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

Black patinas are very common biological deterioration phenomena on lapideous artworks in outdoor environments. These substrates, exposed to sunlight, and atmospheric and environmental agents (i.e. wind and temperature changes), represent extreme environments that can only be colonized by highly versatile and adaptable microorganisms. Black patinas comprise a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms hinders their identification by optical microscopy. This study used Next-Generation Sequencing (NGS) (including shotgun and amplicon sequencing) to characterize the black patina of the travertine embankments (muraglioni) of the Tiber River in Rome (Italy). Overall, the sequencing highlighted the rich diversity of bacterial and fungal communities and allowed the identification of more than one hundred taxa. NGS confirmed the relevance of coccoid and filamentous cyanobacteria observed by optical microscopy and revealed an informative landscape of the fungal community underlining the presence of microcolonial fungi and phylloplane yeasts. For the first time high-throughput sequencing allowed the exploration of the expansive diversity of bacteria in black patina, which has so far been overlooked in routine analyses. Furthermore, the identification of euendolithic microorganisms and weathering agents underlines the biodegradative role of black patina, which has often been underestimated. Therefore, the use of NGS to characterize black patinas could be useful in choosing appropriate conservation treatments and in the monitoring of stone colonization after the restoration interventions.

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 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.