that the bioavailability of iron is reduced which would make more sense if this is inorganic. Pleas rewrite this section

Buck, K. N., P. N. Sedwick, B. Sohst, and C. A. Carlson (2018), Organic complexation of iron in the eastern tropical South Pacific: Results from US GEOTRACES Eastern Pacific Zonal Transect (GEOTRACES cruise GP16), Mar. Chem., 201, 229-241.
Kleint, C., J. A. Hawkes, S. G. Sander, and A. Koschinsky (2016), Voltammetric investigation of hydrothermal iron speciation, Front. Mar. Sci., 3, 75.
Sander, S. G., and A. Koschinsky (2011), Metal flux from hydrothermal vents increased by organic complexation, Nature Geoscience, 4(3), 145-150.
Citation: https://doi.org/10.5194/egusphere-2023-2017-RC1
- AC1: 'Reply on RC1', Colleen Hoffman, 01 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2017/egusphere-2023-2017-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2017-AC1
RC2:
'Comment on egusphere-2023-2017', Eva Stueeken, 19 Dec 2023

The findings are impressive and important for our understanding of global biogeochemical cycles. I also imagine that this work will carry weight in the Precambrian community where transition metal cycles may have been quite distinct from today. This work is therefore timely and likely to have significant impact.
I’m not an expert in the methods and can therefore not comment on those aspects in detail. The results sound plausible; however, I strongly suggest reorganizing the structure of the paper. Separating results from discussion would go a long way in making the paper more accessible to an interdisciplinary audience. In its current form, the text is often difficult to follow, because it seems to jump between topics unexpectedly, at least for reader who is somewhat outside of this field.
Line comments:
l .52: Please define how this K value is defined with an equation. Otherwise, this notation is not understandable.
l. 67: comma after ‘ocean’
l. 101: This sentence that all known L1 sources are biological needs a reference.
ll. 106-129: This paragraph is out of place. It should be moved to the end of the introduction, because it includes background information (xxx has never been measured) and methods. It’s confusing to read about the method in the middle of the results & discussion section. Please move this upwards.
l. 118: Does total L1 ligands refer to ligands for Fe only? Or are other metal ligands included in this pool? Please clarify.
ll. 132-133: This statement about energetic costs and Fe-regulation of siderophore production needs a reference. It is not something that is evident from the data.
Section 2.2 (identifying ligands) should probably come before Section 2.1 (the role of ligands). It would feel more logical to first discuss what was found before discussing the implications.
ll. 151-153: Elaborate on this. How do siderophores change with distance and vent type?
ll. 200-202: This was already said earlier. Please streamline the order of sections in the manuscript.
Methods: I’m not familiar with most of these and won’t comment in detail. However, I think, it would be helpful for the reader to briefly summarize at the end of the introduction which methods were used. For example, I found myself being surprised when suddenly in the results & discussion section genomic data were brought up. It would have helped if I had known from the beginning that this was coming.
Fig. 2b: Explain in the caption or legend what the star next to point 35 means.
Fig. 3: Does ‘depth’ mean water depth? Please clarify.
Eva Stüeken

Citation: https://doi.org/10.5194/egusphere-2023-2017-RC2
- AC2: 'Reply on RC2', Colleen Hoffman, 01 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2017/egusphere-2023-2017-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2017-AC2

Status: closed

RC1:
'Comment on egusphere-2023-2017', Anonymous Referee #1, 06 Dec 2023
Review of "Microbial strong organic ligand production is tightly coupled to iron in hydrothermal plumes" by Hoffman and coauthors:
The manuscript employs a combination of classical electrochemical methods (CLE-AdCSV) to measure iron ligand concentrations in solution with novel developments in chromatographic recognition and quantification of siderophores to shed further light in our understanding of iron speciation in the ocean, in this case in the plumes of hydrothermal vents. The goal of the chromatographic section is to detect a broader range of these compounds for which a careful description of the level of confidence in the assignment of molecular formulae is described. The manuscript is well-written and accessible to specialists.
However, although the experiment planning and the resulting database are extremely valuable for the scientific community, I have strong reservations about the interpretation of the results, particularly in the electrochemical part and I am going to suggest major changes. Whole sections should be thought through using the arguments hereafter and followed by rewriting of many sections. Accordingly, I would only consider this manuscript as a letter if those changes are implemented.
My major concern with the current version is the assumption that the outcome of CLE-AdCSV represents "organic" ligands' concentration and stability constant. Unfortunately, a wrong assumption, once established, might be challenging to reverse. In waters with high terrestrial dissolved organic matter (DOM) input or significant biological activity (resulting in ligand exudation), this assumption might be close to the actual situation. However, it cannot be universally granted. The technique involves introducing an artificial ligand that equilibrates with the sample (CLE step), extracting a fraction of the iron in solution, and then measuring the concentration bound to the artificial ligand (the electrolabile fraction, the AdCSV step). If there are no sequestering natural ligands, the artificial ligand recovers the entire iron concentration, and electrolabile iron equals total. If all iron is so refractory bound that it does not exchange at all overnight in contact with the artificial ligand, the concentration of electrolabile iron is zero, and all iron is considered complexed with a stability constant that should be infinite (K~∝) but that the analytical window limitation of the technique forces to fall inside this window. Real samples exhibit an intermediate situation, with a partition between iron complexed to the original ligands and the added ligand, dependent on the concentration and stability constant of both ligands.
It is evident from this description that CLE-AdCSV cannot discriminate between organic and inorganic fractions, only between "labile" and "refractory" fractions competing for exchange with the added ligand. Historically, since the oceanographic studies in the 80s, researchers assumed that the partially exchanged fraction should mostly be of organic origin, especially since the technique was initially used for copper, which has no solubility issues. As the field started the study of iron in the 90s, this assumption was carried over and it was assumed that inorganic oxyhydroxides do not contribute to the partially exchangeable fraction, as their concentration is presumed to be very small and their stability very small compared to organic ligands. However, this is hardly compatible with the well known low solubility, formation of aggregates characterized by very different reactivity as a function of aging, estuarine trapping and open ocean scavenging suffered by iron.
It is certain that the manuscript's assumption about the organic complexation of iron in hydrothermal fluids relies on the literature. However, this stems from the assumption that CLE-AdCSV experiments exclusively determine "organic" ligands (Buck et al., 2018; Kleint et al., 2016; Sander and Koschinsky, 2011). This assumption is highly unlikely in hydrothermal waters with surges of Fe(II) at substantial concentrations. Fe(II) quickly oxidizes (hours-days at the local temperature and pH), creating stable colloids that continue to grow until reaching a size that induces precipitation. And no local nanomolar concentration of ligands can prevent this precipitation of micromolar iron. In this scenario, if the sample for CLE-AdCSV contains any inorganic phase smaller than 0.2 µm, that does not undergo complete solubilization overnight by the extracting effect of the artificial ligand, it will be counted as a ligand and labelled as "organic”. This is very unlike in this case. Some ligand concentrations presented here are of the same order of magnitude as those measured in cultures using cell growth media and an order of magnitude higher than iron ocean fertilizations. This is very suspitious.
In my opinion, considering that part of the ligands may be inorganic would explain 1) the high ligand concentrations, 2) the high correlation of ligands and dissolved iron independent of any biological variable, 3) and the high concentration of ligands in the absence of primary producers that could support the ligand production of bacteria. Actually arguing fot the presence of inorganic fractions in the definition of ligands, would drift from the current paradigm and add extra relevance to the manuscript.

Other concerns:
There is a non explicit, but implicit, argument pointing to siderophores as a “major” part of the so-called L1 (my interpretation). This is not based in literature where there is no evidence of such, it seems very unlikely (siderophore production is very energy demanding and bacteria requirements are not that high, and the bibliography used is very one sided to this argument. In my opinion the ecological importance of siderophores is paramount but, since the fraction of iron complexed by siderophores in minor (<2% with our current analytical tools), siderophores should not be considered a major driver of iron cycling. No author has found important concentrations of siderophores to justify this relevance (Bundy, Boiteau, Gledhill, Ahner and so on manuscripts).

The authors do not seem aware of the limitations of the technique and the limitation caused by the existence of an analytical window. In table S3 stability constants (K1 to K3) are spread in almost 5 orders of magnitude. Such accuracy is impossible, it would mean negligible analytical error. The analytical window is approximately 4 orders of magnitude wide (depends on the error, there is related bibliography from Apte, van den Berg, Pizeta, Laglera, Gledhill and Gerringa) and K fall inside the range, cannot fall on the very edges or beyond like in a few cases here. Could this be related to the use of salycilaldoxime as competing ligand (recent Geriinga et al BG manuscript)?

In the methodology section the authors state that “forward” and “reversed” titrations were performed but this is not shown in the results section neither discussed. Is that high K1 coming from reverse and low K3 from forward titrations? Results form both treatments cannot concur if more than one ligand (as it is 100% the case here) are present and a separate discussion and comparison are due. This must be added to a future version

Self citation is excessive and the authors give as settled some particular arguments that are not accepted by the whole scientific community, example the referred prominence of siderophores as source of strong ligands in the ocean. I think that papers should reflect and discuss other visions in the field.

I know that this is quite radical but I would suggest to mend the interpretation of the voltammetric data, commenting on inorganic complexes and discussing forward and reverse and focus on the interesting chromatographic finding. The paper must be put into the context of the ecological relevance of these finding more than in the relevance for iron cycling since the siderophore concentrations found here apparently only binds a very minor fraction of the iron concentration in solution.

As I was reading I took some note that should be of interest to the editor and authors. I attach them, part are a repetition of what I stated above:

48 I find here that self-citation is a bit excessive, there are more people involved in this type of studies
For being a L1 it must be a L2. This is based in results from a particular technique that do not match results from other CLE-AdCSV protocols. I mean that other analytical approaches do not always measure L1 and L2 using a different artificial ligand. Moreover, when log K is outside the analytical window, the limitation of the analytical window brings the value inside. The classification of ligands as a function of log K is not a sensible strategy.

53 again self-citation. There are many more studies about transition of iron from estuarine waters to the sea that so not concur with this vision. The importance of humics (that I assume from the authors’ previous publications that they consider weak ligands) has been well established in many studies (Laglera/van den Berg, Slagter, Yang and Muller studies by CLE-AdCSV and many other studies using fluorescence, coprecipitation). Other studies have found that transport is a function of the molecular weight of the ligand with prevalence of smaller fractions. The process and visions of different research groups are quite more diverse than simplified here.
60-64 please revise grammar
66 see my previous comment about organic ligands and hydrothermal fluids
71 word repetition
72-76 impressive range of sampling sites with different physicochemical conditions. This gives relevance to the manuscrit

Appendix 226……. Methods
245 the concentration of buffer is possibly wrong. It should be millimolar and not micromolar. This concentration would not buffer at all against the bicarbonate natural buffer, let alone against the huge formation of hydroxides inevitably associated to the polarographic analysis of oxygen saturated solutions. If the buffer was settled at such concentration, the analysis was carried out at pH close to 9 (Laglera et al 2016)
10 micromolar SA seems a compromise solution between the concentration suggested by Abualhaija and van den Berg (5 uM) and the concentration traditionally used by Buck and collaborators (25 uM). Since doubts about the use of SA increase (Gerringa et al BG paper) it is not clear that the effect of the Fe(SA)2 complex has been removed and not counted as L3. It would be good to show a linear titration of UV digested seawater in this condition to rule out such effect.
254 here the boric acid is at the same low concentration which makes me think that perhaps the buffer was not correctly implemented and the analysis was carried out at a very basic pH (at the surface of the electrode). For instance Hawkes et al (2013) 5 mM in each aliquot (50 mM in the paper is wrong).
What was the pH of the solution here? NN is supposed to work only at low pH (8 or less) according to the intial Gledhlii/van den Berg papers. This pH is so far away from the pK of the buffer (close to 9) and the buffer concentration so low that its buffering effect would be null. This is usually detected by changes in the peak potential. Can the authors compare peaks for this work with peaks obtained in studies with higher concentrations of buffer?
260 I could not find in Hawkes et al (2013) any reference to χmin = 0.8, χmax = 0.9, and c1high = 0.75. What are these constants and what is the implication of fixing them at this value and not other? I found them in the R script and although I am not expert in R it seems that the authors of the R routine suggested to use 0.9 or 0.8 as maximum value reached during the RV if the shape of the curve was not that of a double michaelis-menten. I know the topic of this manuscript is not to criticize such but it all looks very arbitrary to me and not sure how much change in L and K would bring a change of value here. I do not suggest to recalculate anything but the method used is a bit arbitrary in the assignment of constants.
265 onwards: I congratulate the authors for the effort to apply an internal quality criterion. This is randomly the case and improves greatly the relevance of this work.

266 why the result is called L1? Is there L2 in samples?
310 “siderophore concentrations reported here are estimates of siderophore concentrations in these environments based on ferrioxamine E.” although this is obviously a strong limitation, possibly this is the only way to move forward. In cases like this applied to concentrations obtained by means of other techniques, cconcentrations of other siderohores are reported in DFOE equivalents and not simply as nM. Hopefully, at the time to evaluate total siderophore concentrations, overestimations and underestimation may compensate but it would be interesting to evaluate whether DFOE gives sensitivities around the average for all compounds commercially available. Because if DFOE is particularly more or less sensitive to the detector, the authors would incur in substantial over or under estimations of concentrations for other siderophores. Was this considered at the time to select DFOE? A comment should be added

80 this sentence is 1) not based in a prior understanding of CLE-AdCSV in the case of 2018 Buck; as I referred before, the technique does not discriminate organic or inorganic ligands 2) not based in any experimental evidence in the case of the other two papers that are one a review and the other one a model where CLE-AdCSV ligand have been added.
83 onwards. Although the argument about complexation seems right and coherent, again a concentration of 10-90 nM ligands are 1 to two orders of magnitude higher than observed in very concentrated cultures or fertilization at any growth stage. Since hydrothermal plumes are not watermasses especially abundant in biomass, the biological release of tens of nanomols per litre of “organic” ligands is extremely unlikely. This would be energetically absurd, to release ligands for concentrations that are well over the iron requirement. That some aged/stabilized oxyhydroxides and/or iron sulphides are part of the sample is a more likely explanation.

Log K3 values around 8.8 are difficult to reconcile with what we know about analytical windows and CLE-AdCSV. This is especially true if the authors claim that can resolve ligands of log K 12-13 and 9-10 (separated 3 orders of magnitude) from the same titration. It would be a mathematical artifact
94-96. Again there is only self citations about rivers where there is no consensus about and there are available results from other groups that differ substantially with the processes described here. In any case it is good that the adjective organic dropped in this paragraph.
100-101 Again self citation. Recent evidence shows that a fraction of humics of riverine origin compete with siderophores for dFe (Slagter and Laglera papers in Arctic waters). Moreover, I insist that stabilized/growing inorganic fractions (of no biological origin) could be found in the L1 fraction and in the physicochemical conditions described here, constitute most of L1.
101-103 all these processes are no doubt present, but very unlikely to produce L1 ligands in the order of tens of nM.
107 None of the Cowen references include ligands measurements or even include the word ligand. The Lauderdale paper is a modelling paper and does not constitute empiric evidence. The bibliography does not support the argument
118-129 this section is very speculative and as such should be remarked. please remove significant since this term implies some statistics behind and this is not the case, it is just a speculation. The Hider and Kong reference is a review and only speculates about whether more products are expected. My problem here is that the paragraph is based in repeating a speculation. Other sources of L1 referred to in the bibliography do not deserve even a mention (humics, EPS, etc)
126 this calculation is addressed to increase the relevance of the paper but again is very speculative. A factor of 10 was found for overall ligands but the factor for siderophores following the evidence presented here should be 2.5. If the range in line 118 is increased ten fold, the range is 0.2-4% but it would be fairer to use a range about 0.03 to 0.1 %.
132-133 I agree but a reference would be nice here.
138-139 apart of bringing back again the argument that the technique cannot measure “organic” L1, since the contribution of siderophores to L1 is estimated by authors as 4% tops. This is less than the CLEAdCSV error, that it is very difficult to bring down to ~5%, there are simulations at different error level of copper titrations in the literature. This uncertainty and low contribution would impede any statistically robust contribution of siderophores to the coupling of L1 and dFe. For that, siderophores should be a substantial contribution to L1 and their concentrations be well above the analytical error in the determination of both dFe and L..
142-153 I like this paragraph and its finding, implying somehow more biodiversity in on-axis locations (assuming a wider variety would imply more bacterial species). The problem is that the relevance would be diminished if the last paragraph stands as it is. If the fraction of siderophores found is a minimum fraction of the total, these variabilities of small fractions would be irrelevant. I suggest to reduce the number of previous speculations and leave this paragraph as it is.
160-161. Not so surprising if most of L1 is very refractory/low bioavailable inorganic iron released by the vent and stabilized in the oxic environment. Bacteria would need to solubilize a fraction of such iron and the likely mechanism would be siderophore release.
169-170 this paragraph fits with the explanation that part of what is interpreted here as L1 is inorganic (<0.2 um) refractory iron.
175-182. I assume there were no bacteria counts in particles or free living. Particles in the ocean are hot spots of bacterial activity. It could be that this difference here in siderophore producers it is simply a matter of bacteria density.
202 In my opinion tis argument that concentrations of units to tens of nM of iron cannot be enough to suppress siderophore production. It is clearly a matter either of passive siderophore production (continuous production, and not a response to low iron concentrations) or that the bioavailability of iron is reduced which would make more sense if this is inorganic. Pleas rewrite this section

Buck, K. N., P. N. Sedwick, B. Sohst, and C. A. Carlson (2018), Organic complexation of iron in the eastern tropical South Pacific: Results from US GEOTRACES Eastern Pacific Zonal Transect (GEOTRACES cruise GP16), Mar. Chem., 201, 229-241.
Kleint, C., J. A. Hawkes, S. G. Sander, and A. Koschinsky (2016), Voltammetric investigation of hydrothermal iron speciation, Front. Mar. Sci., 3, 75.
Sander, S. G., and A. Koschinsky (2011), Metal flux from hydrothermal vents increased by organic complexation, Nature Geoscience, 4(3), 145-150.
Citation: https://doi.org/10.5194/egusphere-2023-2017-RC1
- AC1: 'Reply on RC1', Colleen Hoffman, 01 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2017/egusphere-2023-2017-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2017-AC1
RC2:
'Comment on egusphere-2023-2017', Eva Stueeken, 19 Dec 2023

The findings are impressive and important for our understanding of global biogeochemical cycles. I also imagine that this work will carry weight in the Precambrian community where transition metal cycles may have been quite distinct from today. This work is therefore timely and likely to have significant impact.
I’m not an expert in the methods and can therefore not comment on those aspects in detail. The results sound plausible; however, I strongly suggest reorganizing the structure of the paper. Separating results from discussion would go a long way in making the paper more accessible to an interdisciplinary audience. In its current form, the text is often difficult to follow, because it seems to jump between topics unexpectedly, at least for reader who is somewhat outside of this field.
Line comments:
l .52: Please define how this K value is defined with an equation. Otherwise, this notation is not understandable.
l. 67: comma after ‘ocean’
l. 101: This sentence that all known L1 sources are biological needs a reference.
ll. 106-129: This paragraph is out of place. It should be moved to the end of the introduction, because it includes background information (xxx has never been measured) and methods. It’s confusing to read about the method in the middle of the results & discussion section. Please move this upwards.
l. 118: Does total L1 ligands refer to ligands for Fe only? Or are other metal ligands included in this pool? Please clarify.
ll. 132-133: This statement about energetic costs and Fe-regulation of siderophore production needs a reference. It is not something that is evident from the data.
Section 2.2 (identifying ligands) should probably come before Section 2.1 (the role of ligands). It would feel more logical to first discuss what was found before discussing the implications.
ll. 151-153: Elaborate on this. How do siderophores change with distance and vent type?
ll. 200-202: This was already said earlier. Please streamline the order of sections in the manuscript.
Methods: I’m not familiar with most of these and won’t comment in detail. However, I think, it would be helpful for the reader to briefly summarize at the end of the introduction which methods were used. For example, I found myself being surprised when suddenly in the results & discussion section genomic data were brought up. It would have helped if I had known from the beginning that this was coming.
Fig. 2b: Explain in the caption or legend what the star next to point 35 means.
Fig. 3: Does ‘depth’ mean water depth? Please clarify.
Eva Stüeken

Citation: https://doi.org/10.5194/egusphere-2023-2017-RC2
- AC2: 'Reply on RC2', Colleen Hoffman, 01 Feb 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2017/egusphere-2023-2017-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2017-AC2

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Latest update: 20 Nov 2024

In their study, Hoffmann and co-workers provide new exciting data on the role of organic ligands in supporting elevated dissolved iron in hydrothermal vent plumes. The authors were able to detect siderophores (including amphiphilic types) in hydrothermal plumes at different sites along a 1,700 km section of the Mid-Atlantic Ridge, pointing to microbial utilization of siderophores to access particulate hydrothermal iron.

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Hydrothermally-derived iron can be transported thousands of kilometers away from deep-sea vents, representing a significant flux of vital micronutrients to the ocean. However, the mechanisms that support the stabilization and transport of dissolved iron remain elusive. Using electrochemical methods, advanced mass spectrometry techniques, and genomic tools we demonstrate that strong microbially-produced ligands appear to exert an important control on plume iron biogeochemistry and dissemination.


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Microbial strong organic ligand production is tightly coupled to iron in hydrothermal plumes

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