Diversity of NC10 Bacteria and ANME-2d Archaea in Sediments of Fault Zones at Lake Baikal
, Gennady Kalmychkov 2, Svetlana Chernitsyna 1 and Tamara Zemskaya 1Round 1
Reviewer 1 Report
This is an interesting manuscript and the authors have presented valuable data. However, the manuscript needs to be revised so that readers will be able to follow everything that was done. See following:
Abstract
Line 14, change 'reservoir' to 'reservoirs'.
Lines 20-22; Change sentence to: We used different molecular biology methods including high-throughput sequencing and PCR analysis, using 16S rRNA genes as well as different functional genes of AOM (mcrA and pmoA).
Line 24; change to 'which were different from the composition of the discharged fluid.'
Introduction
Line 30; (also line 97) change to (CH4)
Materials and methods
Line 120; change 'Sediments' to 'Sediment'
Line 124; this makes no sense: '- in those the methane seep PB.' ???? This needs to be fixed.
Line 126-127; The authors state that 'Chemical analysis of pore waters was carried out onboard immediately after sampling.' However, they present no information about which chemicals were analyzed and they present absolutely no methods as to how the analyses were done. Readers will need to have the information for these analyses. The authors should not just list a previous paper they worked on, but the original methods. This is so the readers do not need to search out multiple papers to get the methods.
Results
In several places the authors have included sentences that belong in the Discussion in the Results. For example:
Line 203; 'which were recorded only in the surface layers at the reference sites [59].' This is not Results and should be in the Discussion.
Line 209 and Lines 210-212; 'compared to reference values [59]. etc.' Again, this is all Discussion material.
Line 238; 'of mud volcanoes previously indicated [43].' belongs in the Discussion
Lines 250-251; Sentence belongs in the Discussion.
The authors may want to combine the Results and Discussion into one section.
Author Response
Response to Reviewer 1 Comments
Abstract
Line 14, change 'reservoir' to 'reservoirs'.
Line 13 Changed
Lines 20-22; Change sentence to: We used different molecular biology methods including high-throughput sequencing and PCR analysis, using 16S rRNA genes as well as different functional genes of AOM (mcrA and pmoA).
Line 19-21 Changed
Line 24; change to 'which were different from the composition of the discharged fluid.'
Introduction
Line 23 Changed
Line 30; (also line 97) change to (CH4)
Line 30 and Line 99 Changed
Materials and methods
Line 120; change 'Sediments' to 'Sediment'
Line 123 Changed
Line 124; this makes no sense: '- in those the methane seep PB.' ???? This needs to be fixed.
Line 127 Corrected
Line 126-127; The authors state that 'Chemical analysis of pore waters was carried out on board immediately after sampling.' However, they present no information about which chemicals were analyzed and they present absolutely no methods as to how the analyses were done. Readers will need to have the information for these analyses. The authors should not just list a previous paper they worked on, but the original methods. This is so the readers do not need to search out multiple papers to get the methods.
Line 153-172 Corrected and added. On board we carried out only chemical analysis of anions.
Results
In several places the authors have included sentences that belong in the Discussion in the Results. For example:
Line 203; 'which were recorded only in the surface layers at the reference sites [59].' This is not Results and should be in the Discussion.
Line Ok, moved in the discussion
Line 209 and Lines 210-212; 'compared to reference values [59]. etc.' Again, this is all Discussion material.
Line Ok, moved in the discussion
Line 238; 'of mud volcanoes previously indicated [43].' belongs in the Discussion
Line Ok, moved in the discussion
Lines 250-251; Sentence belongs in the Discussion.
Line Ok, moved in the discussion
The authors may want to combine the Results and Discussion into one section.
I moved materials from chemical results in the discussion section.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments
In the manuscript, Lomakina et al. reported 16S rRNA sequences of the recent identified anaerobic methantrophs, ANME2d and NC10 bacteria, and their respective mcrA and pmoA gene sequences from Lake Baikal. From the sediments of the same site, ambiguous activity of anaerobic oxidation of methane (AOM) was reported by the same author previously. The pore water chemistry does not support the presence of NC10 bacteria, which use nitrite as the electron acceptor for AOM. It was also very surprising that ANME1,2,3 groups were not detected from the sediments, although sulfate was the dominant electron acceptor. In the phylogenetic analysis, besides some environmental sequences, the authors need to include more recently reported Methylomirabilis spp. sequences as references, for instance, 16S and pmoA of M. oxyfera, M. lanthanidiphila, M. limnetica and M. sinica, could be added to Figure 6 and 7, respectively. To better define the presence of NC10 methanotrophs, the use of nitric oxide dismutase (nod) gene as a marker also recently became available.
Suggestions:
Change the title. The title does not reflect the MS content, other denitrifying microorganisms were not addressed, and ANME2d convert nitrate to ammonia, so they are not denitrifying microbes. Please quantify the relative abundance of NC10 bacteria and ANME2d to general bacteria and archaea, respectively. line 176, in this section, please provide key information of the sequencing, how many sequences were obtained, after quality control, how many sequences remained, etc… Is the authors’ previous publication (Ref. 46) in Russian?
Author Response
Dear Review!
The authors are very grateful to you and the reviewer for competent, thorough and kind revision of our manuscript. Authors are grateful for the careful editing, valuable comments and recommendations of the Reviewer. We took into account all the comments and amended the text of manuscript, figures and captions.
Author Response File: Author Response.pdf
Reviewer 3 Report
A presented study is of considerable interest and add to the findings that could be important for our understanding of anaerobic methane oxidation in freshwater systems and Lake Baikal specifically. The researchers increased the level of sensitivity of molecular DNA surveys by designing a set of very specific primers allowing the detection of Archaea different from sulfate-dependent ANME-1, ANME-2 and ANME -3. Using specific primers they detected 16s RNA gene sequences affiliated with archaeal ANME-2d subcluster and Candidatus M.oxyfera (Phylum NC10) bacteria. Despite that anaerobic methane oxidation (AOM) has been demonstrated in the respective sites of the Lake Baikal by conclusive experiments using radiolabeled isotopes, the biological agents involved in this process have not been detected previously. I think this is an important contribution in the study of AOM in freshwater environments, and the paper should be published.
However, I have some comments and questions that could be easily incorporated and could improve the writing.
First and foremost, it should be stated strongly that AOM has been demonstrated in the vicinity of the studied sites. It should be added started from “Abstract” right after “gas hydrate were found” (Line 15). For instance, “Despite that AOM has been demonstrated….”
In “Abstract” too:
Line 17. The sentence is awkward. Re-phrase:
“A search for representatives ….. has been carried out in the different types of Lake Baikal methane enriched sediments”
Line 21 “of” should be removed
Line 22 “found divers” did you mean “diverse”?
Introduction
Line 29 “stores” did you mean “storages”?
Line 35 “ key enzyme” add please “of a methylotrophic pathway”
Line 42 “very widespread” give examples, please. In marine environments?
Line 49 Remove “on the example” use “demonstrated in the enrichment cultures”
Line 56 “using these primers” What primers? Primers targeting pMMO gene? It should be stated clearly.
Line 72 It’s better to remove “to be used for a more” Leave just “for refined …”
Line 84 add “and “ after “nitrate ions”
Line 86 use “coupling “ instead of “to couple”
Line 89 there is no iron-rich ions. Perhaps you want to say “Minerals”?
Line 96 it should be added “anaerobic” if you mentioned the rates of anaerobic CH4 oxidation.
Add the references regarding AOM in Lake Baikal: Pimenov et al., 2014.
Line 99 use “detected” instead of “revealed” and also add “by the shape of methane profile with depth typical for AOM”
Line 100 instead of “prove” use “reveal”
Line 101 “and significant sulfate ion concentrations (15000 μM) in contrast to the site of Kukuy Canyon “ The verb is missing. It is not clear: are there a lot of sulfate 15mM or no sulfate. Re-phrase, please.
Line 104 remove “members”
Line 207 add “content” after “pore water NO3”
Line 209 Use “analyzed ions”not “analyses ions”
Line 243 You need to provide the minimum concentrations too.
Line 253-254. Finish the sentence. Use a dot instead of semi-colon. Then in the next sentence say what happened there? A verb is missing. For instance,” Also, we detected DNA signatures of….
Line 260 a typo in “analysed” use z instead of s here and throughout the text.
Line 263 do not use a dash (-), use a verb please
Line 281 add “to” in front of 1%
Line 289 remove “minor”
Line 289-90 insert “sequences of” in front of “archaea”
Paragraph from Line 324-329. It is not clear have been all the sequences from three different sampling sites 100% identical to coal-rich sediments, iron-rich freshwater mat and Lake Orn, OR they have been 100% identical respectively: from surface layer of the methane sip was 100% identical to coal rich sediments, from oligonite layer 100% identical to the sequences from the freshwater mat and from the mud volcano to those from Lake Orn??
Clarify this, please.
Line 332. It will be friendlier to a reader if you add the name of the freshwater lake
Line 334. Again, it will be friendlier to a reader if you add “typical for marine environment” after ANME 1, 2, 3
Line 337. A typo. Remove dot after ANME-2d subcluster.
Line 349. A reference for a publication on a bioreactor where AOM was driven by iron reduction, please.
Line 350 a phrase “phylotype ANME-2d.mcrA-OTU3 had sequences from the surface sediments of the methane seep” is not clear. Re-phrase, please.
Line 373 add “above mentioned” in front of ANME
Line 377. Remove “investigated”
Line 380. A reference for the specific primers, or to the table if they are placed there.
Lines 388-395. Put references, please, for sequences from Lake Constance.
Line 421 add “using primers targeting pmoA gene” and provide a reference for these primers, or table.
Discussion
Line 456. Remove “Therefore”
Line 457 add “most likely”in front of “carry out”. Only in vitro analysis in cultivation experiments can prove that. Molecular sequencing is giving merely a potential.
Line 474. Add “marine” ANME
Line 477. Is it “should be no less”? Otherwise, it is not clear what did you want to say
Line 478. “this site” What site? Is it Lake Baikal site or Lake Cadagno? Not clear.
The same lines. The write term is not “intensity”. It is either sulfate reducing activity or rates of sulfate reduction. In both cases it is per unit of time. Thus, 0.07 nM is a concentration. The rate is 0.07 nM/day. Correct please everywhere.
Line 480. sulfate reducing bacteria accounted for. Do not use “were” in this case
Line 483 “the obvious activity”. Did you mean detectable?
Line 486-487. Here you need briefly explain, why do you think that AOM in Lake Baikal occurs by different mechanism and not coupled with sulfate reduction. For instance: “As far as sulfate content is mostly very low and no ANME 1,2,3 known for coupling AOM to sulfate reduction sequences were detected, it is possible that other mechanisms of AOM are functioning”
Line 500. Was there enough ammonia in the studied sites?
Line 503 use “were” instead of “are”
Line 528 Use “nitrogen oxides “ instead of “oxidized nitrogen”
Line 533-537 Interestingly, you did not detect much of nitrate/nitrite in the studied sites. It’s also unclear is it enough ammonium there. Wetlands and paddy soils are dramatically different from Lake Baikal, because they are very rich in organic. Organic matter decomposition is a source of ammonium. What is a source of ammonium in your opinion in the pristine Lake Baikal?
Line 538-539 “We did not find them at depths of 70 and 140 cm near the methane seep PB, which also confirms a possible coexistence of these microorganisms” How it confirms?
Line 543 “Lower nitrite values compared to nitrate ions can be due to the likely toxicity of nitrite in a concentration of 1.5 μМ _for the NC10 bacteria” Did you mean that because of toxicity the bacteria were reducing it to N2O and N2? If they can do denitrification, they will do it anyway. If nitrite were toxic for NC10, it could be inhibitory and would rather accumulated.
Line 551 “admit” is a wrong word. Use “hypothesize”, because it is a hypothesis.
Line 558 use “by” instead of “of”
Line 558 Can potentially occur. You did not show the process, just a potential
Line 567 Again ‘admit” is a wrong word. “We think it could be plausible that…”
Author Response
Dear Reviewer!
The authors are very grateful to you and the reviewer for competent, thorough and kind revision of our manuscript. Authors are grateful for the careful editing, valuable comments and recommendations of the Reviewer. We took into account all the comments and amended the text of manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors have successfully addressed my previous comments. However, there is one issue that needs to modification: on lines 196 and 197 can you change 'rpm' to 'X g' (i.e., times gravities).
Author Response
We thank the Reviewer for the appreciation of our manuscript.
We changed rpm on g.
Reviewer 2 Report
the manuscript has been greatly improved. The questions and suggestions raised were adequately addressed. Well done!
Author Response
We thank the Reviewer for the appreciation of our manuscript.
