Evaluation of Selenium-Based Biocides with Biocidal Potential for Cultural Heritage Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal Title                   : Heritage

Article Type                   : Research article

Manuscript Title           : Evaluation of Selenium-Based Biosimilars with Biocidal Potential for Cultural Heritage applications

 

Dear Editor, My suggestions regarding article evaluation are below.

 

The manuscript focused on the Selenium-Based Biosimilars with Biocidal Potential for Cultural Heritage applications. The synthesis and characterization of selenium-based biosidal agent was performed using NMR spectroscopy and gas 24 chromatography–mass spectrometry (GC-MS). The biocidal efficacy of these compounds was evaluated via algal growth inhibition tests (OECD 201), employing Raphidocelis sub-capitata microorganisms as a model organism.

The results indicate that certain seleno-sugars exhibit dose dependent inhibition of algal growth, suggesting superior biocidal activity compared to conventional agents. Notably, one compound demonstrated an optimal balance of efficacy and chemical stability and was selected for subsequent in vivo testing.

 

The results of the research are presented in a visual and understandable way and are well organized. I am not an English expert. It appears that the article has been pre-edited by assistant editors. The article can be accepted for publication in its current form.

 

 

Best wishes and regards.

 

Author Response

We thank Reviewer 1 for the positive comments.

Reviewer 2 Report

Comments and Suggestions for Authors

General notes:

The introduction is lacking citations (see comments below listed by line number). There are issues with some of the references in the document, these are listed in the Specific notes on references section at the end. Sorry that I did not have time to point you towards some suitable references.

In the document you make claims of enhanced cell permeability for the selenosugars but provide no data, it is also notable that the solvent used in the study if DMSO which facilitates transport across cell membranes. Please add in the methods used to confirm the cell permeability of SD1-5 and the associated data. If DMSO was used as part of this then the ability of DMSO to facilitate transport across cell membranes should be addressed.

 

Comments by line number:

Line number(s)	Comment
49-53	This section needs citations for both the pioneer organisms and the colonisation of established microorganisms
59-69, 71-73	All facts in these sections should have citations
73-74	Provide examples of the biological methods used to remove biological patina and make sure that these are adequately cited
74-75	Provide examples of the chemical methods used to remove biological patina and make sure that these are adequately cited. This is also a good opportunity to discuss the increasing regulation of chemical biocides, how this has resulted in a need for more environmentally friendly alternatives (again both need to be adequately cited) and how your study helps to address this need.
81-83, 84-88	All facts in these sections should have citations
89-98	In lines 78-80 you mentioned that the selected molecules should not be harmful to the environment or restorers. In this section you need to expand on the evidence for this being the case for selenosugars ensuring that you have cited all facts/examples
100-103	Incomplete sentence
107-109	Add in the fact that this algal species is referred to it’s former strain designation, Pseudokirchneriella subcapitata, in OECD 201 and that it is one of the approved species for this protocol.
114-202	Throughout the whole methods section please provide the source and catalogue number of any materials which were purchased to ensure that the work is repeatable by others. For example DMSO might be DMSO (Merck:D2650).
179	Also reference OECD 201 here
180-181	Include the strain catalogue reference number for R. subcapitata and include a citation for ACUF – a web page citation will suffice
185-192	How were the samples laid out in the 24 well plates? Was the position randomised to prevent edge effects influencing the results? This needs to be addressed in the methodology.
193-202	Please include the equations (and citations for where you found them) which you used to calculate the concentrations of the pigments and the cell density. Also note that this is an accepted deviation from OECD 201 which normally requires cell mass as the final measurement, please include the technique you used to calibrate these against cell mass!
294	You state that there was a high degree of correlation between the experimental data and the IC50. Please include the result and the p-value for the calculation and add the method by which the correlation was calculated to the methods section.
298-321	As SD5 did not demonstrate the desired effect it would be better to integrate this section into the main results, i.e. talk about SD1-5 all together.
325-367	The conclusions section would work better if restructured to discuss molecules SD1,2,4 & 5 together and then to go into more depth on SD3 as this is your candidate for a potential biocide.
355	List the compounds in brackets after the word ‘compounds’
351-356	Include what the IC50 was here and make it clear that this was for compound SD3 (note this will be easier if the conclusion is restructured as previously mentioned.

 

 

Specific notes on references:

Reference 1 should be directly referencing the ICROM report, not a review paper which references it.

Reference 3 this paper is primarily on biodeterioration of surfaces and as such you are referring to the references used in the introduction. Please replace with papers on the bioreceptivity or microbial colonisation of stone surfaces.

Reference 8 is not a good reference for this paper as it is not referring to the removal of biological growth from stone. This needs to be changed to a source from a study on stone conservation.

References 10, 11, 13 & 14 are all from this research group. Your arguments will be better supported if you can show that researchers from outside of your group are also finding the same things. Please review these references and replace them.

Reference 19 the reference to this paper in the methods section does not seem appropriate, the paper does not explain why the maximum temperature is 325C and as with references 10,11,13 & 14 appears to be an attempt to inappropriately self cite. If this is the maximum temperature that the machine can reach then please cite the manufacturers literature, if not then please remove the citation and include an explanation as to why this is the maximum temperature.

Author Response

We thank the reviewer for the meticulous analysis of our manuscript. Following our responses, we have carefully revised the text to address all the comments and suggestions. Below, we provide a point-by-point reply, highlighting the changes made in the revised version.

Reviewer 2:

The introduction is lacking citations (see comments below listed by line number). There are issues with some of the references in the document, these are listed in the Specific notes on references section at the end. Sorry that I did not have time to point you towards some suitable references.

In the document you make claims of enhanced cell permeability for the selenosugars but provide no data,

Answer: We thank Reviewer for this comment. We agree that the manuscript did not report any data on the cell permeability of the selenosugars. To address this point, we have now included reference 24, which provides the relevant data supporting the permeability of our compounds.

it is also notable that the solvent used in the study if DMSO which facilitates transport across cell membranes. Please add in the methods used to confirm the cell permeability of SD1-5 and the associated data. If DMSO was used as part of this then the ability of DMSO to facilitate transport across cell membranes should be addressed.

Answer: To the best of our knowledge, DMSO is conventionally employed when the compounds under investigation exhibit poor solubility in aqueous/biological media, at concentrations that are not cytotoxic ( Kollerup Madsen B, Hilscher M, Zetner D, Rosenberg J. Adverse reactions of dimethyl sulfoxide in humans: a systematic review. F1000Res. 2018 Nov 5;7:1746.). Nevertheless, its presence does not play a decisive role in transmembrane transport, which is primarily governed by cellular and structural recognition mechanisms that are not yet fully elucidated and will be the subject of our future investigations.

"Comments by line numbers":

49-53: This section needs citations for both the pioneer organisms and the colonisation of established microorganisms.

Answer: Citations were added to provide context for the stated information about microbial colonization.

• Ferrari, C.; Santunione, G.; Libbra, A.; Muscio, A.; Sgarbi, E.; Siligardi, C.; Barozzi, G. S. Review on the influence of biological deterioration on the surface properties of building materials: organisms, materials, and methods. IJDNE 2015, 10(1), 21-39. DOI: 10.2495/DNE-V10-N1-21-39
• Tiano, P. Biodegradation of cultural heritage: decay mechanisms and control methods. In Seminar article, new university of Lisbon, Department of Conservation and Restoration 2022, 7-12.

59-69, 71-73: All facts in these sections should have citations.

Answer: Citations were added to provide context for the stated information about the mitigation of biodeterioration.

• Gaylarde, C. C.; Morton, L. G. Deteriogenic biofilms on buildings and their control: a review. Biofouling 1999, 14(1), 59-74. DOI: 10.1080/08927019909378397
• Koul, B.; Upadhyay, H. Fungi-mediated biodeterioration of household materials, libraries, cultural heritage and its control. In Fungi and their role in sustainable development: current perspectives (pp. 597-615). Singapore: Springer Singapore 2018. DOI: 10.1007/978-981-13-0393-7_32
• Borderie, F.; Alaoui-Sossé, B.; Aleya, L. Heritage materials and biofouling mitigation through UV-C irradiation in show caves: state-of-the-art practices and future challenges. ESPR 2015, 22(6), 4144-4172. DOI: 10.1007/s11356-014-4001-6
• Chaudhuri, A.; Bhattacharyya, S.; Chakraborty, A.; Mukherjee, S.; Sudarshan, M.; Ghosh, C. K.; Chaudhuri; P. Inhibitory effect of UV and gamma radiation for fungal biodeterioration of concrete: A short-term study for sustainable conservation. J. Cult. Herit. 2024, 66, 316-325. DOI: 10.1016/j.culher.2023.12.005
• Mascalchi, M.; Osticioli, I.; Riminesi, C.; Cuzman, O. A.; Salvadori, B.; Siano, S. Preliminary investigation of combined laser and microwave treatment for stone biodeterioration. Stud. Conserv. 2015, 60(sup1), S19-S27. DOI: 10.1179/0039363015Z.000000000203

73-74: Provide examples of the biological methods used to remove biological Patina and make sure that these are adequately cited.

Answer: Citations were added to provide context with regards to biological/green approaches to biodeterioration’s mitigation.

• Essential oils: Komar, M.; Derese, N.; Szymczak, K.; Nowicka-Krawczyk, P.; Gutarowska, B. Natural Plant Oils as Anti—Algae Biocides for Sustainable Application in Cultural Heritage Protection. Sustainability 2025, 17(15), 6996. DOI: 10.3390/su17156996
• General plant extracts: Rotolo, V.; Barresi, G.; Di Carlo, E.; Giordano, A.; Lombardo, G.; Crimi, E.; Costa, E.; Bruno, M.; Palla, F. Plant extracts as green potential strategies to control the biodeterioration of cultural heritage. Int. J. Conserv. Sci. 2016, 839.

74-75: Provide examples of the chemical methods used to remove biological Patina and make sure that these are adequately cited. This is also a good opportunity to discuss the increasing regulation of chemical biocides, how this has resulted in a need for more environmentally friendly alternatives (again both need to be adequately cited)

Answer: Citations were added to provide context with regards to chemical approaches.

• Rajkowska, K.; Koziróg, A.; Otlewska, A.; Piotrowska, M.; Nowicka-Krawczyk, P.; Brycki, B.; Kunicka-Styczyńska, A.; Gutarowska, B. Quaternary ammonium biocides as antimicrobial agents protecting historical wood and brick. Acta Biochim. Pol. 2016, 63(1), 153-159. DOI: 10.18388/abp.2015_1134
• Kakakhel, M. A.; Wu, F.; Gu, J. D.; Feng, H.; Shah, K.; Wang, W. Controlling biodeterioration of cultural heritage objects with biocides: A review. Biodeterior. Biodegrad. 2019, 143, 104721. DOI: 10.1016/j.ibiod.2019.104721
• Li, T.; Zhang, H.; Tan, X.; Zhang, R.; Wu, F.; Ma, Q.; Zhang, B.; Su, B. Cu nanoparticles, a candidate biocide for the conservation of stone monuments against biodeterioration. J. Build. Eng. 2024, 98, 111043. DOI: 10.1016/j.jobe.2024.111043

and how your study helps to address this need.

Answer: Thank you for your valuable comments. However, regarding the ecocompatibility of our compounds, we are unable to provide the supporting data within the present manuscript, as this is the subject of a separate study currently under revision, recently submitted in collaboration with our colleague Marco Guida (University of Naples, Federico II). We would like to reassure the reviewer that these results will soon be available to the scientific community, thereby complementing the findings presented here.

81-83, 84-88: All facts in these sections should have citations.

Answer: We thank Reviewer for this comment. We have now included reference 21.

• Mangiavacchi, F.; Coelho Dias, I.F.; Di Lorenzo, I.; Grzes, P.; Palomba, M.; Rosati, O.; Bagnoli, L.; Marini, F.; Santi, C.; Lenardao, E.J.; et al. Sweet Selenium: Synthesis and Properties of Selenium-Containing Sugars and Derivatives. J. Pharm. 2020, 13, 211. DOI: 10.3390/ph13090211

89-98: In lines 78-80 you mentioned that the selected molecules should not be harmful to the environment or restorers. In this section you need to expand on the evidence for this being the case for selenosugars ensuring that you have cited all facts/examples.

Answer: Thank you for your valuable comments. However, regarding the ecocompatibility of our compounds, we are unable to provide the supporting data within the present manuscript, as this is the subject of a separate study currently under revision, recently submitted in collaboration with our colleague Marco Guida (University of Naples, Federico II). We would like to reassure the reviewer that these results will soon be available to the scientific community, thereby complementing the findings presented here.

100-103: Incomplete sentence.

Answer: The sentence (now 114-118) have been edited according to Referee suggestions.

107-109: Add in the fact that this algal species is referred to it’s former strain designation, Pseudokirchneriella subcapitata, in OECD 201 and that it is one of the approved species for this protocol.

Answer: The sentence was edited according to the reviewer’s recommendation, and a citation was added.

• Machado, M. D.; Soares, E. V. Features of the microalga Raphidocelis subcapitata: physiology and applications. Microbiol. Biotechnol. 2024, 108(1), 219. DOI: 10.1007/s00253-024-13038-0

114-202: Throughout the whole methods section please provide the source and catalogue number of any materials which were purchased to ensure that the work is repeatable by others. For example, DMSO might be DMSO (Merck:02650).

Answer: Thank you for your valuable comments. At the beginning of the Materials and Methods section, we included the elements that are customarily reported in manuscripts of organic synthesis.

179: Also reference OECD 201 here.

Answer: OECD 201 Guidelines were cited at the start of the paragraph.

180-181: Include the strain catalogue reference number tor R. subcapitata and include a citation tor ACUF - a web page citation will suffice.

Answer: The sentence was edited accordingly and both strain number and ACUF website were cited.

185-192: How were the samples laid out in the 24 well plates? Was the position randomized to prevent edge effects influencing the results? This needs to be addressed in the methodology.

Answer: The precise location of each repetition was specified.

193-202: Please include the equations (and citations tor where you found them) which you used to calculate the concentrations of the pigments and the cell density. Also note that this is an accepted deviation from OECD 201 which normally requires cell mass as the final measurement, please include the technique you used to calibrate these against cell mass!

Answer: Mass of pigments was not calculated. As we’ve been using a Victor microplate reader to measure the ODs related to the optical peak of each the biological parameters, and the data confrontation is between samples which constitute all products of this specific experiment, the data was expressed as simple Optical densities. For each, the same conditions and techniques were respected. OECD 201 states that spectrophotometric techniques might be used to account for cell density, which is the primary parameter considered for that protocol.

294: You state that there was a high degree of correlation between the experimental data and the IC50. Please include the result and the p-value for the calculation and add the method by which the correlation was calculated to the methods section.

Answer: The dose-response curve was fitted using a non-linear four-parameter logistic (4PL) regression model. The IC₅₀ value was estimated by non-linear least squares optimization using the curve fit function from scipy.optimize. The associated p-value for the IC₅₀ estimate was calculated based on the t-statistic derived from the parameter standard error and degrees of freedom of the fit. The goodness of fit was confirmed by R² = [value] and visual agreement between predicted and experimental values. This specification was added to the last section of Materials and Methods, and p-value and a new IC50 graph reporting CI was added to the Results.

• Straetemans, R.. Nonlinear modeling of dose-response data. In Modeling Dose-Response Microarray Data in Early Drug Development Experiments Using R: Order-Restricted Analysis of Microarray Data (43-66). Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. DOI: 10.1007/978-3-642-24007-2_4

298-321: As SD5 did not demonstrate the desired effect it would be better to integrate this section into the main results, i.e. talk about SD1-5 all together.

Answer: We respectfully disagree with this suggestion, as we believe that in this alternative presentation the rationale for selecting this molecule, and for introducing the OAc group, is not clearly conveyed.

325-367: The conclusions section would work better it restructured to discuss molecules SD1, 2, 4 & 5 together and then to go into more depth on SD3 as this is your candidate tor a potential biocide.

Answer: We respectfully disagree with this suggestion, as we believe that in this alternative presentation the rationale for selecting this molecule, and for introducing the OAc group, is not clearly conveyed.

355: List the compounds in brackets after the word 'compounds'.

Answer: Amended in the Manuscriptfile

351-356: Include what the IC50 was here and make it clear that this was for compound SD3 (note this will be easier it the conclusion is restructured as previously mentioned).

Answer: The sentence was modified accordingly.

Specific notes on references:

Reference 1 should be directly reterencing the ICROM report, nota review paper which references it.

Answer: Amended

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Your study focusing on new selenium-based biosimilars with biocidal potential for preserving cultural heritage is an important contribution to the field.

The scientific methods used are clearly and exhaustively described as the presentation of the results and their discussion.

I consider this manuscript to be well written, very interesting and innovative. For these reasons, I will propose it for publication. The only thing I would suggest is to increase the number of bibliographical references.

Best regards

Author Response

We thank Reviewer 3 for the comments, moreover new bibliographical references have been added.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript presents a novel investigation into selenium-based biocides for inhibiting microbial growth on cultural heritage materials. The synthesis, characterization, and algal toxicity testing are technically sound. However, the study’s applicability to cultural heritage conservation is overstated.

- The synthesized compounds are synthetic seleno-sugars, not biologically derived biosimilars - a term that misleadingly suggests equivalence to complex biologics like monoclonal antibodies. It is recommended to use selenium-based biocides or organoselenium compounds instead.
- The study exclusively uses Raphidocelis subcapitata (algae) for testing. Cultural heritage biodeterioration involves fungi, bacteria, and lichens-algae alone is insufficient. Without data on these key biodeteriogens, claims about efficacy in heritage conservation are unsubstantiated.
- Figures 3–10: Embedded tables within graphs are cluttered and unreadable. Raw data should be moved to supplementary tables.
Overall, I consider the article a report suitable for publication, but it is essential that the above points in the text be corrected or clarified.

 

Author Response

We thank Reviewer 4 for the comments, following our responses:

Reviewer 4:

The synthesized compounds are synthetic seleno-sugars, not biologically derived biosimilars – a term that misleadingly suggests equivalence to complex biologics like monoclonal antibodies. lt is recommended to use selenium-based biocides or organoselenium compounds instead.

Answer:  We agree with the Reviewer that the term “biosimilars” is too ambitious to be included in the title. To address this concern, we have replaced “biosimilar” with “selenium-based biocides” in the title. This modification has been implemented consistently in both the Manuscript and the Supplementary Materials.

 

The study exclusively uses Raphidocelis subcapitata (algae) tor testing. Cultural Heritage biodeterioration involves fungi, bacteria, and lichens-algae alone is insufficient. Without data on these key biodeteriogens, claims about efficacy in heritage conservation are unsubstantiated.

 

Answer: Thanks for pointing out this issue. Lane 417-422 have been added.

"This experimental work represents a preliminary study in the toxicity and biocidal activities of similar substances to specific strains of biodeteriogen organisms. Given that most of the chromatic alteration phenomena inherent to biodeterioration of outdoor cultural heritage involves photoautotrophs, the first step was to check the activity of the substances against a susceptible standard organism belonging to the green algae group (phylum Chlorophyta), which share many cellular features and physiological traits, thus the choice of Raphidocelis subcapitata as one of the most studied model organisms for these kind of tests. Next steps would see the application of the studied compounds on a selection of strains isolated from stone heritage in the Archaeological Park of Baia’s Thermal Baths."

 

Figures 3-10: Embedded tables within graphs are cluttered and unreadable. Raw data should be moved to supplementary tables.

Answer: Amended in the Manuscript file

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your changes to the manuscript. Please could you add a citation to the conclusions (line 425) for the source of the information of the algal isolates being associated with biodeterioration (note this is mis-spelled on line 424).

In response to your comments on the ecocompatibility of the compounds please add a sentence or two at the end of the conclusion to let readers know that this work will be coming shortly.

Author Response

We sincerely thank Reviewer 2 for his/her valuable observations. Our manuscript has been revised accordingly (with added sentences highlighted in magenta), and it now more clearly outlines the pathway for our future projects and publications.