Phyto-Synthesis and Characterization of Silver Nanoparticles Using Box-Behnken Design and Its Anti-Alternaria Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

- The manuscript titled by “Phyto-synthesis and Characterization of Silver Nanoparticles using Box-Behnken Design and its Anti-Alternaria Activity” discusses the Phyto-synthesis and Characterization of Silver Nanoparticles and the potential use of Ag-NPs as antifungal agent to fight against fungicide-resistant Alternaria alternata in order to reduce the environmental impact of synthetic fungicides.

The main idea isn’t new, silver nanoparticles phyto-synthesis and its characterization have been used for many years as antifungal agent.

The bio-statistical design is acceptable, scientific flow is also acceptable but there are few comments as follows:

- The manuscript requires verification of guidelines (for references for example: non-homogeneous presentation, some references contain abbreviated journal name such as reference 11 and others are not).

- Editing of microorganisms should appear in italics even in references.

- The Authors separated the results from the discussion. This is not making the reading fluent. I would suggest re-organizing these sections (Results – Discussion) with a single chapter and several thematic sub-chapters.

- The author mentioned in discussion line 452 that “fungal spores’ membrane structure changes significantly” and mentioned that silver nanoparticles effect on cell pathogenicity, viability, however the author didn’t approve that by experiment such as DNA, protein leakage.

- Some sections showing the applications are not enough documented. Just as an example: in discussion line 468 the author is comparing results with other nanoparticles such as zinc oxide and Fusarium spp., although author is recommended to compare results with related references.

- Some of the figures are not appropriate for publication (sharpness and precision; for example: Figure 2, 3 and 5), Figure 5 (e and f) is better to be moved to supplementary section.

- FTIR of syntheized silver nanoparticles is missing and also the assignments for FTIR bands should be supported with appropriate references.

- References should be updated to 2023, only one reference is latest, should be more with relevant articles.

- Conclusions should be restated to be more comprehensive 

Author Response

Reviewer #1 Comments and Suggestions for Authors

The manuscript titled by “Phyto-synthesis and Characterization of Silver Nanoparticles using Box-Behnken Design and its Anti-Alternaria Activity” discusses the Phyto-synthesis and Characterization of Silver Nanoparticles and the potential use of Ag-NPs as antifungal agent to fight against fungicide-resistant Alternaria alternata in order to reduce the environmental impact of synthetic fungicides.

The main idea isn’t new, silver nanoparticles phyto-synthesis and its characterization have been used for many years as antifungal agent.

The bio-statistical design is acceptable, scientific flow is also acceptable but there are few comments as follows:

Comments 1: The manuscript requires verification of guidelines (for references for example: non-homogeneous presentation, some references contain abbreviated journal name such as reference 11 and others are not).

Response 1: We thank the reviewer for the comment and taking the time to evaluate our manuscript. The abbreviated journal names have been written in full in the revised manuscript. Pg 23-27, Lines 584-719.

Comments 2: Editing of microorganisms should appear in italics even in references.

Response 2: We thank the reviewer for the comment. All organism names listed in the manuscript (including reference section) have been revised have and corrected as suggested by the reviewer. Lines 584-719.

Comments 3: The Authors separated the results from the discussion. This is not making the reading fluent. I would suggest re-organizing these sections (Results – Discussion) with a single chapter and several thematic sub-chapters.

Response 3: We thank the reviewer for the suggestion. However, we have compiled the manuscript using the journal template provided in the authors guidelines.  

Comments 4: The author mentioned in discussion line 452 that “fungal spores’ membrane structure changes significantly” and mentioned that silver nanoparticles effect on cell pathogenicity, viability, however the author didn’t approve that by experiment such as DNA, protein leakage.

Response 4: We thank the reviewer for the comment. We have revised the section and corrected it accordingly as we did not perform the experiments in this manuscript. The revision of this section is highlighted in the revised manuscript. Pg 21, Lines 497-504.

Comments 5: Some sections showing the applications are not enough documented. Just as an example: in discussion line 468 the author is comparing results with other nanoparticles such as zinc oxide and Fusarium spp., although author is recommended to compare results with related references.

Response 5: We thank the reviewer for the comment. The discussion section in question has been duly corrected and proper comparison was made within the context of the manuscript and referenced accordingly.

Comments 6: Some of the figures are not appropriate for publication (sharpness and precision; for example: Figure 2, 3 and 5), Figure 5 (e and f) is better to be moved to supplementary section.

Response 6: We thank the reviewer for the comment. We have revised the figures pointed out and significantly improved their quality in line with the expectation of the journal for publication. Pg 11-16, 18

Comments 7: FTIR of syntheized silver nanoparticles is missing and also the assignments for FTIR bands should be supported with appropriate references.

Response 7: We thank the reviewer for the comment. However, FTIR characterization of the synthesized nanoparticles does not form part of this manuscript. We are of the opinion that the characterization methods used is sufficient to confirm the synthesis of the nanoparticle presented in this manuscript.

Comments 8: References should be updated to 2023, only one reference is latest, should be more with relevant articles.

Response 8: We thank the reviewer for the comment. All references used in the manuscript were revised and updated where possible to include the latest literature. Page 24, Line 627-634

Comments 9: Conclusions should be restated to be more comprehensive

Response 9: We thank the reviewer for the comment. The conclusion section of the manuscript was revised and improved where possible. Page 23, Line 548-564

Reviewer 2 Report

Comments and Suggestions for Authors

 

In this manuscript entitled “Phyto-synthesis and Characterization of Silver Nanoparticles using Box-Behnken Design and its Anti-Alternaria Activity” (Manuscript Number: cleantechnol-2645289) I think it’s better to discuss about below questions. Therefore, I suggest a revision for the manuscript.

 

Comments:

1. Introduction: should be extended.
2. Experimental: should be extended. Also, the total materials and companies, characterization of all instruments, methodology, synthesis steps and etc. should be presented with more details.
3. The text can be improved by providing a more critical discussion of related literature. For example: RSC advances 4 (72), 37979-37984 (2014) / Materials Science-Poland 35 (2), 368-373 (2017) / Materials Science and Engineering: C 111, 110854 (2020) / Analyst 139 (16), 4064-4072 (2014) / Journal of Nanostructure in Chemistry 9, 29-37 (2019). 
4. All equations should be cited in the text by a number.
5. There are many reports about synthesis of silver nanoparticles. The authors should be explaining about the importance and novelty of the work than the previous works.
6. A comparison table should be presented to show the novelty of the work.
7. The text should be revised about English language.
8. Conclusions: should be supported by the results

 

Comments on the Quality of English Language

 

In this manuscript entitled “Phyto-synthesis and Characterization of Silver Nanoparticles using Box-Behnken Design and its Anti-Alternaria Activity” (Manuscript Number: cleantechnol-2645289) I think it’s better to discuss about below questions. Therefore, I suggest a revision for the manuscript.

 

Comments:

1. Introduction: should be extended.
2. Experimental: should be extended. Also, the total materials and companies, characterization of all instruments, methodology, synthesis steps and etc. should be presented with more details.
3. The text can be improved by providing a more critical discussion of related literature. For example: RSC advances 4 (72), 37979-37984 (2014) / Materials Science-Poland 35 (2), 368-373 (2017) / Materials Science and Engineering: C 111, 110854 (2020) / Analyst 139 (16), 4064-4072 (2014) / Journal of Nanostructure in Chemistry 9, 29-37 (2019).
4. All equations should be cited in the text by a number.
5. There are many reports about synthesis of silver nanoparticles. The authors should be explaining about the importance and novelty of the work than the previous works.
6. A comparison table should be presented to show the novelty of the work.
7. The text should be revised about English language.
8. Conclusions: should be supported by the results

Author Response

Comments 1: Introduction: should be extended.

Response 1: We thank the reviewer for the comment. The introduction section of the manuscript was revised and additional changes are highlighted in revised manuscript. Page 2, Line 62-79

Comments 2: Experimental should be extended. Also, the total materials and companies, characterization of all instruments, methodology, synthesis steps and etc. should be presented with more details.

Response 2: We thank the reviewer for the comment. The experimental section of the manuscript in its current form is detailed and clearly outline all step performed. We have included instrument names and the company names that manufactured these instruments. Page 3, Line 98-101.

Comments 3: The text can be improved by providing a more critical discussion of related literature. For example: RSC advances 4 (72), 37979-37984 (2014) / Materials Science-Poland 35 (2), 368-373 (2017) / Materials Science and Engineering: C 111, 110854 (2020) / Analyst 139 (16), 4064-4072 (2014) / Journal of Nanostructure in Chemistry 9, 29-37 (2019).

Response 3: We thank the reviewer for the comment and suggestion. The revised manuscript has been significantly improved with the addition of relevant literature to support the results presented in the manuscript. Page 2, Line 62-79.

Comments 4: All equations should be cited in the text by a number.

Response 4: We thank the reviewer for the comment. All equations have been numbered and cited in the text accordingly. Page 4, Line 151, 171, Page 20, Line 420-425

Comments 5: Conclusions: should be supported by the results

Response 5: We thank the reviewer for the comment. The conclusion section of the manuscript was revised as suggested by the reviewer. All amendments to the conclusion section have been highlighted in the revised manuscript. Page 23, Line 548-564

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editor,

This article optimize Phyto-synthesis of Silver Nanoparticles using Box-Behnken Design and investigate its Anti-Alternaria Activity. Below I will mention some of the problems seen in the MS text.

------------------------------------------

1. Minor:

--The writing of the text is good, however, check it again for grammar and spelling of the words. for example, “… particle size of 23.75 and 34.36nm respectively was synthesized…“ and , “… potential of Ag-NPs as antifungal agent to fight against…“ and , “… synthesizing nanomaterials is at the heart of…“ and …..

 

-“Please bring all the materials used in the research in the 2.1 section.

 

-You have used the green method. While the first time you mentioned it was section 2.4. Write about it in the introduction and even in the abstract.

 

-Line 119. ..was centrifuged at 900 rpm for 10 minutes and washed... Was the supernatant completely clear after centrifugation? According to my knowledge, this low round and low time does not have the ability to deposit nanoparticles of about 100 nm!

 

-Increase the quality of Figure 2 and 3.

 

-Add the section of the instrumental used in the research to the article.

*****************************

1.      Major:

-I have a problem with your methodology.

You have optimized the reaction time, even though after the reaction, you did not separate the nanoparticles and kept them in the reaction conditions for another 24 hours. This long time eliminates the storage of many small data in time. What was your reason for doing this? If these reasons are obvious, add them in line 429 and after.

 

-You have not entered the extract value in the optimization. Did you have a specific reason for it? Couldn't more extract reduce the reaction time?

 

 

-In Figure 1, the interpretation results of TEM and SEM do not match! I imagine that you have selected images from several optimized examples

Comments on the Quality of English Language

The writing of the text is good, however, check it again for grammar and spelling of the words. for example, “… particle size of 23.75 and 34.36nm respectively was synthesized…“ and , “… potential of Ag-NPs as antifungal agent to fight against…“ and , “… synthesizing nanomaterials is at the heart of…“ and …..

Author Response

Minor:

Comments 1: The writing of the text is good, however, check it again for grammar and spelling of the words. for example, “… particle size of 23.75 and 34.36nm respectively was synthesized…“ and , “… potential of Ag-NPs as antifungal agent to fight against…“ and , “… synthesizing nanomaterials is at the heart of…“ and …..

Response 1: We thank the reviewer for the comment. The grammar and spelling of words in the manuscript has been revised to improve the quality of the manuscript. Page 1, Line 16-17, 28-29, Page 2, Line 66 and 68.

Comments 2: Please bring all the materials used in the research in the 2.1 section.

Response 2: We thank the reviewer for the suggestion. The materials used in this manuscript has been included in section 2.1 as suggested by the reviewer. Page 3, Line 98-101.

Comments 3: You have used the green method. While the first time you mentioned it was section 2.4. Write about it in the introduction and even in the abstract.

Response 3: We thank the reviewer for the comment. The section about the green synthesis has been included in the appropriate sections of the manuscript as per the reviewers’ suggestion. This inclusion has been highlighted in the revised manuscript. Page 1, Line 16-17, Page 2, 69-79

Comment #4: Line 119. ..was centrifuged at 900 rpm for 10 minutes and washed... Was the supernatant completely clear after centrifugation? According to my knowledge, this low round and low time does not have the ability to deposit nanoparticles of about 100 nm!

Response 4: We thank the reviewer for the comment and observation. We agree with the reviewer and acknowledge that this was a typographical error. The speed at which the nanoparticles was centrifuged should by 9000 rpm and not 900 rpm as indicated in the manuscript. The centrifuged speed of 9000 rpm has been corrected in the revised manuscript. Page 3, Line 123.

Comments 5: Increase the quality of Figure 2 and 3.

Response 5: We thank the reviewer for the comment. All the Figures presented in the manuscript was revised to improve their quality for publication. Page 11-16, Line 313-328

 Major

Comments 6: I have a problem with your methodology. You have optimized the reaction time, even though after the reaction, you did not separate the nanoparticles and kept them in the reaction conditions for another 24 hours. This long time eliminates the storage of many small data in time. What was your reason for doing this? If these reasons are obvious, add them in line 429 and after.

Response 6: We thank the reviewer for the comment and observation. The materials were synthesized and centrifuged immediately to separate the nanoparticles. We acknowledge the error in the method reported and have corrected it accordingly in the revised manuscript. Page 3, Line 125-134.

Comments 7: You have not entered the extract value in the optimization. Did you have a specific reason for it? Couldn't more extract reduce the reaction time?

Response 7: We thank the reviewer for the comment. We are of the opinion and supported by literature that different parameters could influence the nature and type of nanoparticles synthesized. The volume of the extract used for synthesis can also affect the size and the rate of synthesis of the nanoparticle. In this study, we kept the volume of the extract constant at a ratio of 1:5 of the extract to silver nitrate solution and vary other parameters as shown in our study. Therefore, the effect of the extract or extract volume is not captured in our study.

Comments 8: In Figure 1, the interpretation results of TEM and SEM do not match! I imagine that you have selected images from several optimized examples.

Response 8: We thank the reviewer for the comment and observation. The section has been corrected and revised accordingly. Page 6, Line 273-276.

Reviewer 4 Report

Comments and Suggestions for Authors

The article of A.I. Daniel et al. is very interesting and contains voluminous research about the synthesis of silver nanoparticles and their use as anti-fungal agent. The synthesis of nanoparticles was performed with an analysis by the Box-Behnken Design method; the exhaustive characteristics of the obtained NPs are presented; and a detailed study of the microbiological effect on the fungi mycelia is performed. In my mind, this is a high-quality article with very interesting research. One question, which remains unclear, is the reason for precise use of the seed extract of Abrus precatorious to prepare NPs. Lastly, I strongly recommend to accept the article to be published in Clean Technologies. 

Author Response

Comments 1: One question, which remains unclear, is the reason for precise use of the seed extract of Abrus precatorious to prepare NPs. Lastly, I strongly recommend to accept the article to be published in Clean Technologies.

Response 1: We appreciate the reviewer for the kind feedback and positive consideration of our manuscript.  The seed of Abrus precatorius is a reservoir of bioactive compounds (Rajaram, N., & Janardhanan, K. (1992). The chemical composition and nutritional potential of the tribal pulse, Abrus precatorius L. Plant foods for human nutrition, 42, 285-290) with known therapeutic (Bhatia, M., Siddiqui, N., & Gupta, S. (2013). Abrus precatorius (L.): An evaluation of traditional herb. J Pharm Res, 3, 3296-315), antioxidant (Pal, R. S., Ariharasivakumar, G., Girhepunjhe, K., & Upadhay, A. (2009). In-vitro antioxidative activity of phenolic and flavonoid compounds extracted from seeds of Abrus precatorius. Int J Pharm Pharm Sci, 1(2), 136-40) and antimicrobial activity (Bobbarala, V., & Vadlapudi, V. (2009). Abrus precatorius L. seed extracts antimicrobial properties against clinically important bacteria. International journal of pharmtech research, 1(4), 1115-1118).  In light of this, we explored the use of the seeds for the synthesis of nanoparticles as there are reports of the use of leaves and roots for nanoparticle synthesis (Gaddala, B., & Nataru, S. (2015). Synthesis, characterization and evaluation of silver nanoparticles through leaves of Abrus precatorius L.: an important medicinal plant. Applied Nanoscience, 5, 99-104).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All corrections have been revised and accepted 

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript entitled “

 Phyto-synthesis and Characterization of Silver Nanoparticles using Box-Behnken Design and its Anti-Alternaria Activity” (Manuscript Number: cleantechnol-2645289) is rather satisfactory and can be accepted in the present form.