Examining the Potential of Enzyme-Based Detergents to Remove Biofouling from Limestone Heritage

Round 1

Reviewer 1 Report

1. Line 44. Reference is required.
2. Enzyme based detergents have been used in earlier studies. How this study is different from earlier work?
3. Please specify the novelty in an introduction.
4. Line 225. Please discuss the phenomenon in involved in color change and biological pigmentation.
5. Fig. 5 is not clear. Please improve.
6. What about the cost effectiveness and environmental imoact of proposed treatment?

Author Response

Dear Reviewer:

Thank you for reading and commenting on our manuscript. Your comments are very valuable to improve it, and we tried to implement these as well as possible. In here, we copied all the comments (in bold) and answered starting with “RESPONSE TO REVIEWER COMMENT NO.”. In our answers, we refer to the line numbers of the Revised manuscript.

1. Line 44. Reference is required.

RESPONSE TO REVIEWER 1 COMMENT NO. 1: We added the reference: (Crispim and Gaylarde, 2005) to justify this statement (Line 44).

Crispim, C.A., Gaylarde, C.C., 2005. Cyanobacteria and Biodeterioration of Cultural Heritage: A Review. Microb. Ecol. 49, 1–9. https://doi.org/10.1007/s00248-003-1052-5

2. Enzyme based detergents have been used in earlier studies. How this study is different from earlier work?

RESPONSE TO REVIEWER 1 COMMENT NO. 2: This study differs from previous work because it is the first study showing the effect of enzyme-based treatments to clean natural building stone in-situ, in environmental conditions. Previous work focused on laboratory experiments. Moreover, glucose oxidase or lipase was used, while in this manuscript, we worked with commercially available enzyme-based detergents (Line 71-79).

3. Please specify the novelty in an introduction.

RESPONSE TO REVIEWER 1 COMMENT NO. 3: The novelty of this paper is that for the first time, a large field study was conducted to verify if commercially available enzyme-based detergents can clean biocolonised headstones. (Line 71-79).

4. Line 225. Please discuss the phenomenon in involved in color change and biological pigmentation.

RESPONSE TO REVIEWER 1 COMMENT NO. 4: The color change occurred in the samples treated with the enzyme-based detergent. The local spectral reflectance minima linked to biological pigments became obscure or disappeared, and thus this suggests that the detergent caused a decrease in biocolonisation (Line 196-203). The interaction of the product with the microorganisms was beyond the scope of this project but should be included in future research (Line 370-373).

5. Fig. 5 is not clear. Please improve.

RESPONSE TO REVIEWER 1 COMMENT NO. 5: There were indeed some problems with the readability of Figure 5. We added extra information in the caption to make it clearer (Line 287-289).

6. What about the cost effectiveness and environmental imoact of proposed treatment?

RESPONSE TO REVIEWER 1 COMMENT NO. 6: This was not studied in this manuscript, but can be included in future work. It was also addressed in the Conclusions and future research section on Line 374-375. Nevertheless, it is necessary to explore alternatives for cleaning with pesticides due to the EU sustainable use directive on pesticide reduction and banning (Line 68). Enzymes are one of the options, and the cost are reasonable, as we used commercially available products for which prices can be found online. We prefer not to include any price, as we do not want to advertise any product.

Thank you for your constructive comments. It enabled us to improve the quality of our manuscript.

Yours Sincerely,

Laurenz Schröer, Géraldine Fiers, Maxim Deprez, Nico Boon, Veerle Cnudde, Lander Soens and Tim De Kock

Reviewer 2 Report

The manuscript Schröer et al describes the use of Enzyme-based detergents for the removal of biofouling on headstones. The authors used a commercial detergent for both lab studies and two detergents for in situ studies. The manuscript is well written and it is very interesting. 

However, the reviewer has the following points that should be taken into consideration: 

• It is not clear why the authors used 2 detergents in the in-situ studies while the second one was not tested in lab studies.
• What is the mechanism for removal of biofouling? Is it really an effect of the enzyme or is it more the basic pH that can affect the growth of microorganisms? Different tests should be carried out in lab scale to identify that solutions with the same pH as the enzyme-based detergent used will not give the same results. 
• How would these detergents react with the soil? Would there be some kind of influence on microorganisms and other plants in the soil? It is not clear from pictures 6-8 if with longer exposure to the enzyme based the grass at the bottom of the headstone is disappearing because of the enzyme or because of the colder weather. Lab experiments should be carried out with the mixtures to identify any effect. 

 

Minor: 

• Please add company and country of each chemical used in the work
• in row 105, change +- with circa or ca. 
• mistakes in the description of the A, B, C, and E areas, AB are in northwestern Belgium, while CD are in northern France

 

Regarding SI, the reviewer did not receive the SI figures to be able to review them 

Author Response

Dear Reviewer:

Thank you for reading and commenting on our manuscript. Your comments are very valuable to improve it, and we tried to implement these as well as possible. In here, we copied all the comments (in bold) and answered starting with “RESPONSE TO REVIEWER COMMENT NO.”. In our answers, we refer to the line numbers of the Revised manuscript.

The manuscript Schröer et al describes the use of Enzyme-based detergents for the removal of biofouling on headstones. The authors used a commercial detergent for both lab studies and two detergents for in situ studies. The manuscript is well written and it is very interesting. 

However, the reviewer has the following points that should be taken into consideration: 

1. It is not clear why the authors used 2 detergents in the in-situ studies while the second one was not tested in lab studies.

RESPONSE TO REVIEWER 2 COMMENT NO. 1: Within this research, we did not aim to find the best commercially available enzyme-based detergent. We aimed to determine if we could use them to clean biofouled natural building stones. The in-situ experiments gave no difference in effectiveness in one product compared to the other. Moreover, we only had a limited number of samples with biofilm to perform the lab experiments. For these reasons, we were convinced that testing only one of the products was sufficient.

2. What is the mechanism for removal of biofouling? Is it really an effect of the enzyme or is it more the basic pH that can affect the growth of microorganisms? Different tests should be carried out in lab scale to identify that solutions with the same pH as the enzyme-based detergent used will not give the same results. 

RESPONSE TO REVIEWER 2 COMMENT NO. 2: We did not test if it was the enzyme or the pH that affected the growth of the microorganisms. However, the literature showed that enzymes could achieve this (Valentini et al., 2012, 2010). Moreover, the products we use are not experimental but commercially available enzyme-based detergents, so it is assumed that the enzyme is here the active substance (Line 170-172).

Valentini, F., Diamanti, A., Carbone, M., Bauer, E.M., Palleschi, G., 2012. New cleaning strategies based on carbon nanomaterials applied to the deteriorated marble surfaces: A comparative study with enzyme-based treatments. Appl. Surf. Sci. 258, 5965–5980. https://doi.org/10.1016/j.apsusc.2012.01.076

Valentini, F., Diamanti, A., Palleschi, G., 2010. New bio-cleaning strategies on porous building materials affected by biodeterioration event. Appl. Surf. Sci. 256, 6550–6563. https://doi.org/10.1016/j.apsusc.2010.04.046 

3. How would these detergents react with the soil? Would there be some kind of influence on microorganisms and other plants in the soil? It is not clear from pictures 6-8 if with longer exposure to the enzyme based the grass at the bottom of the headstone is disappearing because of the enzyme or because of the colder weather. Lab experiments should be carried out with the mixtures to identify any effect. 

RESPONSE TO REVIEWER 2 COMMENT NO. 3: The effect of the enzyme-based detergents on the environment should be investigated in detail. This was added to the Conclusions and future research section on Line 370-373. However, it was out of scope for this research, and we did not include such experiments.

During the experiments, we did not see any effect of the enzymes on the surrounding plants. The grass was not disappearing. The yellowing of the grass in summer was not synchronous with the product application. The deterioration of the biofilms on the headstone occurred much earlier. However, cleaning continued due to the rain wash as can be seen in Figure 6-8. In Figure 8, there is even more grass visible on 03/10. However, the grass was often mowed, which could make it seem sometimes that grass was disappearing. Moreover, the plants just next to the headstone grew very well, and e.g. in Figure 6, you can see them flowering on 27/05 one month after treatment.

Minor: 

4. Please add company and country of each chemical used in the work

RESPONSE TO REVIEWER 2 COMMENT NO. 4: Biomix ATM is a product from Bionova (Belgium) and Enzylan from AlgaVelan (The Netherlands). This was added on Line 86 and 164.

5. in row 105, change +- with circa or ca. 

RESPONSE TO REVIEWER 2 COMMENT NO. 5: +- was replaced by ca. in Line 102, 104, 143, 144, 145. 

6. mistakes in the description of the A, B, C, and E areas, AB are in northwestern Belgium, while CD are in northern France

RESPONSE TO REVIEWER 2 COMMENT NO. 6: There was indeed a mistake here, and we replaced it to: “Site A and B are located in western Belgium, near the French border. Site C and D are in northwestern France, close to the Belgian border (Figure 2).” (Line 153 – 154).

7. Regarding SI, the reviewer did not receive the SI figures to be able to review them 

RESPONSE TO REVIEWER 2 COMMENT NO. 7: We are sorry to read this. We included these as supplementary information.

 

Thank you for your constructive comments. It enabled us to improve the quality of our manuscript.

Yours Sincerely,

Laurenz Schröer, Géraldine Fiers, Maxim Deprez, Nico Boon, Veerle Cnudde, Lander Soens and Tim De Kock

Round 2

Reviewer 1 Report

Manuscript is well revised and can be considered for publication in Coatings.

Author Response

Dear Reviewer,

Thank you for your time and efforts to review the article. It made it possible to improve our manuscript.

Greetings,

The authors

Reviewer 2 Report

The authors have improved the manuscript by adding specific sentences in the manuscript that would reply to some of the points of the reviewer.  

 

Author Response

Dear Reviewer,

We appreciate the careful review and suggestions for minor editing. We hope that our previous explanation and adaptations improved the document and that we allayed your concerns. Thank you for your time and efforts to review the manuscript.

Greetings,
The authors