Cu(II) Biosorption and Synthesis of CuO Nanoparticles by Staphylococcus epidermidis CECT 4183: Evaluation of the Biocidal Effect

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study highlights the unique abilities of Staphylococcus epidermidis CECT 4183, a bacterium that not only cleans copper from contaminated water but also turns it into powerful antimicrobial nanoparticles. The researchers found that this bacterium efficiently removes copper and then uses it to create tiny particles that can kill harmful microbes. By adding a simple agent, they made the nanoparticles even more effective. This approach offers a green and innovative solution for both pollution cleanup and developing new antimicrobial treatments.

Before further consideration, please follow the below points: 

1. The introduction has no proper context about the 2. problem/solution. The introduction must be improved.

2. What is intended to be this part of the text? "Featured Application: Staphylococcus epidermidis CECT 4183 presents a good capacity for its 11 application in the bioremediation of Cu(II) in contaminated water, and at the same time its cellular 12 extract has a good capacity to synthesize CuO-NPs with potential applications in nanomedicine". Follow the author's guidelines.

3. Extra keywords are needed

4. In section 2, missing subsections for Materials used and Characterization techniques employed. Separate this information for better reliability and include details. 

5. The study does not provide sufficient details about the specific conditions under which the bacterium Staphylococcus epidermidis CECT 4183 was cultured.

6. The incorporation of polyvinyl alcohol (PVA) to improve nanoparticle dispersion is mentioned, but the method of its use is not fully detailed. The concentration of PVA, the method of mixing, and its potential impact on the results need to be clarified. There is also no discussion on whether PVA itself could influence the biocidal efficacy of the nanoparticles, which could be a confounding factor.

7. The discussion around the FT-IR results could be enhanced by comparing the observed shifts with similar studies.

8. The authors report that no characteristic peaks of CuO-NPs were observed after biosorption, attributing this to low concentrations. However, they do not explore alternative explanations or quantify the concentration threshold at which detection would be possible.

9. While the morphological changes and elemental composition are described, the discussion is somewhat superficial. The authors should delve deeper into how these structural changes relate to the biosorption efficiency and the formation mechanism of nanoparticles.

10. The discussion of the isotherm models is technical but lacks a critical analysis of why the Langmuir model fits better than others.

11. The discussion of the biocidal activity could benefit from a more detailed comparison with other studies on CuO nanoparticles. The authors claim that their nanoparticles are more effective, but they do not thoroughly compare their results with the wider body of literature.

Comments on the Quality of English Language

English is generally good but could be improved by simplifying complex sentences, ensuring consistent tense, and correcting minor punctuation errors.

Author Response

Reviewer 1

The authors have submitted a very detailed article, which mainly reports Cu2+ biosorption and synthesis of CuO nanoparticles by Staphylococcus epidermidis CECT 4183. The manuscript is rich in data, especially the characterization before and after incubation with Cu2+. This work seems very meaningful and I suggest minor revision before publication.

 

1. Section 3.4, It is best to have a comparison of similar reports on the biological adsorption capacity of Cu2+.

Thank you for your feedback. Additional text and a new table (Table 3) have been included in section 3.5. The table details results obtained by other authors. All changes have been marked in red.

2. Section 3.6, the antibacterial ability of PVA should also be added as a control. Moreover, please analyze the reasons for the enhanced antibacterial ability after adding PVA.

Thank you for your comment. New text has been added to section 3.6 to clarify these aspects of the research.

 

3. When calculating the adsorption capacity, the author used the dry mass of biomass. I would like to know what the bacterial concentration is equivalent to during the experiment, i.e. OD600 of the tested S. epidermidis.

The bacterial concentration used during the investigation was expressed in g/L. Table 2 lists all the biomass concentrations tested during the design of the experiments. Finally, after adjusting the experimental data, the optimal concentration was 0.2 g/L and the biosorption isotherms were obtained with this concentration.

That is, the biomass concentration was not calculated by the optical density method, the cell concentration was calculated by determining the dry weight of the biomass contained in the cell suspension. This method is highly reproducible and for this reason it was chosen. The procedure is described in section 2.1 and additional text has been incorporated to clarify this aspect.

4. All bacteria names need to be italicized.

Thanks for the comment. The errors have been corrected.

5. Fig.3, it’s better to add elemental map results, even if provide as supporting information.

Thank you for your comment. A new figure (Figure 1S) showing the elemental maps has been incorporated into the supplementary material. New text has also been included in section 3.3.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have submitted a very detailed article, which mainly reports Cu²⁺ biosorption and synthesis of CuO nanoparticles by Staphylococcus epidermidis CECT 4183. The manuscript is rich in data, especially the characterization before and after incubation with Cu²⁺. This work seems very meaningful and I suggest minor revision before publication.

1. Section 3.4, It is best to have a comparison of similar reports on the biological adsorption capacity of Cu²⁺.

2. Section 3.6, the antibacterial ability of PVA should also be added as a control. Moreover, please analyze the reasons for the enhanced antibacterial ability after adding PVA.

3. When calculating the adsorption capacity, the author used the dry mass of biomass. I would like to know what the bacterial concentration is equivalent to during the experiment, i.e. OD₆₀₀ of the tested S. epidermidis.

4. All bacteria names need to be italicized.

5. Fig.3, it’s better to add elemental map results, even if provide as supporting information.  

Author Response

Reviewer 2

The author presents “Cu(II) biosorption and synthesis of CuO nanoparticles by Staphylococcus epidermidis CECT 4183: Evaluation of the biocidal effect”. Some results are interesting and presented well, which would be good contributions to the research community. However, I suggest major revisions on the following points of the manuscript.

 

1. Section 3.4, It is best to have a comparison of similar reports on the biological adsorption capacity of Cu2+.

Thank you for your feedback. Additional text and a new table (Table 3) have been included in section 3.5. The table details results obtained by other authors. All changes have been marked in red.

2. Section 3.6, the antibacterial ability of PVA should also be added as a control. Moreover, please analyze the reasons for the enhanced antibacterial ability after adding PVA.

Thank you for your comment. New text has been added to section 3.6 to clarify these aspects of the research.

 

3. When calculating the adsorption capacity, the author used the dry mass of biomass. I would like to know what the bacterial concentration is equivalent to during the experiment, i.e. OD600 of the tested S. epidermidis.

The bacterial concentration used during the investigation was expressed in g/L. Table 2 lists all the biomass concentrations tested during the design of the experiments. Finally, after adjusting the experimental data, the optimal concentration was 0.2 g/L and the biosorption isotherms were obtained with this concentration.

That is, the biomass concentration was not calculated by the optical density method, the cell concentration was calculated by determining the dry weight of the biomass contained in the cell suspension. This method is highly reproducible and for this reason it was chosen. The procedure is described in section 2.1 and additional text has been incorporated to clarify this aspect.

4. All bacteria names need to be italicized.

Thanks for the comment. The errors have been corrected.

5. Fig.3, it’s better to add elemental map results, even if provide as supporting information.

Thank you for your comment. A new figure (Figure 1S) showing the elemental maps has been incorporated into the supplementary material. New text has also been included in section 3.3.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The author presents “Cu(II) biosorption and synthesis of CuO nanoparticles by Staphylococcus epidermidis CECT 4183: Evaluation of the biocidal effect”. Some results are interesting and presented well, which would be good contributions to the research community. However, I suggest major revisions on the following points of the manuscript.

-        Need to rewrite the title of the manuscript.

-        In abstract: Authors need to highlight the important findings, opinions, numerical results and its contribution accurately.

-        Reconstruct the keywords.

-        Add the recent literature review related to the present work in the introduction section.

-        A crucial issue is the novelty of the CuO nanoparticles synthesized from microorganism. The authors should explain the unique advantages of the bioengineered CuO nanoparticles compared to other reported works for biosorption and antibacterial applications.

-        In Figure 2: Around 80- and 85-degree XRD peaks are not indexed; why?

-        The authors should calculate the structural parameters such as lattice constant, lattice strain, and dislocation density of the prepared samples and explain them in the text.

-        The authors should provide the FESEM images of both samples with two images (low and high magnifications only) of each sample. The same scale and magnifications for all images would be necessary. Here, they provided four of each, which creates confusion.

-        Need to provide the EDAX analysis composition table of the prepared samples (Figure 3 e-f).

-        Should add the UV-Vis analysis of the prepared samples and correlate with antibacterial activity results.

-        Page 12, lines 300 & 302: Bacterial pathogens should be in Italics.

-        In Table 3: Add the positive control, negative control, and load concentration details.

-        How does the CuO nanoparticles morphology affect the antibacterial properties? Explain it.

-        Should provide the antibacterial activity mechanism diagram using the prepared CuO nanoparticles and explain them in the text.

-        Cited references are not enough to discuss the results biosorption and antibacterial applications in the text.

-        The authors can compare this research with other similar studies in a tabular format to highlight the advantage of their work.

-        The authors include their outlook on the future of this research in the conclusion section.

-        The text is not free of grammatical, formatting, and punctuation errors. Ask a native English speaker to revise and proofread their revised manuscript before resubmission.

Comments on the Quality of English Language

Ask a native English speaker to revise and proofread their revised manuscript before resubmission.

Author Response

Reviewer 3

The author presents “Cu(II) biosorption and synthesis of CuO nanoparticles by Staphylococcus epidermidis CECT 4183: Evaluation of the biocidal effect”. Some results are interesting and presented well, which would be good contributions to the research community. However, I suggest major revisions on the following points of the manuscript.

 

1) Need to rewrite the title of the manuscript.

Thank you for your comment. The authors consider that the title fits well with the content of the manuscript, although we are open to possible suggestions.

2) In abstract: Authors need to highlight the important findings, opinions, numerical results and its contribution accurately.

Thank you for your comment. The text of the abstract complied exactly with the journal's instructions regarding format and structure, without exceeding the required 200 words. The authors consider that the data shown are the most significant of the work. However, in order to respond to the reviewer's suggestion, the information in the abstract has been expanded to include new data.

3) Reconstruct the keywords.

Thank you for your comment. The word “wastewater” has been changed to “biosorption” and new keywords have been included.

4) Add the recent literature review related to the present work in the introduction section.

Thank you for your comment. The authors consider that the citations included in the Introduction section are sufficient and up-to-date. Some of these citations are very complete reviews. This section includes 9 recent bibliographic citations: 2024 (6), 2023 (2) and 2021 (1). However, a new citation (2022) and additional text have been added that includes aspects related to the following comment.

5) A crucial issue is the novelty of the CuO nanoparticles synthesized from microorganism. The authors should explain the unique advantages of the bioengineered CuO nanoparticles compared to other reported works for biosorption and antibacterial applications.

Thank you for your comment. The Introduction section has been reworded and new text has been included.

6) In Figure 2: Around 80- and 85-degree XRD peaks are not indexed; why?

Thank you for your comment. The main reason for the lack of identification of the peaks located at 80° and 85° is that these peaks are not relevant for the identification of CuO-NPs. Generally, the most characteristic peaks are located between 30° and 70°. Sometimes peaks located at 72° and 76° also appear, as in this case.

7) The authors should calculate the structural parameters such as lattice constant, lattice strain, and dislocation density of the prepared samples and explain them in the text.

Thank you for your comment. We understand the importance of these parameters, but we consider that the work represents a first approach to the synthesis of CuO-NPs from S. epidermidis cell extract and that the ultimate goal of the work is to analyze their potential as biocidal agents. For this reason, we consider that, in line with what other authors do, at this stage of the research it is not necessary to determine these microstructural parameters. Nevertheless, we gratefully accept your suggestion for future work in which we will delve deeper into the mechanisms of interaction between NPs and microbial cells.

8) The authors should provide the FESEM images of both samples with two images (low and high magnifications only) of each sample. The same scale and magnifications for all images would be necessary. Here, they provided four of each, which creates confusion.

Thank you for your comment. The inclusion of 4 images in Figure 3 is intended to provide the maximum visual information obtained from the FE-SEM analysis. If the reviewer considers it strictly necessary to remove two images, they will be removed. On the other hand, it is true that the comparative images do not have the same scale but it is a similar scale that allows definitive conclusions to be drawn. The images of the same scale did not offer the same image quality on this occasion and for that reason it was decided to replace them. In the meantime, and in response to your comment, the text of Figure 3 has been rewritten for a better understanding of the figure.

9) Need to provide the EDAX analysis composition table of the prepared samples (Figure 3 e-f).

Thank you for your comment. Authors do not usually present these results because the analysis is semi-quantitative. However, in response to your suggestion, a new table with this information has been included in the supplementary material. Table 1S has been cited in section 3.3, the text of which has been rewritten. At the same time, a typo was detected in section 3.1 and therefore part of the text has also been rewritten.

10) Should add the UV-Vis analysis of the prepared samples and correlate with antibacterial activity results.

Thank you for your comment. A new figure has been added to the supplementary material. Figure 2S shows the growth curves obtained for the 5 microorganisms during the biocidal tests. The graphs include the curves corresponding to the positive control that incorporated 10% PVA and confirmed that microbial inhibition did not occur at that concentration. The curves of the negative control are also included, which ruled out contamination of the nutrient medium used in the tests. To clarify these aspects of the research, text has been added to section 2.4.

11) Page 12, lines 300 & 302: Bacterial pathogens should be in Italics.

Thank you for your comment. The errors have been corrected.

12) In Table 3: Add the positive control, negative control, and load concentration details.

Thank you for your comment. This information has already been incorporated into section 2.4 following your previous recommendations.

13) How does the CuO nanoparticles morphology affect the antibacterial properties? Explain it.

Thank you for your comment. Text has been added to section 3.6

14) Should provide the antibacterial activity mechanism diagram using the prepared CuO nanoparticles and explain them in the text.

Thank you for your comment. The authors understand the importance of determining the biocidal mechanism of nanoparticles, however, this work did not address this aspect because it was a first approximation to the biocidal efficacy of CuO-NPs synthesized by S. epidermidis. This interesting aspect will be addressed in future works.

15) Cited references are not enough to discuss the results biosorption and antibacterial applications in the text.

Thank you for your comment. Two new referenced tables have been incorporated into the text, in which the results of our work are compared with those obtained by other authors. Table 3 has been incorporated into section 3.5 and Table 5 has been incorporated into section 3.6.

16) The authors can compare this research with other similar studies in a tabular format to highlight the advantage of their work.

Thank you for your comment. In line with your previous comment and for comparative purposes, a new table (Table 3) has been included in section 3.5 and an additional one (Table 5) in section 3.6.

17) The authors include their outlook on the future of this research in the conclusion section.

Thank you for your comment. A paragraph has been included in the conclusion section to clarify this aspect of the research.

18) The text is not free of grammatical, formatting, and punctuation errors. Ask a native English speaker to revise and proofread their revised manuscript before resubmission.

Thank you for your comment. The text has been reviewed and corrected.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors uploaded another response letter. 

Comments on the Quality of English Language

English can be improved 

Author Response

Reviewer 1

 Dear Reviewer 1, I am very sorry, I accidentally sent you the answers to another reviewer. I am sending you the answers to your 11 comments.

 

This study highlights the unique abilities of Staphylococcus epidermidis CECT 4183, a bacterium that not only cleans copper from contaminated water but also turns it into powerful antimicrobial nanoparticles. The researchers found that this bacterium efficiently removes copper and then uses it to create tiny particles that can kill harmful microbes. By adding a simple agent, they made the nanoparticles even more effective. This approach offers a green and innovative solution for both pollution cleanup and developing new antimicrobial treatments.

 

Before further consideration, please follow the below points:

 

1. The introduction has no proper context about the 2. problem/solution. The introduction must be improved.

Thank you for your comment. New information (in red) has been added to the Introduction section.

2. What is intended to be this part of the text? "Featured Application: Staphylococcus epidermidis CECT 4183 presents a good capacity for its 11 application in the bioremediation of Cu(II) in contaminated water, and at the same time its cellular 12 extract has a good capacity to synthesize CuO-NPs with potential applications in nanomedicine". Follow the author's guidelines.

Thank you for your comment. Indeed, this section is not common in scientific articles, however, the instructions for authors of the journal Applied Sciences recommend that they include it in the manuscript. It is an optional section that appears in the Word template of the journal itself.

3. Extra keywords are needed

Thanks for the comment. New keywords have been added.

4. In section 2, missing subsections for Materials used and Characterization techniques employed. Separate this information for better reliability and include details.

Thank you for your comment. 4 new sub-sections have been included for a better understanding of the text.

5. The study does not provide sufficient details about the specific conditions under which the bacterium Staphylococcus epidermidis CECT 4183 was cultured.

Thank you for your feedback. This information can be found in the new section 2.1.1

6. The incorporation of polyvinyl alcohol (PVA) to improve nanoparticle dispersion is mentioned, but the method of its use is not fully detailed. The concentration of PVA, the method of mixing, and its potential impact on the results need to be clarified. There is also no discussion on whether PVA itself could influence the biocidal efficacy of the nanoparticles, which could be a confounding factor.

Thank you for your comment. To clarify this aspect, new information has been included in sections 2.4 and 3.6.

7. The discussion around the FT-IR results could be enhanced by comparing the observed shifts with similar studies.

Thank you for your comment. The text of section 3.1 has been improved and an additional citation has been included.

8. The authors report that no characteristic peaks of CuO-NPs were observed after biosorption, attributing this to low concentrations. However, they do not explore alternative explanations or quantify the concentration threshold at which detection would be possible.

Thank you for the comment. A new Figure (Figure 1S) and a new table (Table 1S) have been added to the supplementary material and have been discussed in section 3.3. Indeed, our initial statement is only a hypothesis, but it can be adjusted to reality if the FE-SEM images and EDX spectra are analyzed together with Table 1S and Figure 1S. In any case, we cannot affirm that we are right and in future works we will analyze this interesting question more thoroughly.

Regarding the possibility of quantifying the concentration threshold at which it would be possible to detect CuO-NPs after the biosorption stage, we consider that the signal from the salts present in the biomass will always be a confusing factor, but logically if the concentration of NPs is higher, their characteristic peaks can be identified. In any case, this aspect is not decisive for the final objective of the research and is only mentioned because in the case that NPs are detected in the biomass this would indicate that the cellular extract of the microorganism will be a good candidate for the green synthesis of nanoparticles.

9. While the morphological changes and elemental composition are described, the discussion is somewhat superficial. The authors should delve deeper into how these structural changes relate to the biosorption efficiency and the formation mechanism of nanoparticles.

Thank you for your comment. This information has been incorporated into section 3.3

10. The discussion of the isotherm models is technical but lacks a critical analysis of why the Langmuir model fits better than others.

Thank you for your comment. Text has been included in section 3.5 to clarify this aspect of the discussion of results.

11. The discussion of the biocidal activity could benefit from a more detailed comparison with other studies on CuO nanoparticles. The authors claim that their nanoparticles are more effective, but they do not thoroughly compare their results with the wider body of literature.

Thank you for your comment. In order to compare our results with those obtained by other authors, a new table (Table 5) has been included in section 3.6.

English is generally good but could be improved by simplifying complex sentences, ensuring consistent tense, and correcting minor punctuation errors.

Thank you for your comment. The English and wording have been revised.

Reviewer 3 Report

Comments and Suggestions for Authors

In the revision, the authors addressed all of the concerns that are addressed and the quality has been improved.  

Author Response

Thank you very much for your comments and contributions.

Best regards

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for uploading the correct response report. Most of the comments have been properly considered and others have been clarified. I recommend the publication of the revised manuscript.