Antifungal, Antibacterial, and Interference Effects of Plant-Extracted Essential Oils Used for Mural Conservation at Buyeo Royal Tomb No. 1

Round 1

Reviewer 1 Report

The article expands on the growing attention to essential oils for remediation of biopollution. As such, it is of interest to the scientific community. In particular, mural paintings are targeted.

 

I have few recommendations to strengthen the paper quality:

 

Title is too long and could be shortened with only the essential terms.

 

Essential oils: the authors should provide more accurate and complete literature data regarding the composition and volatility (boiling point, etc.) of the selected oils. This info can go in a SI file, but needs to be provided for conservation scientists to have a clearer idea of what is being applied onto the murals.

 

Stability to painted layers: the authors assessed animal glue as a binder for “secco” murals, but they should also assess oil; that’s where one would expect possible swelling or leaching of the bound pigments by the essential oils.

 

Also, the stability of the essential oils to UV-Vis should be commented further. Some residues of the oils seems to remain based on gravimetric assessment by the authors, and even though tombs are rarely exposed to light, this factor should be discussed somehow by the authors.

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Reviewer 2 Report

The paper is quite interesting and suitable for publication after taking into consideration some comments.

Row 155. “The concentration of 100% pure EO is too high to apply to ancient tomb murals.” Please explain why

Row 185 What about the size if the specimens? How did the authors treat the specimens? Brushing, dipping…

I think the XRD analysis is useless for the assessment, because the investigated stones are very stable, there is not any reason to observe an changing in the mineral composition after a treatment with an oil.

Row 205 The specimens of the painting layer. Is the recipe based on a previous study of the composition of the paintings themselves, not only in terms of pigments, but also in terms of execution technique (binder, stratigraphy and so on)

Table 5. It would be interesting which parameter (L, a, b) is mostly involved into the color changing, in the text authors referred to a yellowing, but it should be quantified.

Row 387 authors stated that they have evaluated the stability, but I think that the stability is something to be assessed during time, for example by exposing treated specimens in the Tombs. That is why I think the authors did not asses the stability, but just the suitability of the EOs on stone and painted layers.

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Reviewer 3 Report

The manuscript by Hyun Ju Lee and Yong Jae Chung is focused on the possibility of the use of EOs in the conservation strategy  of mural paintings at Buyeo Royal Tomb No. 1. 

This is a relevant and interesting topic, and the presented work seems to me useful to improve the knowledge about Eos; if the topic is, not so, new this study is well structured and clearly illustrated, this is -in my opinion- very important to achieve a practical use of Eos in the field of cultural heritage. However, some revisions should be necessary.

As a main suggestion, I invite the authors to add statistical elaborations of their data, so their conclusions can be better supported and the paper clear for the reader. The weight change measurements of granite and gneiss specimens is a bit weak and not well illustrated part, must be clarified.

An improvement can also be obtained by making the captions of tables and figures more complete; a caption must always allow the understanding of the figure/table without having to read the text. The title “Microbial Growth Inhibition and Interference Effects of 2 Plant-Extracted Essential Oils to conserve Mural paintings at 3 Buyeo Royal Tomb No. 1” is accurate. In the References section it is necessary to carefully check the formatting of the text, especially as regards the use of italics.

A list of comments is subsequently reported to detail some points of the text which should/may be re-checked to improve the clarity of the contents.

 

Abstract: 

lines 17-18:  “forming inhibition zones of 3.0–19.3 mm and 2.6–17 19.3 mm, respectively” this kind of detail is not appropriate for the abstract: for the reader at this moment the measures of the inhibition halo are not indicative (moreover, the sentence speaks of 4 EOs but only two measures are given)

 

line 19: “the six microbial strains.” Since the microbials stains have not yet been presented, “the tested microbial strains” is clearer.

 

line 21: “granite and gneiss specimens treated with the EOs at 1–10%.” lithotypes also have not yet been presented, “lithotypes specimens (granite and gneiss)” maybe is clearer.

 

line 23-24: “Our results suggest 3% oregano EO as a natural alter-23 native biocide for conservation of ancient tomb murals with biological damage”. This sentence is perhaps too strong: the system is complex and species-specific sensitivity must be considered, the sentence should be reformulated.

 

Introduction:

line 33: “entry and exit of tourists” written like this seems to refer to some  bureaucratic problem, but probably the authors wanted to indicate the transport of fungal spores or bacteria on tourists' shoes and clothes, perhaps better to clarify.

 

Materials and Methods:

2.2.1. Selection of standard test strains

The paragraph does not explain why precisely these strains were selected (as for the Eos in the paragraph 2.2.2): the addition of a sentence that justifies the choice (are they the most widespread? the most difficult to eliminate on the basis of the scientific literature?) will be useful for the reader…

 

Table 1. The first column's title (Substance) should be changed in “Vernacular name”.

The caption can be changed as “List of selected essential oils tested on microbial strains isolated from Buyeo Royal Tomb No. 1”, more informative.

lines 137-139: “The EOs were applied to a paper disc (8 mm diameter) to be fixed on the medium; the disc was prepared to be able to absorb 8 μL of oil”. How the paper disc was fixed (not just put on the medium?) can the authors add a reference to clarify?

 

lines 150-151: Test for bacteria are also in triplicate?

 

2.2.4. Measurement of antifungal and antibacterial activities of low-concentration essential oils

the description of the cultivation method is redundant with respect to the previous paragraph, it is better to refer to that and highlight only the differences

 

lines 165-166 “The specimen was cultured in an incubator for 5–7 days, and the diameter (mm) of the halo formed around the paper disc was measured (Figure 2A)“ how was the halo measured? manually? on images taken with a camera and stereo microscope?

 

lines 182-187 “The stone material (source: Buyeo area) was washed with distilled water and dried in a 60°C hot-air drier for 24 h. Specimens of 2.5–5.5 g were used for this assay. The EOs prepared in varying concentrations were placed in a 50-mL conical tube containing the specimen and the tube was shaken at ambient temperature (20 ± 2°C) for 180 h. The control was a mixture of distilled water and the solvent used to dilute EOs to low concentrations.” 

Why did the authors choose this protocol? Can they provide a reference? Information on how many samples were treated is missing

lines 188-189 e lines 219-220 “surface observation”

it is not clear how the surface observation between the rock samples and the painting layers pre and post treatments was performed, can the authors also clarify by adding references and images?

 

Results

 

Figure 3. In the caption it is better to add the information that the arrangement of paper disks with different concentrations and controls is shown in figure 2 

 

Table 2. The caption can be changed in “Effects of tested EOs (pure) on microbial strains isolated from Buyeo Royal Tomb No. 1”.

The presentation of the results only with mean and standard deviation is not immediately readable and there is no indication of how statistically significant the variations are. The authors should also do an analysis of variance ANOVA (or equivalent non-parametric test, if the data are not normal) in order to better discussed differences in efficacy.

 

Table 3. The caption can be changed in “Effects of tested EOs (low-concentration) on microbial strains isolated from Buyeo Royal Tomb No. 1”. Also the results of these tests must also be subjected to analysis of variance.

 

lines 289-290: ”The granite specimens did not show a color change after treatment with oregano EO, clove bud EO, thyme EO, cinnamon cassia EO, and the control (Distilled water and solvent) for all low concentrations tested (1–10%).” The “materials and methods” chapter does not mention any colorimetric measurements for samples of stone material, and numerical results are not reported here. Does this comment refer to visual inspections only? 

 

3.3. Interference of low-concentration essential oils with stone materials

The values ​​obtained are very close to those of the standard deviation, this suggests either an insufficient number of samples or some methodological problem. Furthermore, no statistical analyzes are presented here either to indicate whether the differences are significant or not.

Figure 4 and Figure 5

Results of X-ray diffraction shown similar composition before and after the

treatment, but not identical. Authors should better discuss differences in peak profiles are due to instrumental limits? to specimens variability? how many specimens were analyzed? Can EOs have crystalline phases detected by the instrument?

Table 5

I suggest that the authors put the values ​​above the threshold in bold to make the table more readable

 

Discussion

lines 357-360 "The detection of the microbial inhibition of these EOs at lower concentrations in previous studies than found in the present study is resumed to be related to differences in the EO extraction location and method, along with the type and content of emulsifier used in the production of the EO.” 

The authors should also consider the variability of living organisms as a factor: there is a species-specific sensitivity to chemical biocides, why should it be different with EOs?

line 400 “plastering vs. spraying” plastering is application by poultice?

 

Conclusion

lines 424-426 “Further studies are needed to comprehensively investigate the methods of EO treatment, the retention of these effects, and their stability according to the specific characteristics of the ancient tomb murals.” 

And also medium and long term effects should be investigate…

 

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Reviewer 4 Report

1.      The introduction part is well written but author can incorporate some new references like:

·        Biodeterioration Risk Index of Exhibit Present in Museum Galleries of Tropical Climate, Museum Management and Curatorship, DOI:101080/09647775.20151118645

·        Assessment of viable fungi in indoor air: a case study from Tagore’s residence at Jorasanko, India

·        Fumigation of eucalyptus oil for controlling strong room fungi at Jorasanko Museum (Tagore's residence), India: a study for sustainable conservation

2.      It will better if authors would mention the name of bacteria and fungi used for this experiment. They could have been identified up to genes level at least.

3.      In the result and discussion part author has compare with the previous study so they can incorporate the name of their experimental strain.

4.      Author can mention the path way of inhibition of fungal strain.

5.      The conclusion is well explained.

 

As a whole the manuscript can be recommended for publication

 

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Round 2

Reviewer 3 Report

The authors have revised and improved many parts of the manuscript, however they do not have add statistical elaborations of their data. So the main problem I have noticed is still here… 

Authors presented also in this revised version the table 1 data only with mean and standard deviation cause they affirm “it was not possible to perform additional statistical analysis”: if the authors are not able to perform an analysis of variance ANOVA (or equivalent non-parametric test) they must provide raw data as supplementary materials for both data reported in table 1 and table 3. In these cases I agree with authors that the standard deviation of the results is small, and the data are reliable.

 

 In the case of weight change rate of the granite and gneiss specimens however the obtained values of measurements are too close to those of the standard deviation: and remove the standard deviation values is not the solution! This is unacceptable. In this case results are not reliable at all. Authors do not provide any reference for this method: is it really a correct way to assess the interference of low-concentration essential oils with stone materials? If authors are convinced that it is, they must add a reference for the method, restore the previous version of table 4 (with the standard deviation) and discuss the high variability of results in comparison with literature reference (and also in this case provide raw data as supplementary materials). 

 

A minor point: I’ve noticed that in table 1 “only one species is indicated with the complete name: Syzygium aromaticum (L.), authors can delete (L.) or add authors in the other cases, es. Eucalyptus globulus Labill., the important thing is that it is uniform for all the species.

 

 

Author Response

Thank you for your second comment.

The manuscript has been revised to reflect your comments as much as possible. 

Please check the answer and manuscript files. 

 

 

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