General:

I think this manuscript would benefit from some more proofreading by the more experienced authors. It could use improvement on the structure and the writing, to improve the flow and make it more condensed. Please also pay attention to the switching between different tenses, and to improve the clarity of the methods section. Many different experiments have been performed in this study, which is wonderful. It makes it, however, difficult for the reader to keep an overview. Please structure the manuscript in a way that provides the necessary overview and clarity. Present the results in a structured way in the methods section, and don’t be tempted to already interpret them – this belongs to the discussion. Also prevent the use of language that is either too strong (This means..), or is not specific enough (warm, very few etc.) Overall, I think the experiments are cool and valuable, but improvement is needed to bring this across to the reader.

Abstract

Introduction about sediments is too long. Could skip most of it, one or two sentences is enough.

Instead, tell us more about the two stages of incubations and 13C additions, multiple TEA and inhibitors. What did you use, what were the aims? If you don’t want to stress these, give less detail, now it creates more questions than answers.

25-27. This sentence is a bit clunky, with the two words for the same process (oxidation and AOM). Also, here you name it methanic sediments while these were the incubations/reactors right?

The abstract could use re-structuring, please have in mind what are the most important messages you want to convey, stress those and don’t give too much details about other things. It could also be nice to give one or two sentences at the end that place your results into a broader context.

Keywords: I would add mcr and methanotrophs

General textual: Methanic is not a word that is commonly used I think. Methanogenic is the more general term, at least, I think that is what you mean? But this is personal preference, to choose what you want to use.

Methods

98. If you want to say it’s warm, give a temperature.
99. Similar to what?
100. Are there methane profiles?
101. You have not mentioned the central lake or station A yet.
102. which leaves = leaving
103. Did they receive new methane after that?
104. This sentence is weird, ‘in case of’ is not fitting.
105. This seems more like discussion or results, not methods (‘the variations…)
106. The balck coffee comes out of nowhere and the explanation about why only 1 replicate is not fitting.

This whole paragraph is chaotic, try to restructure to make it a bit more schematic and easier to follow, to help the reader understand.

Do you mean real porewater every time you write porewater, or an artificial substitute? It seems like a lot of porewater to extract, which is possible I guess, but I’m just not sure and curious!

192. Don’t switch between past and present tense within a paragraph.

249 I don’t think this paragraph is necessary.

255. Can you start with simply describing your results? You dive in deeply directly, it would be nice as a reader to get a bit of a gentle overview first, of what you measured and what that showed, to start with.
256. No need to note that here.
257. This was not subsequent but different experiments, right? The word first suggests otherwise.
258. Discussion, not results. Stick to just listing the results, so the values that you measured and their patterns, here.

Fig. 2. What is the difference between the colors of the pre-incubated experiment? The legend calls them the same.

Fig. 3. The text is too small and therefore hard to read. Why don’t you merge the replicates of each treatment into one line with error bars? They seem to nicely follow the same trends. Also, it would be nice to have the same y-axis and x-axis for easy comparison between the treatments.

Fig. 4. Similar to Fig 3: please merge the lines of the replicates.

Table 1. The names of the treatments could be improved. What is a typical fresh sediment bottle?

I’d be happy to provide more comments on a next version of the manuscript.

The paper by Vigderovich et al. investigated the pathways of anaerobic methane oxidation in Lake Kinneret sediments by a combination of incubation techniques, lipid and metagenomic analyses.

The authors performed a series of long-term incubations in bottles and semi-bioreactors with an array of added potential electron acceptors and inhibitors for specific metabolic processes in order to track down the dominant processes responsible for AOM. The results obtained from this study were interpreted in combination with results obtained from previous studies on these sediments. All in one, the experimental design was thorough and the use of combinations of electron acceptors/inhibitors feasible for interpretation of possible AOM pathways in these sediments.

The paper is mostly focusing on presentation and interpretation of geochemical data. The authors did perform taxonomic read and metagenomic analyses from several incubations and incubation time points, but I miss the presentation of these results in the paper. The results are briefly mentioned, but I would prefer to see a visual representation of DNA-based results in a separate section in the ‘Results’ section and a more thorough integration with lipid and geochemical analyses.

It was also a little confusing to see a ‘black coffee’ treatment, as there was no introduction or reasoning why this rather unusual substrate was used for AOM incubations. Also there was no detailed protocol on how this treatment was prepared (what fraction, concentration etc). Every treatment should be reproducible from the information provided by the paper, but here any details are lacking. So I would suggest to either remove this from the paper completely or to describe the treatments and reasoning thoroughly.

In general, the results presented in this paper are interesting and will benefit the scientific community investigating AOM in natural sediments. The paper will benefit from a more clear structure and better visual presentation of results.

This work by Vigderovich et al., investigated the key microbial players and electron acceptors that support anaerobic oxidation of methane, methanogenesis and possibly a sulfur cycle in in the top 20 cm of sediments collected from a lake in Northern Israel. They used a variety of sediment slurry incubations amended variety of electron acceptors, electron acceptor analogs and inhibitors along with ¹³C labeled methane and tracked the buildup of ¹³C-DIC over time. Their results indicate that there is methane oxidation occurring by aerobic methane oxidizing bacteria and anaerobic methane oxidizing archaea (ANME) possibly with oxygen or iron oxides. However, later in the experiments the data suggest that humic substances are the most likely culprit for the turnover of ¹³C-methane. Their metagenomic data suggest the presence of methanogens, aerobic methanotrophic bacteria and anaerobic methanotrophic archaea and suggested that there is an interplay between the groups that cycle the carbon in their experiments.

Generally, I find the data to be very interesting and does fit well within the scope of Biogeosciences and should be eventually accepted with major revisions. My main critiques for the manuscript are in the clarity, flow in all sections and a few discussion inquiries I would like to address in this review.

General comments:

The introduction lacks a clear identification of the gap in the knowledge to which scientific questions are based on. The question and hypothesis in L84-86 is rather vague and could be clearer.

The methods unfortunately are riddled with syntax errors and missing study site and methodological details that need to be clarified. This is particularly important as methods sections should be written such that anyone could reproduce the experiment. This review cannot identify and fix all of them but will provide examples of some of the most severe below.

The beginning portions of the results section sound more like a discussion. There is more space being used to repeat the experiments and experimental setup in this section than simply reporting the data from the experiments. The results section would be stronger if the authors would report the data without method explanations or with interpretation. Further into the section there are more numbers that are reported but there are other sections that read crudely. Consider having introductory and conclusion sentences and sub-headers (applies to other sections) to better separate sections which will help with flow.

Figures 2 and 3 could be organized better. There is a lot of data and the scales do not match which could be misleading at a first glance. Furthermore, there is some data in the supplementary material that belongs in the main text. For example, the authors suggest that aerobic methanotrophic bacteria play a role in the overall AOM process. Their geochemical data do not definitively track aerobic methanotrophic bacteria activity (not sure you even could) but the molecular data do indicate their presence.

In the discussion are a lot of interpretations and claims for which are speculative and do not offer enough literature examples that support the interpretations. For example, in L364: I don’t think that the addition of iron-oxides generally inhibits AOM according to your data. Where in the literature do you find an example of iron-oxides acting as an inhibitor to AOM? Yes, there seems to be less buildup of your 13C-DIC but the system still observes a buildup of 13C-DIC after ~450 days in figure 2 and in figure 3A after 450 days. If it was truly an inhibitor then the 13C-DIC would be identical to the killed control trend throughout the whole experiment, just like the BES trend in Figure 4 where you know BES is inhibiting activity. But Figure 3A lacks a killed control for hematite so how does one know that the addition of hematite is truly an inhibitor? Instead, you observe the trend to become slightly depleted in 13C before the full spike, then see a rebound in the hematite trend back to levels closer to the beginning of the experiment, in which case how do you explain that? In addition, according to Fig 3A, all 4 amendment experiments had decreasing 13C-DIC which leads me to believe that there might be something else at play perhaps the organoclastic iron reduction as the authors mentioned or something in the experimental setup that is causing all of your replicates to all have decreasing 13C-DIC.

Specific inline comments and edits:

L44: AOM coupled to other electron acceptors is not a theory anymore.

L49-50: Consider adding equations of the AOM reactions.

L47-L54: Consider joining L47 into next paragraph at L50.

L74-75: Move citation to the end of sentence.

L111-112: “1) Two stage slurry incubations with 1:1 sediment - pore water ratio for three months, followed by a 1:3 ratio and the addition of different manipulations for up to 18 months.” This sentence is far too long and could be split up to be clearer about the analysis.

L113-114: “Semi-continuous bioreactor experiments with freshly collected methanic sediments and porewater with 1:4 ratio, where porewater was exchanged regularly.” Syntax error, do you mean 1:4 sediment to porewater ratio or do you mean the porewater has a 1:4 ratio, in which case what is the 1:4 ratio in the porewater? Same can be said for L115-117.

L97: The methods would also greatly benefit with a couple sentences that explain how the sediments were retrieved from the Lake (i.e. ship/small boat, instruments (mulitcorer, pushcores, gravity cores etc…).

L98-106: This section is rather vague, I think it would be stronger with more details about the lake such as; approximate temperature and size and perhaps a nearby city for reference. A map would also make this section stronger.

L109: Here you mention you are going to assess the different electron acceptors for AOM. It may be wise to have a sentence somewhere either in the methods or in the intro that you are lumping all methane oxidation by archaea (ANME’s) and bacteria (Methylococcales). Many in the community may just be thinking AOM process is being conducted by the ANME’s but it appears (not clear) you are referring to both, correct?  

L118: This section would be also stronger with one sentence explaining what the purpose of the two-stage incubation is.

L119-120: Add more information of where you sampled perhaps on a map. I do not know where “Station A” is. Additionally, do you know precisely when the sediments were collected between 2017 and 2019? How long did it take for the sediments to be processed into sediment slurries? Were they stored and reactivated? If sediment samples that were collected in 2017 and processed in 2019 how do you know those samples are still viable for this study?

L120: What was the container that the sediments were pooled into?

L121: Please add the speed (rcf x g) for the centrifugation.

L133-134: What do you mean “already running experiment” is this separate from the two-stage experiment? If so, this was never introduced.

L138-139: Syntax; this sentence makes it seem like there is a separate experiment within one. Was there a reason not to add acetylene in the beginning like BES?

L145-147: It was mentioned that ¹³C-label was added after the pre-incubation, please add the time you added the label.

L150: Never begin a sentence with numerical, instead spell out two.

L150- What method and instrument was used to measure the Fe(II)?

L152- How was the methane measured?

L153- Is there an equation used to calculate methane?

L154-155: This is the first time the “Black Coffee experiments” was introduced. Please add why you included this

L154: Is there a reason why there is inconsistencies between duplicates or triplicate samples (i.e. not enough sample or prioritized sample for certain treatments of interest?). Please elaborate.

L156-157: This sentence is contradicting and with parentheticals is incomplete.

L156-157: This is the first mention of any killed controls in this part of the experiments. How many were there? How were they prepared? (etc).

L158- What is so special about the lake in Alaska that humic substances had to be extracted from. Why not get them from Lake Kinneret?

L164-165: What was the other bioreactor amended with? Or is it a control?

L165-166: Syntax issues, had to read it several times over to understand that you are trying to describe how 13C methane was added to the headspace free bioreactor.

L170-172: How many total weeks did the bioreactor run for?

L175: This is the first time that a duration of the experiment has been introduced. Consider adding the actual experiment duration somewhere in the method.

L175-177: Good introductory sentence. Please move this sentence to the beginning of the section.

L185: Figure 1 caption should be moved into the text. Particularly you did not describe how you set up the third experiment till the caption.

L192: What kind of autosampler? Was this done at the home lab?

L197-199: should be moved to L150.

L199-202: Which bottle is now being sampled? This is a new section and don’t know which experiment is being sampled. The sentence would be stronger if you indicate the reason why you track methane and ethylene (i.e. tracking methanogenesis and acetylene turnover).

L207 - 208: You list the same variable “x” as two different parameters.

L216: Please indicate which set of experiments the samples come from.

L254: I think it would be better to break 3.1 into sub sections to have better flow. It is difficult for the brain to switch between experimental setups.

L255: Is the pre-incubated long term experiments the same as the two stage experiments? Please be more consistent with the names of experiments.

L255-256: Here is an example of discussion text in the results. How much 13C methane exactly was converted?

L257-258: This sentence sounds like it should be in the discussion. Consider instead to report the actual permit value and leave the microbial population statement for when you report the microbial ecology.

L261-263: This sentence is very confusing and how does this relate to the statement you had about AOM in the previous sentence? Please reorganize.

L260-273: The whole paragraph sounds like it belongs in the discussion. Perhaps move to discussion or add more details about the data.

L278-280: Move to methods.

L274: Was sulfate ever measured in your experiments?

L288: Is the 308 days the end of the experiment?

L284: End of what? How many days was that?

L292: What is PCA? Please spell out acronym. What was the result of the AQDS addition? Not clear.

L296 By how much did the Fe(II) and delta 13C increase? Please report.

L297: What was the slope?

L289-305: The results are very vague and sound more discussion are. Please add in the decreasing and increasing permil and concentrations values for this section.

L301: A lot going on in Figure 2 and legend could be better organized.

L311-312: Figure 3. Consider making all y-axis scales the same

Consider moving the Fig 3 F next to Fig 3 A since they both seem to be the experiments that indicate when 13C label was added. Also I do not recall an exact time when the label was added in the methods.

Fig 3C: Are the NO3 (grey circles) and the Hematite + NO3 1 mM (green triangles) data on top of each other? Please check your graphs.

L319: Please report in text how high of an abundance and which species.

L320 and 321: Hyphen between “sulfate reducing”

L320-321: Please rewrite sentence. Just report which SRB were present.

L322: Please report the number of reads to NC-10.

L339-342: Where are the profiles? Or are you referring to profiles in previous studies? Please clarify.

L342-344: Is this really your previous work, or the current work or is this the work from the citations at the end of the sentence? I think you are trying to compare the three different experiments but it makes it sound as if there are three papers in one.

L342-344: Is the mechanic zone where Fe-AOM the same sediment regions that you obtained for this study?

L344-348: Please clarify, I can’t tell if you are referring to the current study or other works.

L354-359: Add figure references since you have two figures that compare the three setups.

L357-359: But then how do you explain the sharp increase in the 13C DIC in Line 354?

L364: The methods indicate that the preincubation called for a full methane headspace that was half 12C and half 13C, is it not conceivable that the mass balance would lead to a slight depletion of the 13C in a closed system like this?  I would argue that Figure 3A and F shows that before the addition of the 13C label the 13C DIC was similar to the control but after the addition, all trends become heavier. In which case how do you interpret that as an inhibitory response to the addition of label and iron?

L367-368: I think it is conceivable to claim that organoclastic iron reduction could dilute the 13C-DIC signal in these experiments, especially over time but do you have any evidence to support that either by isotopioc analysis of organic matter in this study or previous study that could allow you to make some 1^storder mass balance to explain that?

L372: I agree with your statement of manganese oxide but what do you have to say about the Magnetite additions in Fig. 3A? Those were the most similar to the no electron acceptor control experiment.

L379: I think the result of the addition of molybdate is not super surprising. It appears that the molybdate was added rather late in the experiment when the trends are already supporting magnetite as a potential AOM electron acceptor. Sulfate reduction would be naturally inhibited since metal oxides yield much higher free energy than sulfate does in the redox cascade.

L385-386: This is interesting but also not super surprising because I believe many sulfate reducers are also iron reducers. Dig into the literature and see if any of the sulfate reducers you detect have been shown to conduct iron reduction.

L392-393: I don’t think you can totally confirm that nitrate and nitrite is inhibitory. Fig 3C shows some buildup of the 13C-DIC and again I would be more convinced that there is a true inhibitory effect of the nitrite, if the trends were identical to the killed control. But even then, what evidence is there that nitrate or nitrite inhibits the enzymatic pathway in AOM, like BES does? Does the literature have any suggestions? In addition, you also added hematite to those samples with nitrate in Fig. 3C so how do you know that the buildup of 13C-DIC that you do see is from denitrification coupled to AOM or iron reduction coupled to AOM? Did you measure a buildup of N2 in parallel? Were you able to somehow inhibit iron reduction (if you did that would be cool and would love to know)?

L399: If AQDS has a high electron shuttling capability leading to higher organ clastic turnover then in a closed system like this wouldn’t your 13C-DIC become very deplete (Rayleigh distillation) over time and not just plateau like your data suggests? I rather think AQDS just doesn’t support anything since it looks just like your killed control or else it would have looked like some kinetic process if any biology was involved.

L 406: I really think your Fe(II) data belongs in the main text especially here where Figure 3F really needs S3 to support your claim.

L412: I think it would be worthwhile to have spent a bit more time on magnetite as another potential electron acceptor since Figure 3A is convincing enough, though the scaling in the y-axis is deceiving.

L446-448: This was left open ended. What does trace methane oxidation have to do with your study? Plus your experiments are in slurries over long periods of time with amendments that are probably much different that the natural environment so would trace methane oxidation be a likely occurring thing in a slurry.

L488: You mean aerobic methane oxidation is decoupled from iron reduction right?  

In this study, the role of AOM in Lake Kinneret sediment incubations was explored. Several incubations tested which terminal electron acceptors accounted for AOM activity. The main findings were that:

•    pre-incubation with methane for 3 months significantly increased AOM

•    hematite seemed the most likely iron mineral used as terminal electron acceptor (TEA) for AOM although it did not stimulate AOM and other iron minerals could have inhibited AOM

•    natural humic acids and black coffee could be TEA for AOM

•    sulfate-AOM was determined neglectable

•    BES inhibition indicated that archaea mediated AOM, which was supported by metagenomic and 13C-lipid analyses

Major comments

It will improve the study to have its goals clarified in the introduction by L84-95. What were the specific research questions and knowledge gaps addressed here? What is this study addressing that was not known from your previous studies? This was still not clear to me after reading the entire manuscript. I think this is reflected in the title: “Modification of methane oxidation pathways during long-term incubations of methanic lake sediments” - I could not understand which modification occurred (bacterial methanotrophs did not thrive? TEA changed?). Think about a more specific title that summarizes the main key message of the study.

The materials and methods section needs major improvements for experiment reproducibility - adding amounts, concentrations, units, calculations etc. Sequencing data must be made available and an accession number must be provided. Data that has been already published and is here reproduced must be made clear.

All these incubations were done but no methane oxidation rates are provided in the manuscript, so calculating them and presenting them would add a lot of value.

Metagenomics results were barely used (same goes for lipid data). Consider doing metabolic reconstruction of the MAGs recovered here or use this data for another study that explores metabolic potential and mechanisms of potential taxa responsible for Fe-AOM.

Detailed comments

review grammar of the manuscript

L42 - ANME between parentheses

Intro: add background on the black coffee experiment - what was the hypothesis and the literature background?

2.1 add geographical coordinates of sampling site

2.2 indicate that concentrations of substrates in pre-incubated sediment experiments are provided in table S1 but bring this table to main text given that it is vital for the manuscript and experimental reproducibility. Also, add to this table similar details about the other two types of experiments (semi-bioreactor and incubation with recently collected material) which are so far missing from the methods section. Indicate if substrates were bought or synthesized (especially for minerals) with manufacturers / synthesis protocols.

2.3 Name it “Porewater and gas analyses”?

L202 can you provide methane detection limit in total amount (µmol) instead of concentration (µM)? Also, add the volume of gas injected into the GC?

Eq 1 and 2: provide units for each term, label eq (1) in L205 and (2) in L206; invert eq (1) so it will be 𝐹𝐷𝐼13𝐶𝑓 = 𝑥 × ð¹13𝐶𝐻4 + (1 − 𝑥) × ð¹ð·ð¼13𝐶𝑖

Also, can you add what was the final time used to calculate rates? Were rates derived from the slope or from the difference between T0 and T-final?

L161, section 2.2.2 - add bioreactor volume and manufacturer information?

2.4 at L215 needs more details for experimental reproducibility: what was the sample exactly (sediment? how many g?), concentrations of added compounds and steps - protocol format given that a modification of Sturt et al., 2004 was used. Suggestion: release the step-by-step protocol as supplemental material or zenodo link with doi number. Deposit sequencing data and add a data availability statement.

2.5 How were counts per million reads calculated? Add formula to methods here. Also, can you briefly list all tools that produced data part of this manuscript and are part of the SqueezeMeta pipeline? For instance, what did you use for MAG taxonomic classification? And for genome annotation / gene search?

I could not fully understand if and which results presented in this manuscript are already published (i.e. L115-117, L249-253). Can you please clarify this? Also, given that a number of different incubations were performed, I suggest numbering them consistently in text, tables and figures to facilitate tracking.

L265-273 & Figure 2 = the most useful to me would be a plot of methane oxidation rates as a figure and, in the text, something like this: “treatment X or addition of X increased methane oxidation rates (in nmol/dry g sed/day to allow comparisons with other studies/settings) by X% relative to controls”. Also, in Fig 2, what is the difference between blue, red and yellow? Add this information in the legend.

Fig 2, 3 and 4 = Is it possible to improve the quality? Also, it would be great to have methane oxidation rates in the text or as a figure - from all these different incubations, the only number provided is “3-8 % of the 13C-methane” in L454, which should be presented as a rate - this information I would find most valuable from this study and would allow comparisons with data from other environments, which could be added to the discussion.

3.2 I suggest showing metagenomic results in the main manuscript. My suggestion is to make a heat map with MAG coverage normalized by metagenome size (instead of RPKM values) and add to this figure the info of Table S3. Also, instead of binscore, use MAG completeness and contamination (in %). Would also be good to know how many MAGs were reconstructed and which ones represent candidate iron reducers - FeGenie could be useful for that: https://doi.org/10.3389/fmicb.2020.00037

Table S4 I was surprised that mcrA and pmoA are not in this table! I think including these and iron reduction and extracellular electron transfer genes would be better use of your metagenomic datasets, which could be extensively better explored in this study.

L328-331 The numbers here do not match Table 1, which shows more data than discussed here. Maybe this table is not so important and could go to supplemental materials?

Table 1 = Can you clarify what exactly each incubation is and what are killed controls potentially present here?

MAG coverages indicate Bathyarchaeota could be mediating Fe-AOM or play an indirect important role given that they are more abundant than ANME-1 - here the metabolic reconstruction of these MAGs would be fundamental! No mcrA was found in Bathyarchaeota - did you use an HMM that could find divergent sequences? what about other genes in reverse methanogenesis? what is Bathyarchaeota’s metabolic potential in your incubations?

From table S1 I assume hematite is the dominant iron mineral in lake sediments, is it? Then I find curious that this most promising terminal electron acceptor did not stimulate Fe-AOM while other iron minerals could have even inhibited AOM. Can these results alone be taken as evidence for Fe-AOM? I find them insufficient. More discussion is needed to hypothesize about what is happening and how to improve experimental conditions.

In the semi-bioreactor experiment, why was little methane provided (when the methane headspace was replaced by anoxic liquid)? For how long were these semi-bioreactors operated? ~600 days? Also, any particular reason for calling them “semi” and not simply “bioreactors”? Finally, know that from our experience shaking biomass/sediments disrupts AOM activity (related to L166-7). So, shaken and with little methane, I am not surprised to see in Fig 2 that there was no AOM detected in the bioreactor. In this manuscript, there is no discussion of bioreactor results, so I suggest to add something.

L423 To enrich the discussion on 13C assimilation into lipid, I suggest addressing your results in the context of these findings and potentially more:

Wegener G, Niemann H, Elvert M et al. . Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane. Environ Microbiol. 2008;10:2287–98.

Kellermann MY, Wegener G, Elvert M et al. . Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities. Proc Natl Acad Sci. 2012;109:19321–6.

Julia M Kurth, Nadine T Smit, Stefanie Berger, Stefan Schouten, Mike S M Jetten, Cornelia U Welte, Anaerobic methanotrophic archaea of the ANME-2d clade feature lipid composition that differs from other ANME archaea, FEMS Microbiology Ecology, Volume 95, Issue 7, July 2019, fiz082.

L426 move to results

L426 Just because ANME are not very abundant it does not mean they are not (very) active. Here abundance is expressed as “< 1.5 %” - specify what this number refers to (relative abundance? how was this calculated? add to methods)

L443 I think it’s appropriate to tune this down: “we hypothesize Methanothrix could be involved in Fe-AOM”. High potential when ANME-1 is present and other archaea are more abundant is a bit stretching; but it would be nice to see some actual physiological evidence for the involvement of Methanothrix in Fe-AOM in the future. Here your back flux inferences also support ANME-1’s role being much larger than Methanothrix.

L469 Table S6 is for the first time mentioned here in the discussion. It presents qPCR results that have not been mentioned in the methods, so these must be added and the mention must be moved to results. Methanogenesis rates are expressed in µM/day, which I found cryptic and does not allow comparisons to other studies - please convert to n or µmol/dry g sed/day

L470 I am missing and thus suggest adding a sentence hypothesizing about the key microorganisms (ANME-1) accounting for 3-8 % of 13C-methane oxidation to CO2 in these incubations. Also, what is this number referring to? Hematite-AOM? Humic acid-AOM? I would love to see rate comparisons between those!

L481-8 I find this insufficient to explain why putative bacterial methanotrophs disappeared in long-term incubations if oxygen could be generated via methanobactins. However, this must be stated at hypothesis level, we don’t know if iron reduction and methane oxidation were coupled via methanobactin-produced oxygen. I think it’s better to offer other explanations or simply say it’s unclear why bacterial methanotrophs disappeared.