Sustainable Restoration Guided by Scientific and Archival Investigations: The Bio-Cleaning of Lorenzo Duke of Urbino’s Sarcophagus, a Michelangelo’s Masterpiece in the Medici Chapels

Round 1

Reviewer 1 Report

Comments on heritage-1974507

In this article, a series of bacteria strains were tested for the biocleaning of Lorenzo Duke of Urbino’s sarcophagus. Moreover, a non-planar dye W01 was used for comparison. After a physical-chemical analysis of this masterpiece, deposits of gypsum, calcium oxalate, calcium phosphates, and proteins were found to have a detrimental effect in its aspect. Thus, S. ficaria, P. Stutzeri, and Rhodococcus sp were immobilized in Vanzan or Laponite to create micro-packs which were applied to deteriorate areas removing the deposits within 2 days. In my opinion, this manuscript, which is well-written and organized, will merit its publication in Heritage journal after addressing the following minor points.

 1)       On page 2, please add the following reviews which should give a wider view to the reader “The introduction of these methods that use safe microorganisms and other natural substances is proving to be a feasible way to address problems in restoration phases: disinfection, cleaning, consolidation, and protection (Bietti et al. 2022)”

Molecules 2020, 25(23), 5499; https://doi.org/10.3390/molecules25235499

Appl. Sci. 2021, 11(12), 5695; https://doi.org/10.3390/app11125695

2)       On page 4 the second paragraph has a different font.

3)       I believe Table 1 should be moved to the Results section. Please remember Materials and Methods should only tell us HOW you did it but not WHAT you got from it.

4)       On page 5 section 2.3. it is mentioned that “…The cultures were then centrifuged at 3000 g for 15 min…” Do you mean 3000 rpm?

5)       In the Figure 6 caption, literal c and d are interchanged.

6)       The authors might combine Figure 7 and Figure 8.

7)       Only the more effective results should be presented in Figure 9, because they look too small now, and one cannot localize them easily. The authors might take the rest of the test to the Supporting Material.

8)       On page 11 a part of the fourth paragraph has a different font.

9)       On page 12 in the Conclusion a part of the first paragraph has a different font.

10)   I think the authors should mention once again the most effective bacterial strains in the conclusions.

 

Author Response

Reviewer 1

• On page 2, please add the following reviews which should give a wider view to the reader “The introduction of these methods that use safe microorganisms and other natural substances is proving to be a feasible way to address problems in restoration phases: disinfection, cleaning, consolidation, and protection (Bietti et al. 2022)”. Ortega-Morales BO, Gaylarde CC. Bioconservation of Historic Stone Buildings—An Updated Review. Applied Sciences. 2021; 11(12):5695. https://doi.org/10.3390/app11125695

Answer: The references have been added and in general the references have been improved 

2)       On page 4 the second paragraph has a different font.

Answer: The font has been unified

3)       I believe Table 1 should be moved to the Results section. Please remember Materials and Methods should only tell us HOW you did it but not WHAT you got from it.

Answer: Table 1 has been moved to Result section

4)       On page 5 section 2.3. it is mentioned that “…The cultures were then centrifuged at 3000 g for 15 min…” Do you mean 3000 rpm?

Answer: yes, rpm  has been changed in section 2.3

5)       In the Figure 6 caption, literal c and d are interchanged.

Answer: thank you for noticing, the caption has been corrected

6)       The authors might combine Figure 7 and Figure 8.

Answer: thank you for the suggestion;  Figure 7 was missing the scheme on the right. We have corrected the figure so now we prefer to keep them separated  since they are related to two different analytical steps: the first one corresponding to the characterization of the patinas, the second one to the in-situ analysis of the distribution of the compounds on the surface.

7)       Only the more effective results should be presented in Figure 9, because they look too small now, and one cannot localize them easily. The authors might take the rest of the test to the Supporting Material.

Answer: thank you for the suggestion, Figure 9 was changed and labels were added to make the figure readable. We believe this picture may help in understanding the choice of the dtrains, so we prefer to keep it in the text.

8)       On page 11 a part of the fourth paragraph has a different font.

9)       On page 12 in the Conclusion a part of the first paragraph has a different font.

 Answer: The font has been unified

10)   I think the authors should mention once again the most effective bacterial strains in the conclusions. Answer: The strains were mentioned in the Conclusion section

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Reviewer 2

The introduction correctly introduces the masterpiece's origins, history, and brawn deposit problems. But little is said about the biocleaning state of the art; only one sentence is included, and one reference is in the third paragraph. The contextualization with respect to previous and present background and empirical research of the topic must be improved. It will be better to include more information about biocleaning technology, a brief description of the technique, and other authors' applications on similar stone masterpieces. Advantages compared to the traditional methods could be also useful to include in this section.

Answer: The introduction was improved according to this suggestion by adding references and more infos.

The Materials and Methods section is well described. Just a few specific comments: -In figure 2 authors talk about stereomicroscope, but in the 2.1 subchapters, that instrument is missing; please add and describe the model used, etc.

Answer: the stereomicroscope details were added (Zeiss model Stemi 200 C equipped with an external alogen lamp).

in subsections 2.3 and 2.4 the micro-packs application procedure is missing; how did you apply the micro-packs? By brush? spatula? –

Answer: by spatula, the procedure was detailed in section 2.3 : The micro-pack is usually applied by spreading the gellified bacterial suspension with a spatula over a sheet of Japanese paper (to be easily removed without leaving any residues) then covered with plastic film to protect from evaporation. All the details were added in the revised version and now we hope the procedure is more understandable.

In subsection 2.4 the procedure is not clear enough. How did you apply the 3 selected strains? At the same time, one after the other? Each micro-pack has only one strain, a scheme or graphic or text detailed description of the process could be very useful for future applications

Answer: The micro-packs, each containing a single strain, were applied in sequence on the whole surface of the sarcophagus. The procedure was detailed in section 2.4.

The general scheme is added to supplementary material, but the need for multiple strains action depends on the nature of the deposit: in some cases one single strain is enough to remove the deposit

-also, it is missing if the final cleaning step with cotton or sponge was done with sterile water, dry cleaning -it is missing monitoring of the bio-cleaning, did you check the absence of the microorganisms on the surface after the biocleaning treatment? Please add it to materials and methods and results and discussion.

Answer: the residues were cleaned with cotton or sponge with tap water. In this case, we did not monitor the absence of the microorganisms on the surface after the bio-cleaning, because in previous applications we did confirm the absence of the specific bacteria so we could trust the procedure. The reason had been added to material and method section.

The results section is correct and shows the information correctly, and the images showing the before and after treatment are spectacular. There is only a comment to be clarified by the authors: -in page 10, just after Figure 9, a sentence “the red dots….”, clarify.

Answer: the figure was missing the red dots on the three strains, they were added in the revised version

The discussion is coherent balanced and compelling. Just some comments, the authors said that the cleaning done on Michelangelo's masterpieces between 2013 and 2019 was monitored by colorimetric and reflectance measurements on the marble. Did not the authors do this monitoring on the sarcophagus of Lorenzo Duke of Urbino was biocleaned 2019-2020? If done, please include such results in this manuscript.

Answer: The analytical plan was carried out to monitor and address the cleaning procedures exclusively of the sculptures belonging the two funerary monuments by the combined application of colorimetric and reflectance measurements.

On the sarcophagus, chemical characterization allowed to identify the composition of the dark patinas to be removed with the bio cleaning and to support the selection of the most suitable strains

The discussion and conclusion sections, It is missing a relation with secondary literature that supported the obtained results. The cited references are poor for the content of the manuscript and are not adequately referenced.

Answer: the supporting literature was implemented in Discussion

 The way they are listed in the final reference list by numbering way in the reference list? I would suggest removing the numerical order and just leaving them with alphabetical order

Answer: The reference section was corrected

Reviewer 3 Report

Manuscript ID heritage-1974507 deals with in situ application of several bacteria for bio-cleaning of Michelangelo’s masterpiece in the Medici chapels. The manuscript is well written and structured, research is sound and of interest to the readers of heritage journal, however there are a few things that need to be corrected before the manuscript can be accepted for publication. The biggest concern is the biosafety level of used strains.

Manuscript appears to be submitted before quality check, i.e. author name being highlighted, various fonts and line spacing being used... please uniform all this in the revision.

List of specific comments:  

Abstract:

- Please use full name of used bacteria

- Please use full names for all mentioned abbreviations (ENEA-MIRRI...)

Material and methods:

- Why was Table 1 included in Material and methods section? It is a results and as such should have been incorporated into the Results section.

- It was noted that used strains are non-hazardous, but you only mention selection based on chemical characterization of strains. Did you check and select them as members of Biosafety Level 1 group? It would be a necessary pre-requisite before any in situ application.

- Once mentioned, there is no need to write full name of species. It must be written in shortened form (e.g. Serratia ficaria - S. ficaria).

Author Response

Reviewer 3

Manuscript ID heritage-1974507 deals with in situ application of several bacteria for bio-cleaning of Michelangelo’s masterpiece in the Medici chapels. The manuscript is well written and structured, research is sound and of interest to the readers of heritage journal, however there are a few things that need to be corrected before the manuscript can be accepted for publication. The biggest concern is the biosafety level of used strains.

 

1-Manuscript appears to be submitted before quality check, i.e. author name being highlighted, various fonts and line spacing being used... please uniform all this in the revision.

Answer: The manuscript quality was improved

List of specific comments: 

Abstract:

- Please use full name of used bacteria

Answer: The full name were added

- Please use full names for all mentioned abbreviations (ENEA-MIRRI...)

Answer: The abbreviations were added

Material and methods:

- Why was Table 1 included in Material and methods section? It is a results and as such should have been incorporated into the Results section.

Answer: these problems were all fixed

- It was noted that used strains are non-hazardous, but you only mention selection based on chemical characterization of strains. Did you check and select them as members of Biosafety Level 1 group? It would be a necessary pre-requisite before any in situ application.

Answer: This is a very sensitive issue. The strains we use are all isolated from environmental samples (soil, water, ect.) and are checked in the ATCC and DSMZ collections for their biohazard classification. They belong to biohazard safety level 1 (that is, not recognized as dangerous for human health).  Stenotrophomonas maltophilia TPID9 is classified in  biohazard class 1 by ATCC and 2 by DSMZ. Only the strain Acinetobacter calcoaceticus LAM21 is classified in class 2 by both the collections,  and for this reason we have performed (togheter with a qualified laboratory) a complete set of antibiotic resistance, resulting in a wide sensitivity to the major classes of antibiotics. Also, the complete genomic sequence is under way, to assess the phylogenetic affiliation and the lack of pathogenicity of this particular strain (it will probably belong to a new species). All these facts were added to the text

 Once mentioned, there is no need to write full name of species. It must be written in shortened form (e.g. Serratia ficaria - S. ficaria).

Answer: the short form was used throughout the text