Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing

Federica Antonelli; Alfonso Esposito; Ludovica Calvo; Valerio Licursi; Philippe Tisseyre; Sandra Ricci; Manuela Romagnoli; Silvano Piazza; Francesca Guerrieri

doi:10.1371/journal.pone.0227639

Peer Review History

Original SubmissionJuly 29, 2019
11 Oct 2019 Decision Letter - Ana R. Lopes, Editor PONE-D-19-21352 Tiber’s embankments black patina characterization by Next-Generation Sequencing PLOS ONE Dear Dr Guerrieri, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Although this study is interesting, some of the criteria publication have not been achieved in particular namely criteria 3, 4 and 5 (https://journals.plos.org/plosone/s/criteria-for-publication). Thus, the authors are strongly advised to hire a copyeditor. In particular, sections such as Abstract/Results and Discussion must be improved, as referred by the reviewers. It is important as indicated by reviewers to give more detail in the material and methods section “DNA extraction, library preparation and sequencing”. In addition to the reviewer’s suggestions I would also advise the authors to indicate the range of the concentration of DNA obtained for the samples. It is also important to know the rarefaction values for Beta diversity. Discussion must be improved as mentioned by the reviewers. Additionally, I would like to know if we can exclude handling contamination when you discuss the following results: “Considering the low number of reads and the few identified taxa we cannot say that actual bacterial or fungal communites were present in the W samples. The presence of bacteria is due to a conspicuous contamination from the Tiber’s water or from humans frequenting the river’s bank, while the presence of fungal sequences is just linked to the environmental contamination.”? Because of the negative result mentioned “this allowed, for the first time, to explore the whole structure of a black patina bypassing the culture problem linked to the underestimation of unculturable species and comprising the bacteria that are usually neglected in routinely analyses. The absence in the molecular results of Chroococcus lithophilus, identified through microscope observation, can be linked to the lack of 16S sequences of this species in the databases. Indeed, currently the main limit to the application of NGS to the study of black patinas and, more in general, of subaerial biofilms is linked to the absence in the databases of several environmental microorganisms’ sequences (like cyanobacteria and RIF). This is confirmed by the presence of several unidentified sequences in the obtained results.”. Have you tried to blast/map the unidentified sequences with the 16S sequence of Chroococcus lithophilus from for example ncbi or EzBioCloud? Minor issues: Standardize in the text “(g, Chroo…” or “(g. R..)”, but not both; Standardize in the text “(species A. pullulans)” or “(A. alternata)”, but not both; We would appreciate receiving your revised manuscript by Nov 25 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Ana R. Lopes, PhD Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 1. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the sampling site access and, if no permits were required, a brief statement explaining why. 2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Black patina are common features in monuments and walls around the world. They often associated with substrata where microenvironmental conditions promote the retention of water. They can be broadly classified depending on the dominant process leading to their formation. Earlier studies highlighted the chemical, pollutant-related origin of black crusts, often associated with Sulphur-laden atmosphere and the formation of gypsum layers that harden on the stone upper layers. On the other hand, on the Tropics, the occurrence of biologically-dominated black crust have been recently reported to be predominantly linked to microbial colonization by pigmented organisms synthesizing compounds such as scytonemins and mycosporine-like substances. It appears that the authors have dealt with in this study with latter type of black crusts; they should highlight this in the title. Secondly, I find that the abstract fails to display the main findings. NGS studies about subaerial communities are comparatively less studied than other terrestrial habitats, the authors should take advantage of this gap of knowledge and exploit more their data. Third, the introduction could certainly be improved by adding relevant references that highlight the biological composition of black crust in stone surfaces. I feel that the authors could improve their study by providing more details about the nature on substratum (mineralogy of stone, from bibliographic data), the prevailing microenvironment (from nearly meteorological stations) and orientation of surfaces. I could notice that samples were taken from either Black or White areas (replicates). A this point I am not sure the white-derived samples are originated from a “white platina” or non-colonized (at least by visual inspection) surfaces. A patina normally refers to surface alteration by a number of processes that result on modification of the upper layer, sometimes pure staining (aesthetic) but also chemical transformation of the upper layer. Please clarify this point. Also, please state if they were taken randomly. In addition, indicate how you managed with graffiti that is obvious on the image. Adding arrows to the sampled areas would have the reader to clearly identity the origin of samples. The apparent lack of correspondence of certain organisms not identified by NGS but seen by microscopy could be explained by the non-efficient extraction of nucleic acids, we have seen this in the past with thick-sheathed cyanobacteria. Overall, I find that the results need be better contrasted with published studies based on both non-culture dependent and culture-dependent studies from epilithic habitats and highlight the main findings. Please also state and provide relevant references as to how this type of NGS-based study can provide relevant information regarding conservation issues. In addition, the conclusion section needs to be enhanced to fully be supported by the results. Reviewer #2: The authors present an interesting study by analyzing the microbiota of a black patina often found over travertine embankments of Tiber river in Rome. For this reason, Next-Generation sequencing techniques, through Illumina platform, were applied in order to identify and characterize different communities of bacteria, fungi and algae, as a mean to understand the possible effects of these colonial organisms to the studied material. The study is well organized, presents relevant data, especially the statistics results and graphics, and the manuscript is well written. I have only minor comments that can be found in comments below: Introduction Line 48 – replace “works of art” for “artworks” In this section a final paragraph with the study objectives is missing. Please add the objectives of the work to complete well the Introduction section. Materials and Methods Line 103 – replace the number “1” for number “2”. In this manuscript “Materials and Methods” are the section number 2. Results In sub-sections “3.1 Bacterial Community” and “3.2 Fungal community” please add the percentages of abundance of the described taxa. This is relevant data in such NGS study and is missing on this section. Discussion I advise the authors to add an introductory paragraph to this section, instead of starting immediately with the results discussion. It would be good to start with some statement (3-4 lines) regarding the importance of the used methodology to characterize the microbiota communities of the black patina present in such important Cultural Heritage structure, which was actually the aim of this study. Reviewer #3: I believe that the manuscript by Antonelli et al. “Tiber’s embankments black patina characterization by Next-Generation Sequencing” (ref: PONE-D-19-21352) is a very interesting study concerning the complete metagenomic analysis of black patinas in an important stone monument. In my opinion, the topic is relevant and deserves to be highlighted. I also would like to pinpoint that the application of Shotgun metagenomics is currently rather scarce in the field, thus turning the article highly innovative. I recommend the acceptance of the article after the authors conduct major revisions in the manuscript, and some crucial points are addressed. I´m providing some comments to be taken into consideration by the authors: (1) Comment: The article should be proofread by an English native speaker. (2) Comment: The term 18S ITS should be replaced for ITS2-rDNA sub-region, since from my understanding the 18S region (SSU) was not considered during the course of this study. (3) Introduction section, Lines 49-60 and Lines 75-76. Comment: Please consider rephrasing these sentences. They are too long and their structure could be improved. (4) Line 71 and Line 284. Comment: In line 71. the reference for Pentecost (1992), in this case [45], is missing. In line 284, the reference for Albertano (2012), in this case [18], is also missing. I advise the authors to double check their references along the manuscript and in the references. (5) Introduction section, Line 64-84. Comment: This part of the introduction section is only focussed in Phototrophic microorganisms and Fungi. I believe that the role (if any) and presence of bacteria in black patinas (if previously studied), should also be highlighted in this part. (6) Introduction section, Line 93-95. Comment: The aims of the study should be clearer. (7) Introduction section, Line 95-99. Comment: I believe this part should be moved to the Materials and methods- Sample collection and description sub-section. (8) Materials and methods section, Line 103. Discussion section, Line 274. Comment: In line 103, this section should be: 2. Materials and methods. In the current form is displayed as 1. Materials and methods. In line 274, this section should be: 4. Discussion. In the current form lacks numbering. (9) Materials and methods section; 2.1 Sample collection and description. Comment: I believe that the manuscript could benefit from the addition of a table displaying the distinct samples IDs and further metadata. The table could also display which samples were able to be studied through the distinct metagenomic methodologies applied. (10) Materials and methods section; 2.1 Sample collection and description. Comment: For the microscopical analysis, were the samples randomly selected? What were the criteria for the selection of these samples? Which samples (ID) were studied? (11) Materials and methods section; 2.2 DNA extraction, library preparation and sequencing. Comment: Please provide further details regarding the DNA extraction, library preparation and sequencing. (12) Results section; 3.2 Bacterial community, Lines 204-208. Comment: This part should be moved to the discussion section. (13) Results (e.g. Lines 227-230), Discussion (e.g. Lines 341-383) and Figures 4 and 8. Comment: These parts highlight my main concern with the manuscript. In general, the application of the Illumina MiSeq methodology targeting the ITS2 rDNA sub-region does not allow a proper and accurate taxonomic annotation to the species level. I believe that these parts as well as the figures above mentioned, need to highlight a taxonomic annotation at the genus level, and therefore require to be updated. I don´t feel that the discussion bulk will be affected by this decision. However, I do acknowledge that this change will require several parts of the manuscript to be updated. (14) Figure 6 legend needs further information, namely the distinct COG categories. (15) Discussion section. Comment: Given the quality of the results, some parts of the discussion section could be deeper debated. For example, the authors state that some bacteria taxa can be linked to the presence of substances of anthropogenic origin. Pollution is known to act as an ecological pressure on cyanobacterial communities. Could the presence of these cyanobacterial taxa be linked to extremotolerance profiles allowing them to thrive in these conditions? MCF are also known for their tolerance to various environmental factors, could their presence in the B samples also be explained by their high metabolic capacities? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227639.r001
Revision 1
25 Nov 2019 Author Response Point-by-point response to reviewer’s comments Reviewer # 1 We appreciated the challenging quality of the Reviewer 1’s comments that prompted us to carefully revise the manuscript to clarify experimental details and results in order to increase its readability and to better support our conclusions. Specific points Question 1. Black patina are common features in monuments and walls around the world. They often associated with substrata where microenvironmental conditions promote the retention of water. They can be broadly classified depending on the dominant process leading to their formation. Earlier studies highlighted the chemical, pollutant-related origin of black crusts, often associated with Sulphur-laden atmosphere and the formation of gypsum layers that harden on the stone upper layers. On the other hand, on the Tropics, the occurrence of biologically-dominated black crust have been recently reported to be predominantly linked to microbial colonization by pigmented organisms synthesizing compounds such as scytonemins and mycosporine-like substances. It appears that the authors have dealt with in this study with latter type of black crusts; they should highlight this in the title. Answer 1. We apologize if the description of black patina, and its differences with black crusts, were not clearly defined in the introduction. Reviewer 1 raises an important point and, albeit we feel that the definition “black patina” is already a clear statement on the biological origin of the alteration, in the new MS we highlighted that black crusts and black patinas are two distinct deterioration patterns of stone artefacts. Black crusts have a chemical origin and are defined as “Kind of crust developing generally on areas protected against direct rainfall or water runoff in urban environment. Black crusts usually adhere firmly to the substrate. They are composed mainly of particles from the atmosphere, trapped into a gypsum (CaSO4.2H2O) matrix” (Vergès-Belmin V. Illustrated glossary on stone deterioration. ICOMOS. 2008). The term patina refers to several kinds of alterations, of organic and inorganic origin, tightly adhering to the substrate. As reported in the manuscript’s introduction, since the 1990s the term is also used to define an aesthetic change of rock surfaces linked to the biological colonization. Black patinas are a well-known biological alteration of stone surfaces studied since the beginning of 1900 in natural environments. In the field of the conservation of Cultural Heritage several studies covered this topic in the last decades. Question 2. Secondly, I find that the abstract fails to display the main findings. NGS studies about subaerial communities are comparatively less studied than other terrestrial habitats, the authors should take advantage of this gap of knowledge and exploit more their data. Answer 2. We thank the Reviewer 1 for this suggestion. The abstract was modified. Question 3. Third, the introduction could certainly be improved by adding relevant references that highlight the biological composition of black crust in stone surfaces. Answer 3. The biological composition of the patina is now better described in the introduction and further information were added concerning the bacteria composition. Question 4. I feel that the authors could improve their study by providing more details about the nature on substratum (mineralogy of stone, from bibliographic data), the prevailing microenvironment (from nearly meteorological stations) and orientation of surfaces. Answer 4. We agreed with the Reviewer 1 and the new materials and methods section contains the data concerning the composition of travertine and thermo-hygrometric trends registered in the city. Question 5. I could notice that samples were taken from either Black or White areas (replicates). A this point I am not sure the white-derived samples are originated from a “white platina” or noncolonized (at least by visual inspection) surfaces. A patina normally refers to surface alteration by a number of processes that result on modification of the upper layer, sometimes pure staining (aesthetic) but also chemical transformation of the upper layer. Please clarify this point. Answer 5. The Reviewer is right: The term “white” could be confusing. We collected this powder from apparently not colonized area and not from “white patina”. We modified the legend of the samples changing “white” in “uncolonized” area and we added a new table with the ID of the samples and corresponding DNA extraction values (Table S1). Question 6. Also, please state if they were taken randomly. In addition, indicate how you managed with graffiti that is obvious on the image. Adding arrows to the sampled areas would have the reader to clearly identity the origin of samples. Answer 6. The materials and methods section was clarified as requested and arrows were added in the figure. Question 7. The apparent lack of correspondence of certain organisms not identified by NGS but seen by microscopy could be explained by the non-efficient extraction of nucleic acids, we have seen this in the past with thick-sheathed cyanobacteria. Answer 7. We welcomed the comment of the Reviewer and we agree that difficulties in lysis of specific bacterial sheaths and consequently DNA extraction could be responsible for the not complete correspondence with the microscopic analyses results. For this reason we added a specific note concerning this point in the revised manuscript. Question 8. Overall, I find that the results need be better contrasted with published studies based on both non-culture dependent and culture-dependent studies from epilithic habitats and highlight the main findings. Answer 8. The text was modified as requested. Question 9. Please also state and provide relevant references as to how this type of NGS-based study can provide relevant information regarding conservation issues. Answer 9. We would like to emphasize that this is the first study that apply NGS to the characterization of black patinas, so no references are available about the relevance of this technique in the field of conservation. The importance of the obtained data has been underlined in the text. Question 10. In addition, the conclusion section needs to be enhanced to fully be supported by the results. Answer 10. The conclusion section was extensively revised and we think that now it’s better supported by the results. Reviewer # 2 General comment The authors present an interesting study by analyzing the microbiota of a black patina often found over travertine embankments of Tiber river in Rome. For this reason, Next-Generation sequencing techniques, through Illumina platform, were applied in order to identify and characterize different communities of bacteria, fungi and algae, as a mean to understand the possible effects of these colonial organisms to the studied material. The study is well organized, presents relevant data, especially the statistics results and graphics, and the manuscript is well written. We have been very happy to learn that the Reviewer 2 found the MS “an interesting study”, “well organized”, that “presents relevant data” and “well written”. Specific points Question 1. Introduction. Line 48 – replace “works of art” for “artworks” Answer 1. The text was modified as indicated. Question 2. In this section a final paragraph with the study objectives is missing. Please add the objectives of the work to complete well the Introduction section. Answer 2. The introduction was improved and the objectives of the work were added Question 3. Materials and Methods. Line 103 – replace the number “1” for number “2”. In this manuscript “Materials and Methods” are the section number 2. Answer 3. The text was modified as indicated. Question 4. Results. In sub-sections “3.1 Bacterial Community” and “3.2 Fungal community” please add the percentages of abundance of the described taxa. This is relevant data in such NGS study and is missing on this section. Answer 4. We thank the Reviewer 2 for the suggestion. Two tables (one for bacteria and one for fungi) containing the percentages of abundance for each taxon (averages and standard deviations) in black patinas and uncolonized stone have been produced as supplementary inserts. Question 5. Discussion. I advise the authors to add an introductory paragraph to this section, instead of starting immediately with the results discussion. It would be good to start with some statement (3- 4 lines) regarding the importance of the used methodology to characterize the microbiota communities of the black patina present in such important Cultural Heritage structure, which was actually the aim of this study. Answer 5. We agree with the Reviewer 2 and an introductory statement was added to the paragraph. Reviewer # 3 General comment I believe that the manuscript by Antonelli et al. “Tiber’s embankments black patina characterization by Next-Generation Sequencing” (ref: PONE-D-19-21352) is a very interesting study concerning the complete metagenomic analysis of black patinas in an important stone monument. In my opinion, the topic is relevant and deserves to be highlighted. I also would like to pinpoint that the application of Shotgun metagenomics is currently rather scarce in the field, thus turning the article highly innovative. I recommend the acceptance of the article after the authors conduct major revisions in the manuscript, and some crucial points are addressed. We would like to thank the Reviewer 3 for considering our study “highly innovative” and for his/her comments/suggestions that prompted us to improve the quality of the MS. Specific points Question 1. The article should be proofread by an English native speaker. Answer 1. The manuscript has been proofread by an English native speaker Question 2. The term 18S ITS should be replaced for ITS2-rDNA sub-region, since from my understanding the 18S region (SSU) was not considered during the course of this study. Answer 2. The word "18S ITS" has been replaced with " ITS2-rDNA " in the revised manuscript Question 3. Introduction section, Lines 49-60 and Lines 75-76. Comment: Please consider rephrasing these sentences. They are too long and their structure could be improved. Answer 3. The text was modified as suggested. Question 4. Line 71 and Line 284. Comment: In line 71. the reference for Pentecost (1992), in this case [45], is missing. In line 284, the reference for Albertano (2012), in this case [18], is also missing. I advise the authors to double check their references along the manuscript and in the references. Answer 4. The manuscript was checked and all the missing references were added. Question 5. Introduction section, Line 64-84. Comment: This part of the introduction section is only focused in Phototrophic microorganisms and Fungi. I believe that the role (if any) and presence of bacteria in black patinas (if previously studied), should also be highlighted in this part. Answer 5. We agree with the Reviewer and, although the role of the bacteria in subaerial biofilms had not been clarified jet, we added a section in the introduction regarding these microorganisms. Question 6. Introduction section, Line 93-95. Comment: The aims of the study should be clearer. Answer 6. The introduction was modified and the aims of the study were clarified Question 7. Introduction section, Line 95-99. Comment: I believe this part should be moved to the Materials and methods- Sample collection and description sub-section. Answer 7. The text was modified as indicated. Question 8. Materials and methods section, Line 103. Discussion section, Line 274. Comment: In line 103, this section should be: 2. Materials and methods. In the current form is displayed as 1. Materials and methods. In line 274, this section should be: 4. Discussion. In the current form lacks numbering. Answer 8. The text was modified and the paragraph numbers were removed as requested by the Plos One author’s guidelines Question 9. Materials and methods section; 2.1 Sample collection and description. Comment: I believe that the manuscript could benefit from the addition of a table displaying the distinct samples IDs and further metadata. The table could also display which samples were able to be studied through the distinct metagenomic methodologies applied. Answer 9. Reviewer 3 is right. We added a table (Table S1), displaying the distinct sample IDs, specific amount of DNA extraction, corresponding obtained library and sequencing quality checkpoint. Question 10. Materials and methods section; 2.1 Sample collection and description. Comment: For the microscopical analysis, were the samples randomly selected? What were the criteria for the selection of these samples? Which samples (ID) were studied? Answer 10. We apologize if the description of this part of the material and methods section was not clear. The section “Sample collection and description” was revised and now we believe that we address the specific concerns regarding the selection of the samples. Question 11. Materials and methods section; 2.2 DNA extraction, library preparation and sequencing. Comment: Please provide further details regarding the DNA extraction, library preparation and sequencing. Answer 11. The section “DNA extraction” in material and methods was added with more detailed information, whereas the sections “Sample collection and description” and “Sequencing Data Analysis” were revised. Question 12. Results section; 3.2 Bacterial community, Lines 204-208. Comment: This part should be moved to the discussion section. Answer 12. The text was modified as suggested. Question 13. Results (e.g. Lines 227-230), Discussion (e.g. Lines 341-383) and Figures 4 and 8. Comment: These parts highlight my main concern with the manuscript. In general, the application of the Illumina MiSeq methodology targeting the ITS2 rDNA sub-region does not allow a proper and accurate taxonomic annotation to the species level. I believe that these parts as well as the figures above mentioned, need to highlight a taxonomic annotation at the genus level, and therefore require to be updated. I don´t feel that the discussion bulk will be affected by this decision. However, I do acknowledge that this change will require several parts of the manuscript to be updated. Answer 13. The figures were modified as requested and the Results and discussion sections were updated Question 14. Figure 6 legend needs further information, namely the distinct COG categories. Answer 14. The revised version of Figure 6 contains the descriptions for each COG category. Question 15. Discussion section. Comment: Given the quality of the results, some parts of the discussion section could be deeper debated. For example, the authors state that some bacteria taxa can be linked to the presence of substances of anthropogenic origin. Pollution is known to act as an ecological pressure on cyanobacterial communities. Could the presence of these cyanobacterial taxa be linked to extremotolerance profiles allowing them to thrive in these conditions? MCF are also known for their tolerance to various environmental factors, could their presence in the B samples also be explained by their high metabolic capacities? Answer 15. The discussions were modified as requested. Two paragraphs concerning the cellular and metabolic peculiarities of cyanobacteria and MCF were added Attachments Attachment Submitted filename: Point by point response Antonelli et al.pdf https://doi.org/10.1371/journal.pone.0227639.r002
18 Dec 2019 Decision Letter - Ana R. Lopes, Editor PONE-D-19-21352R1 Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing PLOS ONE Dear Dr Guerrieri, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Although the authors addressed most of the previous reviewers’ comments there are still some minor issues that need to be addressed, in order to fulfill the criteria to publish in PLOS ONE. In particular, improve the manuscript keywords, avoid redundant information. The second aim described in the introduction does not need a new paragraph, include it in the previous paragraph. Please also indicate the place/laboratory where the Illumina sequencing was performed (in line 165). The sentence given in lines 374-376 “The fact that these microorganisms have never before been reported in black patinas on stone artifacts is most likely due to the techniques routinely used in the field of cultural heritage, which are often not appropriate for their detection or identification.” must be properly supported, add a reference please. Minor issues: Include the sentences in lines 208-209 and 210-211 in the previous paragraph; Line 140, Please replace “Twenty samples of black patina (B) and twenty controls (U) were used..” by “Twenty samples of black patina (B) and twenty controls (uncolonized region, U) were used…”; Figure 3 and 4, include the color representing each sample in the PCoA biplot; Figure 5 the species identified should be in italic; Improve the graphics presented in figure 7 and 8, avoid the background colors; The sentences from line 491-494 could be included in the previous paragraph. Moreover the sentence must be improved, it is not clear what the authors are reporting. We would appreciate receiving your revised manuscript by Feb 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Ana R. Lopes, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: The authors have made several improvements to the manuscript and I believe it is now ready for publication. All suggested comments have been fully addressed, as well as other important issues have been corrected and improved. I have no further comments on this work. Reviewer #3: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227639.r003
Revision 2
20 Dec 2019 Author Response Object: Resubmission Manuscript PONE-D-19-21352R1 Dear Editor Ana R. Lopes, We thank you for accepting with minor revision our manuscript entitled “Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing” to PLOS ONE. We feel that we have addressed all the minor issues and in particular: - The manuscript keywords are now improved - The laboratory where the Illumina sequencing was performed was added - Two new references were added to support the conclusions (Sterflinger K, Piñar G. Microbial deterioration of cultural heritage and works of art - Tilting at windmills? Appl Microbiol Biotechnol. 2013;97: 9637–9646. doi:10.1007/s00253-013-5283-1; Ricci S, De Leo F, Urzì C, Guerrieri F, Antonelli F. Advantages of a multidisciplinary approach in the study and the characterisation of black patinas. In: Macchia A, Masini N, La Russa MF, Prestileo F, editors. Dialogues in Cultural heritage, Books of Abstracts of the 6th YOCOCU Conference. Matera: YOCOCU, CNR – IBAM (Istituto per i Beni Archeologici e Monumentali); 2018. pp. 277–280) - Figure 7 and 8 were re-drawn The authors all concur with this submission and affirm that the material presented in this manuscript has not been previously reported and is not under consideration for publication elsewhere. Additionally, the authors have no conflicting financial interests to disclose. Looking forward to hearing from you at your earliest convenience, Sincerely, Francesca Guerrieri Federica Antonelli https://doi.org/10.1371/journal.pone.0227639.r004
26 Dec 2019 Decision Letter - Ana R. Lopes, Editor Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing PONE-D-19-21352R2 Dear Dr. Guerrieri, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Ana R. Lopes, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227639.r005
Formally Accepted
27 Dec 2019 Acceptance Letter - Ana R. Lopes, Editor PONE-D-19-21352R2 Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing Dear Dr. Guerrieri: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ana R. Lopes Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227639.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .