In this paper, Petrash et al. used aqueous chemistyry profiles, analysis of stable isotopes of S, C and O, microbial community analyses, and sediment characterizations to develop a model for processes occurring in suboxic portion of a pit lake resulting from coal mining activities. This is an interesting system in that it has overlapping S, N, Fe, and C cycles all in the anoxic portion of the water column. This is unusual, b/c in many systems at least one of these is a functionally absent, a minor component, or undergoes minimal redox transformations because of the dominance of (an)other component(s). The paper is mostly observational, but I think that’s fine, because the authors have developed a nice model for the processes occurring that’s shown in Figure 8. The paper presents a lot of results. I almost wish the authors could tie everything together a little more succinctly. I think Figure 8 gives a nice summary of the processes involved. Maybe a little more time in the discussion focused on this model and a little less on the “paleo” implications would help the reader synthesize all of the observations into the process model.

Below, I provide some specific comments and suggestions that I hope can help improve the manuscript:

Ln. 11. Do the authors mean “reductive Fe(III) dissolution”?

Ln. 16. “sustained” how?

Ln. 17-18. What is the electron acceptor for sulfide oxidation? And if sulfide is oxidized all the way to sulfate, at which point does the sulfur disproporationation happen?

Ln. 104. Change to “DNA extraction and MiSeq”

Ln. 105. Do you mean ICM-MS? Does mass spec work for specific ions? I don’t think this is an adequate description of the analytical methods. I’m also al little bit leery of putting the entire description of nucleic acid-based microbial community analysis in the supplement.

Ln. 106. Change to “measurement of dissolved”

Figure 2. Can the authors make the axes the same on panels above and below the redoxcline? My first impression was that there was no difference between any of the organic acids, but there are actually rather dramatic differences.

Ln. 150. VFAs were a minor fraction of the total DOC. What is likely the rest? How labile might it be, and how does that inform the biogeochemical model?

Ln. 167-168. Wouldn’t sulfate reduction induce increase in pH? You’re producing carbonate alkalinity and reducing a strong acid (sulfate) to a weak acid (sulfide).

Ln. 221. Please be consistent in including the charge for nitrate

Ln. 223. Please change “sequenced” to “detected”

Ln. 232. By “abundance peak” do you mean maximal relative abundance?

Ln. 210-256. Did the authors try to quantify nitrite? If there are nitrogen transformations occurring in this system I would expect it to be important, and perhaps the ultimate oxidant for Fe2+ in reactions 1 and 2.

Ln . 282. Are the authors referring to Fe(II) oxidation by Mn(III/IV)? Please clarify.

Ln. 293. Please change “[Fe]” to “Fe concentration”; also here and throughout, please check tense agreement.

Ln. 301. I don’t know about this. Attributing metabolism when you only have 91% similarity is tough.

Ln. 329. Please remove “significantly”

Ln. 336 and throughout this section. Why does diversity matter. Wouldn’t relative abundance be more informative with respect to S transformations? There could be a whole lot of diversity of sulfate reducers, but they’re only a minor fraction of the community. A later use of the term “diversity” leads me to believe the authors are referring to diversity of S metabolisms (e.g. oxidation, disproportionation, reduction of different redox states, etc.), but I’m not sure. Please revisit the use of this term and clarify.

Ln. 372-374. If there’s evidence of S metabolizing organisms and some aqueous chemical evidence of S transformations, why no change in del34S-sulfate?

Reactions 4-6. Is Mn-dependent S disproportionation from the Bottcher et al. 2001 paper? What about the siderite-dependent disproportionation? I am unsure how this reaction might occur.

Ln. 520, 534, 559. Why are these minerals italicized?

Overall quality of the preprint ("general comments")

Introduction

It is well-written and summarizes all principal aspect of the pit lake, as well as the importance of the study and how in general was conducted. There were few specific comments that I would the authors to take into considerations.

Methods

This section is nicely organized in subsections as supplementary information (S1), but there were specific details that would enhance the reproducibility of the applied methods.

Results and Discussion

The authors did a good job discussing all the results and its significance. This section requires more work specially with respect to enhancing the clarity of the figures and their description in the text.

Individual scientific questions/issues ("specific comments")

introduction

It was not clear to me when was the lake flooded, 2005 according to figure 1a? If so, please state this in line 43 where it is written: “This newly formed,…”. Do you have supporting info of when did the lake become meromictic, or how long has it been meromictic? Do the meromictic conditions of the lake vary seasonally? Please present supporting information about this too.

In line 95, the authors talk about the present oligotrophic stratified conditions of the lake as a topic sentence. First, I would like to see physico-chemical profiles at this point to ease the understanding of such conditions for the reader. I also would like the authors to describe in more detail such conditions in all stratified layers. Finally, the last sentence of the paragraph, starting in line 100, deserved more written description too.

Methods

From S1:

In line 13, when the authors refer to ~11 mL water samples, is 11 mL the aliquot amount referred to in line 11? In addition, wow many samples were collected?, were they collected along depth? where exactly?

In section SM1.1.3, please clarify the number of samples taken. The same applies for SM1.1.4 and include information about samples from which depths (or layers) were considered for the cation and ion concentration analyses.

In section1.1.5, please clarify the following questions: were the 11 water samples (line 29), the same as described in section SM1.1.3? If so? why eleven? does this number include biological replicates? are these only from two depths? it is important to clarify, where these samples were taken along depth. More details of the PCR and sequencing protocol would benefit future researchers and reproducibility of the methods.

Were the mineralogical (SM1.2.2), isotopic (SM1.2.3), and SEM (SM1.2.4) analyses applied to all sliced sediments?

Results and Discussion

Subsection 4.1:

I am little confused about what is shown in Figure 2a. What is happenning above 48 m? To what depth are you referring to? depth of the lake water, or depth of the whole lake? Based on what is presented in Figure 2a, I interpret that the depth of the water column is only ~10 m? I am sorry if it is not that obvious to me, but it might be worth to clarify.

In line 121, authors refer to several previous profiles of the lake. Do you have previous profiles? Are they published somewhere? or included in the supp info?

In line 135, authors wrote: “The hydrochemically different monimolimnion persists in the deepest depressions of the lakebed throughout the year; although with slight variations in the monitored Eh range that could be accompanied by minor (±1 m) shifts in the vertical position of the redoxcline.” Can you show profiles of this on the supp info?

In caption of figure 2, authors refer to dysoxic (n=4) and anoxic (n=3): at which depth were these samples collected, respectively?

Authors included a separating line referring to the redoxcline in Fig2B. Does this mean that the upper part of Fig 2b corresponds to the myxo-hypolimnion and the lower part to the monimolimnion? If so, please clarify it in the figure too. In addition, what are the red crosses? Could you also include an explanation in the caption?

Section 4.2

In line 150, the given DOC concentrations correspond to an average value of the 7 samples refer in figure 2b?

Authors said: “A six- to ten-fold increase in concentrations of acetate, oxalate, and formate occurred towards the increasingly saline and O2-depleted bottom waters.” This might be better to visualize in a profile. Could you please include one in the supp info?

In line 163, when authors refered to “[ΣCO2] were inversely correlated with the δ13C values”, are they referring to figure 3d.

Paragraph starting in line 169 should have included a reference to Table 1 somewhere.

About Figure 3d referred in line 189, I thought this figure was referring to the water samples. Please, clarify or correct accordingly.

Section 4.3

Colors in Fig 5 must be changed. In 5a, there are two yellows, two greens, two light blues corresponding to different organisms, making it hard to interpret the figure and correlated with the written text. Fig 5b is even harder to differentiate colors and organisms.

While describing the microbial community, authors shoud be more quantitative (avoid low or high and refer to percentage). How much does “increases significantly” or “the abundance peak” mean?  In addition, please be specific if what is shown in Fig 5. corresponds to normalized abundance in percentage with respect to the whole community or only among each microbial group shown separately in a b and c.

In section 4.3.2, the subtitle refers to dissolved Mn and Fe, are they total concentrations? otherwise please be specific and in accordance with what is shown in Fig 4b: MnII and FeII. In addition, do you have concentrations of Mn(IV)? How do authors know Mn(IV) is settling down from the upper layer? Or Fe(II) is difussing upwards?

In line 291, do authors have mineralogical evidence of this fact” “Dissolved phosphate is re-complexed back onto nanocrystalline and amorphous ferrihydrite-like phases precipitating at the redoxcline.” The same comment for mackinawite mentioned in line 295.

In line 303, when referring to Pseudomonas spp., do authors have any control showing that Pseudomonas was not part of the extraction kits, or sampling material?

In 310, include a reference for “anoxygenic phototrophic and nitrate

Reducing species (Magnetospirillum and Ferrigenium; Fig. 5b, Supplement 2), and Azospira-like species.”

In line 323, when referring to the peak of Geobacter, include where specifically and how much?

There are some names of organisms in Fig 5b that are not mentioned in the text. Should you better remove them from the figure and include them, as other lesss abundant taxa, or mentioned them in the text.

In line 345, in Fig 5c is only as Thioalkalivibrio...should you add the species name too as you did in the text?

In line 349, authors mentioned “The abundance of S. hydrogenivorans increases in parallel to a decrease in the T. paradoxus-like bacterium, which suggests that the latter may be at a disadvantage and limited

by organic C fixation under the specific hydrochemical conditions prevailing at the redoxcline” With the current colors in Figure 5, it is difficult to see what you are showing in the text.

In line 360, which bar corresponds to D. acetoxidans in Figure 5c. The same comment for Desulfaticacillum in line 365 and Sulfitobacter in line 366.

In general, with the current colors in Fig 5, it is difficult to agree with the conclusions stated by the authors in section 4.3

Section 4.4

In line 374, do authors have values to support the “weak correlation”?

A reference is needed for the following statement: “It is also within the range observed in studies of S

disproportionation reactions generally proceeding under anoxic conditions” in line 383.

Reference needed for the examples given in line 409.

Section 4.5

Be more quantitative with respect to sentences like in line 445: “…..increase slightly towards the bottom of our 8 cm depth core but their abundance, relative to total iron, decreases downwards” or 451: “a significant increase…”

In line 454, a Sect. 4.6.3 is referred but not found in the current version of the manuscript.

Section. 4.7

In line 595, name which “scarce examples” authors are referring, as well as in line 596: add reference and name which lakes

About figure 8: Nice figure but there are some improvements to be done: 1) a legend is required to understand symbols and colors in the figure. 2) add a depth profile and names of the each layer. 3) why is it necessary the venn diagrams for the microbes, what each color of the circles mean? Add the the biogeochemical role of each microbial group included in the figure.

Technical corrections: typing errors, etc

Introduction

Figure 1. Please describe parts b and c in the caption of the figure.

Methods SM1

Line 126, correct format accordingly.

Results and discussion

Line 119: change this sentence by a better topic sentence that summarizes the results presented in this paragraph instead of starting the sentence with Figure 2a shows....

In line 158, I think it is Figure 3a

In line 209, “with functional annotations on the planktonic prokaryote community” is duplicated.

In line 349, include % after 97.

In line 370, 18(delta)O SO4 is as Osulf., please keep it one way or the other but be consistent.

In Fig. 6a-b include what CDT and V-SMOW mean.

When referring to reactions, authors use Rcs or Rs, use one way or the other but be consistent.

Transform the first sentence starting in line 403 into a topic sentence. Avoid starting a paragraph with Figure.. shows...., this is also the case in line 438.

In figure 7, use the same acronyms in the figure as referred in the text.

Line 516, replace Like for Lake.

Line 637, found duplicated: near the anoxic sediment-water interface near the anoxic sediment-water interface

Petrash et al. presents a largely observational study of a post-mining, low nutrient lake with a unique geochemical setting that may be helpful as an analogue for nutrient cycling in the ancient ocean. They combine geological, biological, and chemical data for their investigation of both the anoxic water column and upper sediments.

Overall, the paper is well written and the results very intersting. I believe the paper should be accepted following a few larger edits that I think will improve the manuscript: 

1) Cutting jargon: this paper is strongest when integrating results across their interdisciplinary dataset. However, the paper is in many places (namely the abstract, introduction, and final disucsion section) unnecessarily complex and looses the non-expert reader. The authors should revisit these parts of the manuscript with an eye for unnecessary jargon - places they will lose interested geobiologists in a complicated explanation of geology, for example. In places where jargon is unavoidable, offering a few more definitions to the reader will help broaden the readership of this really interesting interdisciplinary study. 

2) Improved figures: The figures should be reworked with a goal of consistency across figures. Much of the results/discusison centers around different zones of the lake's water column, but it is difficult to orient yourself across the many figures. Figure 1a panel 4 does a nice job showing the mixolimnion, hypolimnion, and monimolimnion - I think it would be helpful to see these zones in all the figures - either as the different shades of gray like in the Eh figure or with dashed lines and labels. A few of the figures also appear low resolution - namely, figure 5, 6, and 7.  Other comments specific to each figure can be seen below. 

3) Separating results and discussion: It was difficult as a reader to follow the results and disucssion section - I wanted to already be acquanited with much of the data before seeing it synthesized. I found myself jumping back and forth across the sections often. Some parts felt too long and should be more succinct, while other parts begged for more detailed discussion. My suggestion to the authors is to split the results and disucsison and focus the results to be a succinct section, with extraneous details moved to the SI. Then the discussion section can focus on bridging the many types of data together, perhaps starting with Figure 8 which is a nice schematic for the entire ecosystem. I think this will help the readability of the manuscript. 

4) More methods: Currently, there are not enough details for someone to replicate any of the work or think critically about advantages or disadvantages for any method. Much of the SI methods section should be moved to the main text. 

Section specific comments: 

Abstract:

• I would recommend moving the last few lines on the importance of the study more broadly to earlier in the abstract, perhaps as the second sentence   and then expanding/clarifying the point in the current first sentence that this geochemical situation is an unusual but exciting case study

Introduction

• Line 32: It would be helpful for those less familiar with limnology terms to define meromictic briefly, perhaps simply as “indefinitely stratified” or something similar
• Line 33: “common sulfate deficiency” feels unnecessarily complicated, do you mean low in sulfate or absent of sulfate?
• Line 39: If possible, I think it would be helpful to add one more sentence about the importance/relevance to paleo-studies - the connection to me right now is a little weak so would be great to strengthen that point a little more - how exactly do they better inform precambrian ocean redox stratification models? 
• Line 47: Would be helpful to include a mention of the lake’s pH as well (anywhere in this introduction)

Methods:

I think a majority of the SI details should be moved to the main text - because of this I also have line edit suggestions for the SI methods:

• Water sampling: what depths were sampled? Did those change based on the physiological parameters prior to water sampling?
• Line 11: were the exetainers cleaned prior to sampling?
• Line 13: define PES
• Line 22: please define which anions and cations
• Line 70: more details are needed on the IC method used for the VFA analysis - what is the run time, column used, etc?
• Line 77: How much 5M NaOH was added?
• Line 87: should be “quantified gravimetrically” - also can you clarify what you mean here? Just weighed?
• Line 147: Sort of unusual to report 3 sigma, maybe just report 2 sigma as you did earlier to be consistent
• Line 159: there appears to be a typo with an extra ( - maybe a missing reference?
• Supplement figure 3: this appears very pixelated on my download, can you make sure the figure has high enough resolution?

Results and Discussion:

• Line 119: awkward to start the sentence with “Figure 2a”
• Line 150: can you elaborate on the DOC concentrations? What does that tell you about the system - is it typical or unusual? A little more discussion would be great, especially because you mention in the abstract that SR may be limited by low amounts of metabolizable OC
• Line 155: what is your hypothesis for the change in VFA concentration in the different layers of the lake? Can you relate this more explicitly to your 16S data at all?
• Line 159: I think a mention of pH should come much earlier, at least very early in results, if not hinted at in introduction - my initial assumption from hearing about a post-mining lake would be to expect really acidic conditions, so saying that the pH is closer to 7-8 earlier would be helpful
• Line 163: Please put some of the d13C values in the text, such as an average or range
• This discussion on 4.2.2 on total DIC seems very lengthy compared to the other results sections and could be shortened for readability and to better emphasize the main points
• Line 171: Please change “d13C signatures” here and elsewhere to d13C values
• Line 189: Instead of “estimated isotopic C signature of the CO2” say either estimated C isotope composition or estimated d13C value
• Line 370: the title of 4.4.1 is awkward, maybe “Isotopic evidence”
• Line 373: I would suggest avoiding "heavier" and just stick with "enriched in 18O"
• Line 375: a number itself can't be narrow, so maybe change to something like "the bottom waters had a narrow range of d18O values: X to Y"
• Line 410: you say that for the initial sulfate composition it is reasonable to assume its similar to the nearby acidic drainage and the pit lake before flooding - the second seems more reasonable to me but you dont report those values in the text? What are those?
• Line 574: extra parenthesis dangling  - sentence is also not grammatically correct, so should be fixed
• Line 595: more references needed here, overall really enjoyed this section on the paleo implications! 

Figures/Tables

• Figure 1: I really enjoyed this figure! Especially nice to see the lake from 2005 to 2020. The figure caption has a call out to panels b and c, but not to panel a, so that should be added.
• Figure 2: In part b I think the main idea is to compare the VFA concentrations above and below the redoxycline - its currently hard to see that difference and the relative differences between other VFAs -  it would be more clear to show these on all the same plot - so instead of 6 separate figures just one figure
• Table 1: I’m a little concerned by the errors in the ammonium measurements - especially that surface sample, where the error is larger than the measured value - is there also a reason why phosphate doesn’t have concentration brackets - maybe just an error? 
• Figure 3: For panel A can you change it to mM so that the range is not to 12000 in the axis? Also needs more tic marks to see values in between, panel c also needs further tic marks for sulfate concentration. In the figure caption there is a CO2 that needs the 2 to be subscripted and "value" needs to be added after d13C.
• Figure 5 - this figure is pretty hard to read with the colors as they are - I would think about the main point you are trying to make with this figure - instead of having the arrow for the redoxycline I would make the edit I suggested earlier for all figures, having different shades of grey boxes in the background  - its hard to compare the abundance of different microbes against each other because the scale changes aross panels as well. 
• Figure 6: theres is one data point for d34S of sulfate that is much higher value - at ~13.5 permil? Is this an outlier? Should be mentioned in text - 6c would be a bit easier to see if the symbols were also colored if possible 

I hope these comments were helpful and they assist in improving what is already a really interesting manuscript. This is my first time reviewing an article and I very much enjoyed it. Sincerely, Dr Alexandra A Phillips