General comments

The study by Richter et al. describes distributions of BHPs in lacustrine and coastal environments of the Azores. In addition to the environmental samples, an enrichment culture enriched for methanotrophs was also studied. Using LCMS, the authors identified several novel BHPs and report detailed information of their mass spectral identification. The BHP distributions are then discussed in combination with geochemical parameters and the potential as taxonomic markers for different ecological niches is evaluated.

I like the manuscript and think it is well written and structured. The type of figures and the selected statistical methods are chosen well. The detailed identification of the new compounds seems sound and the reported mass spectra in the appendix will be very useful for further research. I am very much looking forward to the final version where the raw data are also accessible. Besides the analytical part, the discussion of the findings in context of previous studies is well balanced and adds important conclusions. Overall, not much should be changed before final publication.

Specific comments

It would be great if the authors could also make the inclusion lists for the analytical method available (see also minor comment). This would help “jump-start” implementation of the LCMS method in other labs.

The appendix is great in explaining in detail how the fragment spectra of the BHPs are used for identification. The main text then only contains a brief mentioning (according to line 212) – this is true for all novel compounds except the formylated-aminoBHPs where lines 262-278 are very similar to section B6 in the appendix. I suggest to shorten the main text version so that it is similar to the summary of findings of the other compounds and keep the details in the appendix B6.

Since there are so many compounds in the NMDS plot of Fig. 4, I wonder if it is possible to use colored font for the compounds clustering together in Fig. 4. For example color A for cluster of amino-containing compounds (referenced in line 323), color B for the nucleoside compounds (line 338) and so on (e.g. in line 466). This should then be indicated in the text (section lines 314-349) and in the caption of Fig. 4.

Section 3.5 (implications for the Rsoil proxy) is not reflected in the abstract and conclusions and should be added.

Minor comments and technical corrections

L67: replace “nor “ with “or”

L154: specify MS model?

L159: resolution does not have unit “ppm”

L159-160: why three separate inclusion lists? Can these lists be provided as supplemental material, other research would benefit a lot from this

L169: how was the normalization between sample runs done? Please explain.

L256: Fig 3e? I might have missed it, but Fig. 3e is not referenced?

L303-305: “Using ANOSIM we find a significant difference…”

Fig. 4: colors are not easy to distinguish (two shades of green, two of blue), choose additional other colors

L311: delete “there is”: “we find no significant difference”

L327: “was previously described”

Fig. 6 caption: RU should be “response units”, not “relative units”

Fig. 9 caption: indicate that structures are tentative structures

L478: instead of “an NMDS” use “the NMDS analysis”. This also reads a bit odd, the NMDS analysis shows many compounds. Do you mean that these compounds, the amino BHP cluster, is close to the sites mentioned in line 482?

L493: suggestion – “observe … in all sediment samples except Cubres East and West (Fig. 2)…”, move outside of parentheses

L538: “BHP distribution”

L603: perhaps “… appear to be produced in the water column…”

Appendix B1 second paragraph last sentence: typo – “…attribute this to a potential co-eluting…are unable to distinguish.”

Appendix B114: m/z 191 was searched in fragment spectra: “…in fragment spectra revealed two…”

Appendix B120: “..is not located in the ring system.”

In an effort to identify BHPs that can be associated with specific ecological niches or bacteria, Richter and colleagues have characterized the BHP distributions in suspended particle matter and sediments in the lakes and lagoons of the Azores Archipelago. The authors are particularly interested in characterizing BHP distributions and their potential as taxonomic markers in lacustrine settings, which have not been as well studied as marine settings. This study identifies multiple novel BHPs, including ethenolamine-BHPs and formylated-aminoBHPs, and proposes potential taxonomic markers for methane-oxidizing bacteria and low-oxygen conditions in lacustrine settings. In addition, they identify nucleoside-BHPs that can be produced within the water column, which has implications for the R_soil proxy, leading the authors to propose a new R_soil-lake proxy. The results of this study will be useful for future work using BHPs to characterize lacustrine settings and I recommend it be published in Biogeosciences with minor revisions. My general recommendation is to consider what additional information may be available, like δ¹³C_TOC or metagenomic data, that could be used to support the potential of certain BHPs as proxies, especially for MOBs.

Specific Comments:

Line 61: Do both type I and type II MOB produce the same diagnostic BHPs or do we know of differences between them? If there are known differences, be more specific when discussing the diagnostic BHPs. Lines 421-422 suggest that they produce different diagnostic BHPs, but this should be consistently clear.

Line 65: The phrase “minor amounts of these BHPs” is vague. Are all of the BHPs mentioned in the previous sentence (aminotetrol, aminopentol, methylcarbamate-aminoBHPs, and 3β-methylated-BHPs) also produced by sulfur-reducing bacteria and nitrite-oxidizing bacteria or just a subset of them? This should be clarified.

Lines 113-114: It is mentioned that Lakes Verde and Funda experience large cyanobacterial blooms in the summer months. Were either of these lakes experiencing cyanobacterial blooms during sampling?

Line 129: What do the given percentages mean for this co-culture? Were there other organisms present and this was the percentage of bacteria that could be identified? If so, could other bacteria present be producing BHPs in the culture?

Line 179: TOC content was measured, but no associated δ¹³C_TOC measurements are reported. This could be a useful additional piece of information to aid in characterizing the MOB input in this environment.

Line 252: Are the acylated versions of aminopentol also commonly associated with methanotrophic activity?

Lines 274-275: Is there any evidence apart from the putative oxazinone-aminotriol for nitrite-oxidizing bacteria in the sediments from Lake Verde? While Elling et al. (2022) identified the same compound in nitrite-oxidizing bacteria, the source of that BHP here is not necessarily the same. Further comment on the potential source of oxazinone-aminotriol in this environment would be useful.

Line 314: The fieldwork was done at the beginning of the stratification season for deep lakes – is there evidence that the lakes had fully stratified at that point? If not, this could potentially explain the finding that there is no significant difference between the BHP distributions in the surface, chemocline, and bottom water.

Figure 4: It would be easier to distinguish between deep lakes, shallow lakes, and coastal lagoons if the sample site markers for type of site had a different shape.

Line 353: Is it clear why methoxylated BHPs are only present in the lagoons?

Lines 444, 452: It is noted that sulfur-reducing bacteria and nitrite-oxidizing bacteria could potentially contribute to the aminotriol, aminotetrol, and aminopentol in the water column, although it is unlikely. Is there any additional data available, like δ¹³C_TOC measurements or metagenomic data, that could support the MOB origin for these BHPs, especially for the potential origin from nitrite-oxidizing bacteria? Confirmation that nitrite-oxidizing bacteria are not making a significant contribution of aminopentol would also support the interpretation that that there is a higher abundance of Type I than Type II MOB.

Line 524: A reference is made to the chironomid community shifting as a result of lake eutrophication. However, in the context of this paper, this line seems unrelated and would require more information on why chironomids would be relevant for understanding MOB contributions to the ecosystem. I would suggest either removing this line or further explaining the connection between chironomids and MOB.

Technical Corrections:

Figure 1C: Sao Jorge Lake is not labeled or obvious in this panel, despite being included in the figure caption. Additionally, in the enlarged map, it would be useful to center and zoom in further on the lagoons of interest because they are currently difficult to see.

Figure 1D: Is the enlarged map different from the map of Flores island? The island map has more lakes than the enlarged panel, which makes it challenging to identify the lakes of interest in the larger geographic context.

Table 1: Formatting issues make this confusing, especially for the pH, NH₄, and NO₂ columns.

Lines 364-366: The caption for Fig. 6 says that dissolved oxygen in plots D and I and pH in plots E and J are pink when they are in black.

Appendix B1, page 31 (no line numbers): General typing errors – the appendix could use a proof-reading. Examples: “is similar to (the) retention time difference” and “we attribute this to potential a potential co-eluting peak.”

Figure B2A: The bottom chromatogram (m/z 718.563) does not have any labeled peaks. Based on retention time, I would assume it to be peak o, as labeled in the same chromatogram in panel B. If so, this should be labeled.