Optimization of the Conditions for the Transformation of a Bacillus subtilis Strain L11 to Prepare Nano Selenium and Its Preliminary Application in Sheep Feed
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors
The manuscript is very well written; all the principle parts are described in precise detail. The manuscript issue is highly specialized, offers new knowledge, and enriches the scientific community.
Sincerely
Comments on the Quality of English LanguageMinor mistakes
The English used in the manuscript is at a good level. In the methodology, it is necessary to unify the person writing. Write uniformly in the passive form and not in one sentence in the third person and in the next in the passive form.
Sincerely
Author Response
Dear Reviewer,
We are deeply grateful for your valuable feedback, which has indeed elevated the academic standards and clarity of our manuscript. Your comments have been meticulously addressed, and the corresponding modifications and supplements have been included in the revised version. You can find the detailed changes in the manuscript.
We appreciate your time and expertise in guiding us towards improving our work. Your suggestions have been instrumental in refining our approach and strengthening the overall argumentation.
Thank you once again for your contributions. We hope that the revised manuscript meets your expectations.
Reviewer 1
Dear Authors
The manuscript is very well written; all the principle parts are described in precise detail. The manuscript issue is highly specialized, offers new knowledge, and enriches the scientific community.
Sincerely
Minor mistakes
The English used in the manuscript is at a good level. In the methodology, it is necessary to unify the person writing. Write uniformly in the passive form and not in one sentence in the third person and in the next in the passive form.
A: I appreciate your feedback. We have made the necessary corrections to the grammar and tense in the Materials and Methods section. Please refer to the revision marks in the manuscript for further details.
Sincerely,
Hua
Reviewer 2 Report
Comments and Suggestions for AuthorsPaper: Optimization of the conditions for the transformation of a Bacillus subtilis strain L11 to prepare nano selenium and its preliminary application in sheep feed
Journal: Microbiology research
Year: 2024
This manuscript presents an interesting study on the production of selenium nanoparticles by a modified Bacillus subtilis L11 screened from a selenium-rich dairy cow breeding base. After selecting this strain, the optimal culture conditions were determined, and XPS, SEM-EDS, and TEM analyses were used to determine the nanoparticles, revealing their synthesis from the transformation of the cell's periplasmic space, cell membrane, and cell wall. This study presents scientific novelty. However, it requires a more in-depth discussion of the results. Therefore, a major review is required before being considered for publication.
Comments to authors
I suggest presenting the time units in abbreviated form: hours in "h" and minutes in "min".
Abstract
-Present the meaning of XPS, SEM-EDS, and TEM in the first occurrence in the text.
Introduction
-Present the meaning of WHO in the first occurrence in the text.
Materials and methods
-Present the meaning of PCR and NCBI in the first occurrence in the text.
-Section 2.1: Replace "culture conditions" with "culture media". Improve the writing of the sentence about cultural media.
-Section 2.2: Which microscope was used? Add information about type, brand, and model.
-Section 2.2: Were the cells cultured in a plate or shake flask? Add this information.
-Section 2.3: Add information about pre-inoculum, working volume, number of replicas, and OD600 nm (present its meaning). Present specific information about reducing Na2SeO3 activity. How was this activity determined? Also, add more information about the experimental planning (type of DoE, factors/variables, coded levels) - this can be done in table form.
-Lines 100, 102, 104, 126, 133, 134, 150: Imperative verbs. Sentences should not be presented as a guide to protocol but rather as a description of what was accomplished.
-Sections 2.4, 2.5, and 2.6: Were the cells cultured in plates? Add this information.
-Lines 121 and 162: Correct “12000 x g” and “3000 x g”. Furthermore, "g" must be presented in italics. Correct throughout the "Materials and Methods" section (lines 115, 121, 132, 135, 162).
-Line 138: Present the meaning of EDS.
-Section 2.7: Define the control group and the experimental group.
-Line 159: Display information about the automated blood analyzer (model and brand).
Results and discussion
In general, there is little discussion of the results. Further comparison with the literature is required.
-Present the meaning of DIS and BLAST in the first occurrence in the text.
-Lines 190, 208: Bacillus should be written in italics.
-Lines 193-194: Avoid repeating information already presented in the "Materials and Methods" section. I suggest rewriting this sentence.
-Section 3.2: Was any design of experiments (DoE) carried out in this part? If yes, present the results of ANOVA, model, and experimental and predicted data from the model. Otherwise, the expression "optimization" should not be used, as it does not result from DoE. Replace with ideal culture conditions. Review the entire manuscript (pay attention to the abstract).
-Section 3.3: Why did the authors not present the results in Fig. 3 as a function of the quantity or conversion of SeNPs? If SeNPs have been quantified, present the methodology in the corresponding section. Otherwise, make it clear that the results were presented depending on the number of cells formed since SeNPs are correlated to OD600 nm.
-Lines 245 and 247: Correct "... range of 20-30 °C..." and "... range of 30-42 °C..."
-Line 249: Wouldn't the ideal temperature be 30 °C? Please review.
-Lines 272-274: As previously suggested, this information should be presented in the "Materials and Methods" section.
-Line 275: Check whether the ideal temperature would not be 30 °C.
-Lines 302-303: Imperative verbs. Sentences should not be presented as a guide to protocol but rather as a description of what was accomplished.
-Lines 302-304 and 339-342: Avoid repeating methodology information in the "Results and Discussion" section.
-Lines 388-389: Avoid bringing up the meaning of the acronyms again.
-Lines 390-393, 409-410, 413-414 and 418-419: Correct p<0.01 and p>0.05 (lowercase p).
Conclusions
-I suggest better highlighting the main scientific contributions of this study.
Figures
-Fig. 1: Improve the quality of images 1a and 1b. Correct the red highlight in the names of microorganisms in 1c. Display the unit of 0.002 in 1c. Indicate the meaning of the numbers (83, 63, 65, 58, 88) in the legend in 1c.
-Fig. 2: 600 must be submitted signed to the OD and with unit nm. Add the meaning of the letters in the legend and their statistical explanation.
In 2a: Bacillus subtillis should be written in italics, and it would be interesting to present the OD600 nm data point by point. To what do the authors attribute the better growth of LB compared to the Bacillus universal culture medium? Add a brief discussion with references to support the hypothesis.
In 2b: Use only "temperature" for the x-axis title.
-Fig. 3: 600 must be submitted signed to the OD and with unit nm. Add the meaning of the letters in the legend and their statistical explanation.
In 3a: Use only "temperature" for the x-axis title.
-Fig. 5: Present the meaning of the circles and arrows in 5g in the caption.
-Fig. 6: Rewrite the title of the figure, as it seems to be a description of what was accomplished and not what the image represents.
Tables
Table 3: Display 0.13±0.001 instead of 0.13±0.008.
Comments on the Quality of English LanguageThe writing also needs to be improved.
Author Response
Dear Reviewer,
We are deeply grateful for your valuable feedback, which has indeed elevated the academic standards and clarity of our manuscript. Your comments have been meticulously addressed, and the corresponding modifications and supplements have been included in the revised version. You can find the detailed changes in the manuscript.
We appreciate your time and expertise in guiding us towards improving our work. Your suggestions have been instrumental in refining our approach and strengthening the overall argumentation.
Thank you once again for your contributions. We hope that the revised manuscript meets your expectations.
Sincerely,
Hua
Reviewer 2
This manuscript presents an interesting study on the production of selenium nanoparticles by a modified Bacillus subtilis L11 screened from a selenium-rich dairy cow breeding base. After selecting this strain, the optimal culture conditions were determined, and XPS, SEM-EDS, and TEM analyses were used to determine the nanoparticles, revealing their synthesis from the transformation of the cell's periplasmic space, cell membrane, and cell wall. This study presents scientific novelty. However, it requires a more in-depth discussion of the results. Therefore, a major review is required before being considered for publication.
A: Thank you for your valuable feedback. We have addressed your comments point-by-point and made the corresponding modifications as follows:
Comments to authors
I suggest presenting the time units in abbreviated form: hours in "h" and minutes in "min".
A: We have followed your suggestion and replaced all time units in the manuscript with 'h' and 'min'. Thank you for your guidance!
Abstract
-Present the meaning of XPS, SEM-EDS, and TEM in the first occurrence in the text.
A: We have added the full names of abbreviations in the abstract where they first appear, as suggested. Thank you for your assistance!
Introduction
-Present the meaning of WHO in the first occurrence in the text.
A: Thank you for referring to the manuscript revision details. We have followed the same approach and updated the abstract accordingly, ensuring that all abbreviations are accompanied by their respective full names when first mentioned. This enhances the clarity and accessibility of the manuscript for readers.
Materials and methods
-Present the meaning of PCR and NCBI in the first occurrence in the text.
A: Added, thank you
-Section 2.1: Replace "culture conditions" with "culture media". Improve the writing of the sentence about cultural media.
A: We have replaced and rewritten this section. Thank you
-Section 2.2: Which microscope was used? Add information about type, brand, and model.
A: The microscope model and manufacturer have been added to the manuscript. Please refer to the revised manuscript for details. Thank you
-Section 2.2: Were the cells cultured in a plate or shake flask? Add this information.
A: For this part of the experiment, the strains were cultured on LB plates, and explanations have been added in the manuscript section. Thank you
-Section 2.3: Add information about pre-inoculum, working volume, number of replicas, and OD600 nm (present its meaning). Present specific information about reducing Na2SeO3 activity. How was this activity determined? Also, add more information about the experimental planning (type of DoE, factors/variables, coded levels) - this can be done in table form.
A: Thank you for your suggestion. This part of the work has been added to section 2.1 of the manuscript on materials and methods. Please refer to the article revision details for details
-Lines 100, 102, 104, 126, 133, 134, 150: Imperative verbs. Sentences should not be presented as a guide to protocol but rather as a description of what was accomplished.
A: Thank you for your suggestion. We have made corrections and modifications to the imperative sentences in this section of the manuscript, as well as grammar and tense
-Sections 2.4, 2.5, and 2.6: Were the cells cultured in plates? Add this information.
A: Thank you for your suggestion. The experimental strains of these three parts were cultured in a triangular flask, and we have added a description of the culture vessel to the manuscript
-Lines 121 and 162: Correct “12000 x g” and “3000 x g”. Furthermore, "g" must be presented in italics. Correct throughout the "Materials and Methods" section (lines 115, 121, 132, 135, 162).
A: We have changed the units of centrifugal force g and significance level p in the text to italics. Thank you
-Line 138: Present the meaning of EDS.
A: The manuscript has added the full name of the abbreviation. Thank you
-Section 2.7: Define the control group and the experimental group.
A: We have added feeding instructions for the control group and experimental group in this section. Thank you
-Line 159: Display information about the automated blood analyzer (model and brand).
A: This section of the manuscript has marked the model and manufacturer of the parameter measurement equipment. Thank you
Results and discussion
In general, there is little discussion of the results. Further comparison with the literature is required.
A: Thank you for your suggestion. We have included research conclusions related to other topics in the corresponding results section of the manuscript and discussed them.
-Present the meaning of DIS and BLAST in the first occurrence in the text.
A: The abbreviation has been annotated in this section of the manuscript. Thank you.
-Lines 190, 208: Bacillus should be written in italics.
A: All parts of the manuscript that involve species names (including references) have been changed to italics. Thank you.
-Lines 193-194: Avoid repeating information already presented in the "Materials and Methods" section. I suggest rewriting this sentence.
A: We have made adjustments and deletions to the content of the materials and methods described in the manuscript results section. Please refer to the revised details of the manuscript for details.
-Section 3.2: Was any design of experiments (DoE) carried out in this part? If yes, present the results of ANOVA, model, and experimental and predicted data from the model. Otherwise, the expression "optimization" should not be used, as it does not result from DoE. Replace with ideal culture conditions. Review the entire manuscript (pay attention to the abstract).
A: In the manuscript, the content of bacterial culture is based on single factor experiments, and the fermentation preparation of nano selenium is based on response surface analysis of three factors and three levels. The experimental descriptions in these two parts have been corrected. Please refer to the revised details of the manuscript for details. Thank you!
-Section 3.3: Why did the authors not present the results in Fig. 3 as a function of the quantity or conversion of SeNPs? If SeNPs have been quantified, present the methodology in the corresponding section. Otherwise, make it clear that the results were presented depending on the number of cells formed since SeNPs are correlated to OD600 nm.
A: Thank you for your reminder. The production of nano selenium here is not only related to the total amount of bacteria, but also to temperature. In the first part, we mainly explore the optimal growth conditions of L11 strain. In the second part, we explore the best nano selenium reduction conditions through three factor and three level response surface analysis.
-Lines 245 and 247: Correct "... range of 20-30 °C..." and "... range of 30-42 °C..."
A: In the manuscript, this section has been revised. Thank you!
-Line 249: Wouldn't the ideal temperature be 30 °C? Please review.
A: In the manuscript, the optimal conditions for bacterial growth are 30-37 degrees, but during the fermentation production of nano selenium, the response surface analysis result shows that the reduction efficiency of sodium selenite at 37 degrees is the highest. Thank you!
-Lines 272-274: As previously suggested, this information should be presented in the "Materials and Methods" section.
A: yes, thanks, We have adjusted the relevant content to the Materials and Methods section. Thank you.
-Line 275: Check whether the ideal temperature would not be 30 °C.
A: Thank you for the reminder. The ideal temperature for fermentation to produce nano selenium is 37 degrees Celsius. Although the reduction rate of nano selenium production is high at 30 degrees Celsius, the bacterial concentration is low, and the total nano selenium production is low.
-Lines 302-303: Imperative verbs. Sentences should not be presented as a guide to protocol but rather as a description of what was accomplished.
A: Thank you for your reminder. The relevant description in the manuscript has been corrected.
-Lines 302-304 and 339-342: Avoid repeating methodology information in the "Results and Discussion" section.
A: Okay, the description of the material method in the Results and Discussion section has been deleted and corrected. Thank you.
-Lines 388-389: Avoid bringing up the meaning of the acronyms again.
A: OK, the duplicates in the manuscript have been corrected.
-Lines 390-393, 409-410, 413-414 and 418-419: Correct p<0.01 and p>0.05 (lowercase p).
A: Thank you for your suggestion. We have corrected the significant level markers P in lowercase and italics throughout the entire text.
Conclusions
-I suggest better highlighting the main scientific contributions of this study.
A: We have removed the wording regarding the application of nano selenium and focused on describing the experimental results and conclusions. We have also corrected some grammar and wording errors in this section.
Figures
-Fig. 1: Improve the quality of images 1a and 1b. Correct the red highlight in the names of microorganisms in 1c. Display the unit of 0.002 in 1c. Indicate the meaning of the numbers (83, 63, 65, 58, 88) in the legend in 1c.
A: We have readjusted the quality of Figure 1, especially Figure 1C, and removed the noisy lines in the figure. The 0.002 scale in Figure 1C represents the relative genetic distance of the evolutionary tree, which does not require units and represents the genetic differences between these substances. We have added relevant descriptions in the Results and Discussion section and the caption of Figure 1. The nodes in the phylogenetic tree in the figure represent values with Bootstrap values greater than 50%, and the superscript "T" represents the mode strain. We have added an explanation in the caption of Figure 1. Thank you!
-Fig. 2: 600 must be submitted signed to the OD and with unit nm. Add the meaning of the letters in the legend and their statistical explanation.
A: We have added OD600nm and its meaning in the image, and also included the statistical significance represented by the letters above the bar chart in the caption. thanks
In 2a: Bacillus subtillis should be written in italics, and it would be interesting to present the OD600 nm data point by point. To what do the authors attribute the better growth of LB compared to the Bacillus universal culture medium? Add a brief discussion with references to support the hypothesis.
A: The species names mentioned here and throughout the text have been changed to italics. Sorry, there was an error in the original manuscript drawing. The blue line in Figure 2A should represent Bacillus culture medium, while the orange line represents LB culture medium. The figure has been corrected. Thank you!
In 2b: Use only "temperature" for the x-axis title.
A: The coordinate title in the image has been modified to temperature
-Fig. 3: 600 must be submitted signed to the OD and with unit nm. Add the meaning of the letters in the legend and their statistical explanation.
A: We have added OD600nm and its meaning in the image, and also included the statistical significance represented by the letters above the bar chart in the caption. Thank you!
In 3a: Use only "temperature" for the x-axis title.
A: The coordinate title in the image has been modified to temperature
-Fig. 5: Present the meaning of the circles and arrows in 5g in the caption.
A: Relevant explanations have been added in the caption. Please refer to the revised content of the manuscript
-Fig. 6: Rewrite the title of the figure, as it seems to be a description of what was accomplished and not what the image represents.
A: We have changed the caption content of Figure 6 to better describe the meaning represented by the image
Tables
Table 3: Display 0.13±0.001 instead of 0.13±0.008.
A: Yes, thank you. It has been corrected here
Comments on the Quality of English Language: The writing also needs to be improved.
A: Thank you for your suggestion. We have made revisions to the entire text, correcting grammar and tense errors. Please refer to the manuscript revision mode for details.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript can now be accepted for publication.
