Dear Drs. Creamean and Rhodes,

Thank you for submitting your manuscript " Evaluating the potential for Archaea to serve as ice nucleating particles" to Biogeosciences. The editorial team and a group of expert reviewers have assessed your submission and feel that it has potential for publication.

We appreciate that your paper addresses gaps in ice nucleation by assessing the role of 4 Archaea species and their cellular components in inducing ice nucleation. I found it particularly interesting that the 4 species varied in their ability to induce freezing and found the discussion of differences in cellular properties of value. Two of the reviewers pointed out that to date there are no studies of ice-nucleation for Archaea which highlights the value of your study. However, all 3 reviewers had concerns about the study design and requested clarification on the methods. In particular, the reviewers raised concerns about the lack of statistical tests, ability to comparing the different species and treatments, and explanation of the methods.

I appreciate your thoughtful and thorough responses to the reviewers and extensive revision of to the paper during the interactive peer review phase. In most cases I agreed with your responses to the reviewers and the revisions made to the revised paper. However, I found some areas that would still benefit from further revisions (see below), and so I would like to invite you to revise the paper and resubmit for further review.

Please feel free to contact me with any questions.

Sincerely,

Dr. Denise M. Akob


Specific Comments:

1. Title: reviewer #2 suggests amending the title by replacing “Haloarchaea” for Archaea. I think this would be a valuable change and would highlight the focus of your study.
2. L. 15-16: consider revising to “nucleation activity of 4 species in the class Haloarchaea”.
3. L. 10: change to “plants”
4. L. 57, 72 and elsewhere: when referring to Bacteria (and Archaea and Eukaryotes) as a domain please capitalize. There are a number of instances where this change is needed for accuracy. Also, if Haloarchaea is a formal taxonomic group name it should always be capitalized.
5. L. 95: change to “these four”
6. Tables 1: It’s not clear from the table and the methods text at which time the cell counts were made. In response to Reviewer 2, text was omitted on shipping the cultures from SC to CO. I disagree with this decision. The reviewer is pointing out a key methodological consideration -- even though you shipped the samples on ice it is important to know if the cultures were still in log-phase growth. It is super important to know what the cell abundance was at the time the experiments were started and how cell abundance changed during shipping. Even though you shipped the samples on ice there could be cell growth or death. Line 130 talks about the cultures being measured for ice nucleating ability within a few days of reaching log-phase but was this before or after shipping? Were the cultures still in log phase? More detail is needed.
7. Table 1: why didn’t you dilute the cultures in media to count them? That would have prevented cell lysis and give you an accurate cell count. Or you could have fixed the cells before diluting for cell counts as suggested by Reviewer #3. Clearly you can’t go back and redo this but consider this for future experiments.
8. Tables 1 and 2: I wonder if you really need 2 tables? The culture medium is already stated in the methods so could be omitted. You could combine the information into a single table and have columns of initial and diluted cell concentrations and salinities. I also suggest putting information in order of action, e.g., you diluted before knowing the cells were intact so switch the column order; otherwise it makes it sound like you diluted lysed cells.
9. L. 105-112: I think this paragraph is of value and should be retained. The authors do a nice job here of identifying how the lab cultures can be environmentally relevant.
10. L. 137-142: a lot of the reviewers’ comments were around the different treatments and use of lysed vs. intact cells. The text here is a great justification on why the treatments make sense and that the different cultures behaving differently has environmental relevance. But, this message really isn’t stated in the introduction. It would improve the paper if the statement about testing cell lysis having environmental relevance was included somewhere in the end of the intro.
11. L. 144-148: the added text is very helpful.
12. L. 142: I’m not sure if I get what is meant by “threshold” and “serially dilution” here – Table 2 doesn’t show data for serial dilutions, just a few select samples. If the cultures were serially diluted that would indicate to me that there is a whole series of dilutions that were assessed for cell survival and the highest dilution that didn’t show lysis was the threshold. Are you really just presenting the final dilution selected based on those factors? Or did you only select these dilutions based on tests with saline solution controls? Please clarify.
13. L. 152: please define the abbreviation CSU
14. Section 3.2: I found the start of this section to be overly focused on the dilutions taking away from the study aim. I suggest revising to start with the main finding and not the methodology. Also, the use of “dilution” seems a bit mis-leading since the focus is really on the cell density and not the salinity of dilution of the sample.
15. L. 216 and elsewhere: the original paper did not have statistics presented. Adding the fisher’s exact test was really helpful for showing the differences in the treatments. Consider adding a supplemental table of the statistics results to compliment the narrative.
16. L. 266: change to “range tested” as it would be a stronger statement
17. L. 269: cite the references on H. morrhuae cells not lysing here.
18. L. 270-272: the statement on the DUF3494 protein domain in the Archaea does not belong here. The reference is not from any of your authors, and you don’t show any data. If you want to include this observation the results and methods need to be included.
19. L. 294: based on this acknowledgement it sounds like the organisms were cultured in Colorado and not just in SC. If they were preserved does that indicate cells were fixed before counting? Clarification would be really helpful per the comments above about the methods for culturing and shipping.
20. Table 2: correct the spelling of pharaonis in the footnotes
21. Figure 3: the revised, track changes version of the paper shows 2 graphs with different data presented. According to a response to Reviewer #1 the data in Figure 3 were originally mislabeled but are now fixed and the text is correct. However, please double check this for accuracy during revision.

Dear Dr. Creamean,

Thank you for revising your paper per my and the reviewers’ comments. I really enjoyed reading the paper again and seeing how the revisions improved the paper and highlight your interesting findings. I’m pleased to accept your paper for publication in Biogeosciences!

Apologies for the delay in getting a decision to you— I had challenges finding a second reviewer at this stage of the process.

Best,
Denise Akob