PONE-D-24-02517Bacterial and zetaproteobacterial communities from iron-rich microbial mats and microbial colonizers in the Mediterranean Sea (EMSO-Western Ligurian Sea Observatory)PLOS ONE

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Agence Nationale de la Recherche (ANR) Grant ANR-21- CE02-0012

Ministère de l'Education Nationale, de l'Enseignement Superieur et de la Recherche (MESR)  Grant Astorch-Cardona PhD scholarship

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This project was funded by the French Oceanographic Fleet through the 2018, 2020 and 2022 cruises within the EMSO Ligure Ouest program (France). This work was supported by a French National Research Agency (ANR) grant as part of the IRONWOMAN project (ANR-21-CE02-0012). A.A.C. was supported by an MESR PhD scholarship and Aix-Marseille University (France). The project leading to this publication has received funding from the European FEDER Fund under project 1166-39417. ]

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This manuscript highlights the SSU amplicon sequencing and dPCR assessment of iron-rich microbial mats and microbial colonizers found at the EMSO-Western Ligurian Sea Observatory with a focus on Zetaproteobacteria. Itemized specific comments:

(1) In the introduction there are several references describing the occurrence of Zetaproteobacteria and various ecosystems, like hydrothermal vents. No included are where microbial growth chambers have been used at hydrothermal vents across the Pacific showing distinct biogeographic signatures of autotrophic communities including Zetaproteobacteria as the initial colonizers at many sites (Fullerton et al., 2024; doi 10.1093/femsec/fiae001). Other habitats like oligotrophic freshwater lakes have included Zetaproteobacteria, like where Zeta OTUs 2 and 6 have also been detected in the benthic hot spring mats at Crater Lake, OR (Stromecki et al., 2022; doi 10.3389/fmicb.2022.876044). Also see lines 343 to 344 and 446 to 448 in the discussion section.

(2) In the introduction (line 94), where EMSO is used for the first time, this should be spelled out for readers not familiar with this abbreviation.

(3) For Figure 1 and 2, can this map (Fig 1) and these photos (Fig 2) be made with more resolution, they seem very pixilated.

(4) It seems rather hard to make any conclusions regarding colonization from colonization modules that were deployed for ~1 year (line 183), how do you reconcile this with your conclusions? Also see lines 435 to 440 in the discussion section.

(5) Regarding dPCR, in line 216, you state that 45 to 75 cycles were used. This seems rather excessive. Can you show any evidence that detections were made in prior to ~25-30 cycles?

(6) Line 219, the word “pression” should read as “pressure”.

(7) Line 429, change “between” to “among”.

(8) Line 440, period at the end of sentence here.

Reviewer #2: This is a review of the manuscript titled “Bacterial and zetaproteobacterial communities from iron-rich microbial mats and microbial colonizers in the Mediterranean Sea (EMSO-Western Ligurian Sea Observatory) by Astorch-Cardona et al. This is a study that analyzed the colonization of various Fe-bearing substrates including basalt and mild steel by bacteria. This work builds on the foundation laid by other recent studies on basalt and mild steel and corroborates for the most part the results of those studies. The value of the paper is in the different geographical and environmental context, with in-situ incubations done in the Mediterranean with Fe levels much lower than in other sites tested (i.e. Juan de Fuca) or at lower temperatures (2 to 4C), compared to shallower marine sites. So, this study is able to corroborate most of those results but also provides some new findings specific to the Zetaproteobacteria.

Although, the paper is overall well-written; there are some areas that need some polishing. Some major/minor comments are included below.

It’s not clear why the Archaea are not included in the analysis. What is the reason? I would also favor replacing Figure 6 with Table S3 in the main text, which clearly shows the values of the abundances of Zetas near 0 in most of the samples. This is hard to see in Figure 5, and the fact that the symbol is shown for the Zetaproteobacteria in the figure is confusing. I would suggest removing the Zeta symbol in Figure 5 (for samples where the abundance is near 0) because it should be accounted for in the “<1%” category. By doing so, and adding Table S3, the reader will have all the important information in clear sight. I suggest moving Figure 6 to the Supplemental section.

L1: The title is somewhat redundant. The word “Bacterial” necessarily includes the Zetaproteobacteria. The word “microbial” is used twice.

L49-50: These lines are verbatim L27-29 in the abstract.

L51-54: These lines are verbatim L29-32.

L55: I believe Dave Karl proposed that concept earlier in 1988 (Loihi Seamount discovery; Nature paper). In that paper he presents evidence.

L72: Reference 10 is more appropriate here; it’s a 2011 paper. Reference 9 is a 2018 paper.

L155: What type of steel is the steel grit used in this study? It is not clear if it is stainless steel or mild steel. Please specify.

L330: Indicate what ASV means here.

L339: Change “Zetaprotobacterial” to “Zetaproteobacteria”.

L373: Is this meant to be stainless steel or mild steel. Also, basalt does contain sulfur, at low % but comparable to mild steel.

L377-379: Sulfurimonas and Sulfurovum can also oxidize hydrogen. Steel produces H2 when it comes in contact with seawater. Barco et al. (reference 14) identified Epsilonproteobacteria on steel as well as hydrogenase proteins belonging to Epsilonproteobacteria on this substrate. This possibility should be discussed in this section. Mori et al. (2017; not cited in the manuscript) also discusses H2 oxidation in Zetaproteobacteria, more specifically ZetaOTU9 (Ghiorsea bivora). Since ZetaOTU 9 is seen in the microbial mat, growing on steel, this possibility should be discussed as well.

L448: This sentence seems unfinished.

L457: Is there an isolated Zeta from ZetaOTU 2? Please specify in the manuscript. Also, what are the Zeta OTUs linked to the different isolated Mariprofundus species? Specifying this in the main text will help interpret some of the data.

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Reviewer #1: Yes: Craig Lee Moyer

Reviewer #2: No

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Diversity and dynamics of Bacteria from iron-rich microbial mats and colonizers in the Mediterranean Sea (EMSO-Western Ligurian Sea Observatory): focus on Zetaproteobacteria

PONE-D-24-02517R1

Dear Dr. ROMMEVAUX,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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