Application of Essential Oils to Control the Biodeteriogenic Microorganisms in Archives and Libraries

Round 1

Reviewer 1 Report

Dear Editor,

thank you very much for the opportunity to review the paper entitled Application of Essential Oils to control the biodeteriogenic microorganisms in archives and libraries. I find this research very interesting, innovative and applicative at the same time. The paper is well-structured and the conducted experiments are newly designed and interesting. Also, the obtained results are promising in the view of protecting the movable cultural heritage such as paper, documents and books from the archives and libraries. I gladly recommend this paper for publication after a minor revision.

- Please, in line 68, start the sentence with Montanari et al. (2012) and at the end put the reference number.

- Line 148. Why using modern blank paper in the experiments instead of ancient one that has similar characteristics to the papers in archives and libraries?

- In line 187, there is a typing mistake, please correct 1x105

- Table 1. Nicely presented composition of the 96-well plates. It is rarely to see such table in literature.

- lines 244-250 - It is very nice of you to share negative results and to present the solution of the problem

- FIg. 4. How would you explain the bigger colonies after 24h treatment than after 0h treatment?

- At the end I would recommend to include in the paper (maybe in conclusion part) your proposition for treatment of large number of books or documents at the same time.

Author Response

We thank the Referee for dedicating His/Her time to our manuscript and acknowledging our work writing that “I find this research very interesting, innovative and applicative at the same time”.

We address each of the comments in detail below.
The Referee’s comments are in red; our answers in black and changes to the manuscript are reported, when appropriate, in Italics.

- Please, in line 68, start the sentence with Montanari et al. (2012) and at the end put the reference number.

“Montanari et al. (2012)” has been inserted at the beginning of the sentence and the reference number [5] has been moved to the end.

- Line 148. Why using modern blank paper in the experiments instead of ancient one that has similar characteristics to the papers in archives and libraries?

We agree with your observation, but to collect numerous results to be compared with a statistical significance, we decided to use modern blank paper that is easily achievable and characterized by a constant grammage, 80 g cm^-2. This has allowed to set-up a high number of replicates of paper blocks, contaminated and subjected to the different proposed treatments (freezing, lyophilization, EOs, etc.).

- In line 187, there is a typing mistake, please correct 1x105

The 1x105 has been corrected with 1x10⁵.

- Table 1. Nicely presented composition of the 96-well plates. It is rarely to see such table in literature.

Authors wish to thank the Reviewer for the comment on the usefulness of Table 1.

- lines 244-250 - It is very nice of you to share negative results and to present the solution of the problem

Authors thank the Reviewer for the comment. The replica plating technique, as reported in the text, has been chosen to guarantee a more accurate microbial detection.

- Fig. 4. How would you explain the bigger colonies after 24h treatment than after 0h treatment?

The growth of R. mucilaginosa was evaluated at 72 hrs of growth. Colonies, treated at t24 with the same amount of EO used at t0, are bigger because, in this case, microorganisms are allowed to grow at a same rate to that of the untreated control.  To explain that, a further comment has been inserted in the text: “…, while for the bacterium differences were not so evident (B and C). Finally, in the case of R. mucilaginosa, after 72 hrs of growth, colonies treated at t24 were larger than those treated at t0 with the same EO amount (E and F); it is possible to observe that, in both cases, colonies are not red pigmented since carotenoids were not produced.”

- At the end I would recommend to include in the paper (maybe in conclusion part) your proposition for treatment of large number of books or documents at the same time.

As suggested by the Reviewer, a sentence related to the possibility to simultaneously treat a large number of book/documents has been inserted in the conclusion section: “As a matter of fact, treatments with the EOs, coupled or not with a lyophilization process, will respectively allow to preserve damaged books or to prevent/reduce the microbial contamination. Moreover, these treatments will allow to simultaneously handle a large number of books or documents provided that a suitable concentration of EO is obtained in the environment where books and documents are stored. The results presented in this paper have to be intended as a proof of concept, and their scale-up to a “real” archive has surely to be addressed, and it is the topic of ongoing studies.”

Reviewer 2 Report

I suggest reviewing sentences that begin with a bibliography, such as in line 58 and 117

Author Response

We thank the Referee for dedicating His/Her time to our manuscript and for the useful comments that help us to improve the manuscript. According to the Referee suggestions we decided to change the order of the Paragraphs in Materials and Methods and Results sections. We hope the revised paper results more concise and easier to follow.

General observations

- Please write the full name without abbreviations when it is used for the first time in the text (ex. lines 12, 13,).

In the Abstract, the full name of the microorganisms has been inserted when reported for the first time: “… Staphylococcus epidermidis, Rhodotorula mucilaginosa and Alternaria alternata, was investigated…”. The extended name of MIC and that of MGI were also added: “Minimum Inhibitory Concentration (MIC)” and “… the inhibition percentage of the mycelial growth, MGI, …”.

- Please indicate which type of concentration you used w/w, v/v, w/v, etc.

The EO concentration has been inserted: “v/v”.

- Please put the name of the author before the reference number when use sentences such as "[5] report...", "according to the work by [12]", etc.

As suggested, names of the author and years have been added before the related number all along the text.

Please modify the Discussion section, moving and adapting some paragraphs for the Results section. It is redundant.

The discussion section was completely revised. The division in paragraphs was removed and the redundant parts with the Results section have been deleted.

It is not clear the necessity of doing experiments on lyophilized paper, and should be better explained, as well as the EOs treatment on this type of specimens. The lyophilization is a method used for preserving delicate materials as well, and it is well known that do not exterminate the possible contaminants present on the support (flooded paper in this case). In my opinion the details of the lyophilization procedure stays better in the Supplementary Materials, while the clarity of results (in the Results section) and additional comments related with some bibliographic insights (in the Discussion section) are more than welcomed. This experiment is tightly related with the title of the paper, as well as the chapter 3.5 "EO application in a contaminated book", which is poorly detailed.

The goal of the study is to evaluate the efficacy of EO on contaminated paper/archival materials, focusing also on the case of flooded documents which undergone freezing and lyophilization processes. Therefore, the lyophilization process was not the main topic of our study while it has been the EO treatment of the dried material.

Other specific observations

- Line 57 - please reformulate "high water activity values", maybe the authors intended need high water values for their activity

The Authors refer to the parameter water activity (a_w) that has a strong influence on microbial growth. The sentence has been reformulated to be more comprehensible: “high values of water activity (a_w) to survive”.

- Line 76 - the freezing is not the unique possibility to control microbial growth and the flooded material biodeterioration

The sentence has been changed as follows: “the freezing represents one of the most efficacious treatment to control biodeterioration of the flooded material”.

- Line 104-105 - please reformulate the sentence, as it is not clear at all

The sentence has been reformulated: “The vaporization of Tea tree EO showed the best results allowing an air contamination reduction equal to 77.3% and 95.0% for fungi and bacteria respectively”.

- Line 122 - please indicate better the type of the experiments, their aim and the differences in between. Here, for example the explanation of related with the lyophilization creates confusion.

This part has been modified to make the text more comprehensive: “Thus, in the present work, a preliminary screening with Thymus vulgaris and Origanum vulgare leaf oils was performed, by means of “broth dilution methods”, on S. epidermidis and R. mucilaginosa. The effect of Thymus vulgaris oil, vaporized immediately after the inoculum or at 24 hours of incubation, was tested on the two unicellular microorganisms grown on agar media. Considering the positive effect of EO, the investigation was also extended to A. alternata, chosen as representative of the filamentous fungi that can be found in archives. Finally, the treatment was extended on a real contaminated book. After that, a system was set-up in which flooding and “artificial” contamination, followed by freezing or lyophilization process, at different time of growth, were realized; contaminated paper sheets, removed from the paper blocks, after lyophilization or freeze-thawing, were treated immediately with the EO or after 24 hours of incubation.”

- Line 126 - sentence incomplete, "on a real contaminated" book.

“book” has been inserted at the end of the sentence: “real contaminated book.”

- Line 130 - please put in the brackets the details related with the strain supply

The details of the strain have been moved into the brackets “(LMG0474, BCCM, Belgium)”.

- Line 145 - I suggest to remove "and"

“and” has been removed as suggested by the Reviewer, the new title sentence is: “2.1. Simulation of flooded contaminated books”.

- Line 158 - which is the ratio between the species of the mixed inoculum?

The ratio is 1:1:1. It has been inserted in the text: “… the three microorganisms (ratio 1:1:1) were also prepared depositing…”.

- Line 166 - remove the space after 200

The space has been removed.

- Line 183 - remove 2020 and 2022, the year is mentioned in bibliography

The two reference years, 2020 and 2022, have been removed.

- Line 193-194, and then Line 245, 272 - what the authors want to say with the "inhibits microbial growth by the unaided eye"? that the microbial growth is not seen by naked eye?

Yes, the meaning is the same. The terms have been substituted in the text. As reported in the EUCAST reading guide for broth microdilution (2022), the first evaluation of the microbial growth inhibition is carried out checking, in liquid culture, the presence or not of turbidity. If turbidity is not observed, microbial growth is absent.

- Line 199 - please modify the caption of the table, here a suggestion: "Composition and amounts (in ml) of the 96-weel plates, where 2xMedium = double concentrated Medium (???) prepared as indicated in ...., Inoculum = single microorganisms (please indicate them) or mixed organisms (name and ratio) with a starting concentration of xxx; EO = thyme and oregano with different concentration (please indicate them)

The caption has been modified as suggested: “Composition and amounts, in µl, of the 96-well plates. “2X Medium” is the double concentrated ME broth; “Inoculum” refers to each single microorganism, S. epidermidis and R. mucilaginosa, 1 x 10⁶ CFU/mL concentrated; “EO” refers to each single suspension of Thymus vulgaris and Origanum vulgaris, concentrated from 0.75% to 0.0059% (v/v), as indicated in the text.”

Line 209 - this chapter could be removed or put in the Supplementary materials. The information that is required could be written in a sentence: g/cm2 of treatment solution and g/cm2 of active compound (you test EOs, and if you apply 6 layers for example you will have a different concentration with respect to 1 layer application). The type of the spray is also important.

The Paragraph was moved to the Supplementary material section.

Lines 335-337 - it is redundant, reduce, remove or unify with what was wrote in Materials and Methods section. The same for Lines 366-369, Lines 432-435. Most of the information gave in the chapters related with the Lines 431, 442,463, 473 are redundant and should be unified with the results section. The Discussion section should be revised and improved.

The Discussion section has been completely revised and improved avoiding redundancies.

- The conclusion: please not extend the conclusion to all the biodeteriogens, please add at line 488 "tested" before "biodeteriogenic organisms".

“tested” has been in the sentence added as suggested: “the growth of the tested biodeteriogenic microorganisms”.

Reviewer 3 Report

The authors performed different experiments for testing the efficacy of Thymus vulgaris and Origanum vulgare EOs on three types of microorganisms: Staphylococcus epidermidis, Rhodotorula mucilaginosa e Alternaria alternata and the best results were obtained by using Thymus vulgaris . The argument of the paper is interesting as many studies in the last period are focused on this argument for its best application in the cultural heritage field as well.

Please find below some comments and suggestions as the paper needs major revision.

General observations

Please write without abbreviation the full name without abbreviations when it is used for the first time in the text (ex. lines 12, 13, ).

Please indicate which type of concentration you used w/w, v/v, w/v, etc.

Please put the name of the author before the reference number when use sentences such as "[5] report...", "according to the work by [12]", etc.

Please do the paper more concise and easy to follow as it is mainly focused on methodology than on results.

Please modify the Discussion section, moving and adapting some paragraphs for the Results section. It is redundant.

It is not clear the necessity of doing experiments on lyophilized paper, and should be better explained, as well as the EOs treatment on this types of specimens. The lyophilization is a method used for preserving delicate materials as well, and it is well known that do not exterminate the possible contaminants present on the support (flooded paper in this case). In my opinion the details of the liophylization procedure stays better in the Supplementary Materials, while the clarity of results (in the Results section) and additional comments related with some bibliographic insights (in the Discussion section) are more than welcomed. This experiment is tightly related with the title of the paper, as well as the chapter 3.5 "EO application in a contaminated book", which is poorly detailed.

Other specific observations

Line 57 - please reformulate "high water activity values", maybe the authors intended need high water values for their activity

Line 76 - the freezing is not the unique possibility to control microbial growth and the fooded material biodeterioration

Line 104-105 - please reformulate the sentence, as it is not clear at all

Line 122 - please indicate better the type of the experiments, their aim and the differences in between. Here, for example the explanation of related with the lyophilization creates confusion.

Line 126 - sentence incomplete, "on a real contaminated" book.

Line 130 - please put in the brackets the details related with the strain supply

Line 145 - I suggest to remove "and"

Line 158 - which is the ratio between the species of the mixed inoculum?

Line 166 - remove the space after 200

Line 183 - remove 2020 and 2022, the year is mentioned in bibliography

Line 193-194, and then Line 245, 272 - what the authors want to say with the "inhibits microbial growth by the unaided eye"? that the microbial growth is not seen by naked eye?

Line 199 - please modify the caption of the table, here a suggestion: "Composition and amounts (in ml) of the 96-weel plates, where 2xMedium = double concentrated Medium (???) prepared as indicated in ...., Inoculum = single microorganisms (please indicate them) or mixed organisms (name and ratio) with a starting concentration of xxx; EO = thyme and oregano with different concentration (please indicate them)

Line 209 - this chapter could be removed or put in the Supplementary materials. The information that is required could be written in a sentence: g/cm2 of treatment solution and g/cm2 of active compound (you test EOs, and if you apply 6 layers for example you will have a different concentration with respect to 1 layer application). The type of the spray is also important.

Lines 335-337 - it is redundant, reduce, remove or unify with what was wrote in Materials and Methods section. The same for Lines 366-369, Lines 432-435.

Most of the information gave in the chapters related with the Lines 431, 442,463, 473 are redundant and should be unified with the results section.

The Discussion section should be revised and improved.

The conclusion: please not extend the conclusion to all the biodeteriogens, please add at line 488 "tested" before "biodeteriogenic organisms".

Author Response

We thank the Referee for dedicating His/Her time to our manuscript and for the useful comments that help us to improve the manuscript. According to the Referee suggestions we decided to change the order of the Paragraphs in Materials and Methods and Results sections. We hope the revised paper results more concise and easier to follow.

General observations

- Please write the full name without abbreviations when it is used for the first time in the text (ex. lines 12, 13,).

In the Abstract, the full name of the microorganisms has been inserted when reported for the first time: “… Staphylococcus epidermidis, Rhodotorula mucilaginosa and Alternaria alternata, was investigated…”. The extended name of MIC and that of MGI were also added: “Minimum Inhibitory Concentration (MIC)” and “… the inhibition percentage of the mycelial growth, MGI, …”.

- Please indicate which type of concentration you used w/w, v/v, w/v, etc.

The EO concentration has been inserted: “v/v”.

- Please put the name of the author before the reference number when use sentences such as "[5] report...", "according to the work by [12]", etc.

As suggested, names of the author and years have been added before the related number all along the text.

Please modify the Discussion section, moving and adapting some paragraphs for the Results section. It is redundant.

The discussion section was completely revised. The division in paragraphs was removed and the redundant parts with the Results section have been deleted.

It is not clear the necessity of doing experiments on lyophilized paper, and should be better explained, as well as the EOs treatment on this type of specimens. The lyophilization is a method used for preserving delicate materials as well, and it is well known that do not exterminate the possible contaminants present on the support (flooded paper in this case). In my opinion the details of the lyophilization procedure stays better in the Supplementary Materials, while the clarity of results (in the Results section) and additional comments related with some bibliographic insights (in the Discussion section) are more than welcomed. This experiment is tightly related with the title of the paper, as well as the chapter 3.5 "EO application in a contaminated book", which is poorly detailed.

The goal of the study is to evaluate the efficacy of EO on contaminated paper/archival materials, focusing also on the case of flooded documents which undergone freezing and lyophilization processes. Therefore, the lyophilization process was not the main topic of our study while it has been the EO treatment of the dried material.

Other specific observations

- Line 57 - please reformulate "high water activity values", maybe the authors intended need high water values for their activity

The Authors refer to the parameter water activity (a_w) that has a strong influence on microbial growth. The sentence has been reformulated to be more comprehensible: “high values of water activity (a_w) to survive”.

- Line 76 - the freezing is not the unique possibility to control microbial growth and the flooded material biodeterioration

The sentence has been changed as follows: “the freezing represents one of the most efficacious treatment to control biodeterioration of the flooded material”.

- Line 104-105 - please reformulate the sentence, as it is not clear at all

The sentence has been reformulated: “The vaporization of Tea tree EO showed the best results allowing an air contamination reduction equal to 77.3% and 95.0% for fungi and bacteria respectively”.

- Line 122 - please indicate better the type of the experiments, their aim and the differences in between. Here, for example the explanation of related with the lyophilization creates confusion.

This part has been modified to make the text more comprehensive: “Thus, in the present work, a preliminary screening with Thymus vulgaris and Origanum vulgare leaf oils was performed, by means of “broth dilution methods”, on S. epidermidis and R. mucilaginosa. The effect of Thymus vulgaris oil, vaporized immediately after the inoculum or at 24 hours of incubation, was tested on the two unicellular microorganisms grown on agar media. Considering the positive effect of EO, the investigation was also extended to A. alternata, chosen as representative of the filamentous fungi that can be found in archives. Finally, the treatment was extended on a real contaminated book. After that, a system was set-up in which flooding and “artificial” contamination, followed by freezing or lyophilization process, at different time of growth, were realized; contaminated paper sheets, removed from the paper blocks, after lyophilization or freeze-thawing, were treated immediately with the EO or after 24 hours of incubation.”

- Line 126 - sentence incomplete, "on a real contaminated" book.

“book” has been inserted at the end of the sentence: “real contaminated book.”

- Line 130 - please put in the brackets the details related with the strain supply

The details of the strain have been moved into the brackets “(LMG0474, BCCM, Belgium)”.

- Line 145 - I suggest to remove "and"

“and” has been removed as suggested by the Reviewer, the new title sentence is: “2.1. Simulation of flooded contaminated books”.

- Line 158 - which is the ratio between the species of the mixed inoculum?

The ratio is 1:1:1. It has been inserted in the text: “… the three microorganisms (ratio 1:1:1) were also prepared depositing…”.

- Line 166 - remove the space after 200

The space has been removed.

- Line 183 - remove 2020 and 2022, the year is mentioned in bibliography

The two reference years, 2020 and 2022, have been removed.

- Line 193-194, and then Line 245, 272 - what the authors want to say with the "inhibits microbial growth by the unaided eye"? that the microbial growth is not seen by naked eye?

Yes, the meaning is the same. The terms have been substituted in the text. As reported in the EUCAST reading guide for broth microdilution (2022), the first evaluation of the microbial growth inhibition is carried out checking, in liquid culture, the presence or not of turbidity. If turbidity is not observed, microbial growth is absent.

- Line 199 - please modify the caption of the table, here a suggestion: "Composition and amounts (in ml) of the 96-weel plates, where 2xMedium = double concentrated Medium (???) prepared as indicated in ...., Inoculum = single microorganisms (please indicate them) or mixed organisms (name and ratio) with a starting concentration of xxx; EO = thyme and oregano with different concentration (please indicate them)

The caption has been modified as suggested: “Composition and amounts, in µl, of the 96-well plates. “2X Medium” is the double concentrated ME broth; “Inoculum” refers to each single microorganism, S. epidermidis and R. mucilaginosa, 1 x 10⁶ CFU/mL concentrated; “EO” refers to each single suspension of Thymus vulgaris and Origanum vulgaris, concentrated from 0.75% to 0.0059% (v/v), as indicated in the text.”

Line 209 - this chapter could be removed or put in the Supplementary materials. The information that is required could be written in a sentence: g/cm2 of treatment solution and g/cm2 of active compound (you test EOs, and if you apply 6 layers for example you will have a different concentration with respect to 1 layer application). The type of the spray is also important.

The Paragraph was moved to the Supplementary material section.

Lines 335-337 - it is redundant, reduce, remove or unify with what was wrote in Materials and Methods section. The same for Lines 366-369, Lines 432-435. Most of the information gave in the chapters related with the Lines 431, 442,463, 473 are redundant and should be unified with the results section. The Discussion section should be revised and improved.

The Discussion section has been completely revised and improved avoiding redundancies.

- The conclusion: please not extend the conclusion to all the biodeteriogens, please add at line 488 "tested" before "biodeteriogenic organisms".

“tested” has been in the sentence added as suggested: “the growth of the tested biodeteriogenic microorganisms”.

Round 2

Reviewer 3 Report

The paper was much improved. I agree with the publication, but the discussions still need to be revised. The authors reported their results again in this section, but it would be great and reccomended to compare/comment the findings obtained by other similar works in the field.

Some other little modifications are suggested: see Line 69 and allover the text where are similar situations.: e.g. "Montanari et al. [5] report" instead of "Montanari et al. (2012) report".

Author Response

We thank the Referee for dedicating His/Her time to revised our manuscript for the second time. We improved the “Discussion” as suggested by the reviewer.

The paper was much improved. I agree with the publication, but the discussions still need to be revised. The authors reported their results again in this section, but it would be great and reccomended to compare/comment the findings obtained by other similar works in the field.

We compared, in Table 3, and commented in the text, the results obtained in the present work with those reported in the Literature. We hope this revision will satisfy the reviewer’s request.

For the Reviewer convenience we report below the revised “Discussion”:

4. Discussion

Results shown in the previous section evidence that both O. vulgare and T. vulgaris EOs exhibited an inhibiting effect on the tested unicellular microorganisms. The MIC value obtained for T. vulgaris EO against S. epidermidis, 7.5 µg/ml, shows a good agreement with values reported in the Literature, as shown in Table 3. As regard O. vulgare EO, a MIC value was not obtained, for this reason such a comparison is not possible.

Table 3. MIC values of different Thymus EOs against S. epidermidis and R. mucilaginosa, reported in literature, compared to those found in the present work.

The tested EOs are able to inhibit, or slow down, the growth of unicellular microorganisms and that of the filamentous fungus, e.g. A. Alternata. In both the cases, the obtained results evidence two important issues:

• The treatment with the EO gives better results when carried out immediately after contamination, although some inhibiting effect was observed in the case of delayed treatments. As an example, in mucilaginosa a depigmentation was noticed (Figure 3), probably related to an influence of EO on the carotenoid synthesis pathway, as reported in a previous work for the fungicide naftifine [26]. The same behavior was observed for all the microorganisms cultured on paper sheets (data not shown), in this case the antimicrobial activity was higher probably in relation to the absorption of the EO on paper which guarantees a longer contact time with the microorganism.
• When “real” contaminated samples have to be managed, a suitable treatment system has to be designed considering that: the EO sprayed on the contaminated book cover gave an inhibition lower than that obtained by means of contact sheets. In the last case, the presence of the paper probably reduces the evaporation rate of the volatile components of the oil (see Paragraph 3.3 and Figure 5).

As concern the EO treatment, after flooding and lyophilization, it has to be pointed out that the lyophilization process did not control the growth of R. mucilaginosa, as previously reported by Fissore et al. [13], while the not sporulated mycelium of A. alternata was inhibited by the lyophilization for a double time with respect to the sporulated one, due to a higher spore resistance to thermal shock and to low a_w values, as reported by Troiano et al. and Lucchese [12, 27]. The treatment with EO is thus mandatory, and T. vulgaris (0.75%) was proven to be the best solution, in the presence of R. mucilaginosa and also of A. alternata. In this last case, the results reported by Salehi et al., Tullio et al., and Soylu et al. were confirmed [24,28,29]. Moreover, the T. vulgaris (0.75%) efficacy was also confirmed in the case of paper sheets with a mixed contamination, in particular when used immediately after the freeze-drying process.

In conclusion, the treatment with T. vulgaris EO represents a real opportunity to control the growth of microorganisms involved in paper-biodeterioration. In this aim, the obtained results were exploited in the risk-based decision-making process for the safety of librarian heritage, adopting EOs as prevention measure but also for protection after accidental events, as discussed in [30].

Some other little modifications are suggested: see Line 69 and allover the text where are similar situations.: e.g. "Montanari et al. [5] report" instead of "Montanari et al. (2012) report".

Requested changes were done.