The Impact of Chicken Manure Biochar on Antibiotic Resistance Genes in Chicken Manure Composting

Round 1

Reviewer 1 Report

General comment:

This article mainly described the biochar effect on antibiotic resistance genes in the chicken manure composting process by adding biochar, heavy metals, and oxytetracycline to solve the problem of drug-resistant gene contamination in the chicken manure composting process. This research was of certain novelty and well designed. But the article has some shortcomings.

 

Detailed comments:

1. The title should be improved, e.g., The impact of chicken manure biochar on antibiotic resistance genes in chicken manure composting.

2. Abstract： It is a bit straightforward for the author to say what technique and treatment he did at the beginning directly. Suggest talking about the purpose of our research briefly at first, then describe the processing.

3. Line 141-142: When analyzing temperature, dissolved organic carbon, and microbial activity, we can see that samples B and BO have big changes in 10 10 20 days respectively. Maybe the author can explore the reasons.

4. Line 33-34, The author's reference in this sentence is not very clear that readers are easily confused about what ARG-carrying microbes are capable of transferring.

5. Line 62-66: The author should illustrate the reason for adding heavy metals, and their amounts. The title should and keywords should also include heavy metal.

6. Line 192: The image of the int1 gene shouldn’t be a slight decrease but a sharp decline.

7. Line 197-198: When discussing tet(X) and tet(A), the author pays more attention to an overall tendency. We can conclude that the sample with biochar has the lowest content of int1, but the sample with heavy metal has a maximum drop of int1.

8. Line 260-261: By using NMDS to analyze the similarities and differences between bacterial communities, the authors believed that there was a certain similarity between B, M, and MO. But firstly, all samples measured at the same time showed a certain similarity. Secondly, by looking at the time of 7, 14, and 28 periods, MO, B showed a certain deviation. Thirdly, the author's focus was to research the effect of adding biochar, heavy metals, and oxytetracycline on the AGR gene, but the analysis of oxytetracycline is not sufficient. Finally, it can be seen from the figure that the similarity of bacterial communities decreases with the increase of time.
The discussion part of this article could be divided into different sections. It not only makes the article more organized but also convenient for readers to read.

Author Response

Response to Reviewer 1 Comments

 

Point 1: The title should be improved, e.g., The impact of chicken manure biochar on antibiotic resistance genes in chicken manure composting.

Response 1: We agree with this comment so the title of article has been changed to the suggested version “The impact of chicken manure biochar on antibiotic resistance genes in chicken manure composting”.

 

 

Point 2: Abstract： It is a bit straightforward for the author to say what technique and treatment he did at the beginning directly. Suggest talking about the purpose of our research briefly at first, then describe the processing.

Response 2: We agree with the comment and corrected the abstract as it was recommended

 

 

Point 3: Line 141-142: When analyzing temperature, dissolved organic carbon, and microbial activity, we can see that samples B and BO have big changes in 10 10 20 days respectively. Maybe the author can explore the reasons.

Response 3: We added the following sentence in the text according this comment : ‘’Since this effect was observed only for the variants containing biochar, it can be assumed that biochar played a role in intensification of microbes enabling exothermic processes of decomposition of organic matter in the composting mixtures’’.

 

Point 4: Line 33-34, The author's reference in this sentence is not very clear that readers are easily confused about what ARG-carrying microbes are capable of transferring.

Response 4: We have corrected the section Introduction according to this comment, as well as the comments of other reviewers

 

 

Point 5: Line 62-66: The author should illustrate the reason for adding heavy metals, and their amounts. The title should and keywords should also include heavy metal.

Response 5: The main goal of our work was to study the effect of biochar on the fate of ARG, and the introduction of heavy metals, like oxytetracycline, in composting mixtures was considered as additional factors. According to this comment, we added an explanation of the reason for the addition of heavy metals to the text and included “heavy metals” in the keywords, and the title of the article was corrected according to the first comment.

 

 

Point 6: Line 192: The image of the int1 gene shouldn’t be a slight decrease but a sharp decline.

Response 6: For a clearer understanding of the dynamics of changes in the content of the int1 gene, we added days 3, 7 and 56 to Figure 3c.

 

Point 7: Line 197-198: When discussing tet(X) and tet(A), the author pays more attention to an overall tendency. We can conclude that the sample with biochar has the lowest content of int1, but the sample with heavy metal has a maximum drop of int1.

Response 7: For a clearer understanding of the dynamics of changes in the content of the int1 gene, we added days 3, 7 and 56 to Figure 3c.

 

 

Point 8: Line 260-261: By using NMDS to analyze the similarities and differences between bacterial communities, the authors believed that there was a certain similarity between B, M, and MO. But firstly, all samples measured at the same time showed a certain similarity. Secondly, by looking at the time of 7, 14, and 28 periods, MO, B showed a certain deviation. Thirdly, the author's focus was to research the effect of adding biochar, heavy metals, and oxytetracycline on the AGR gene, but the analysis of oxytetracycline is not sufficient. Finally, it can be seen from the figure that the similarity of bacterial communities decreases with the increase of time.

Response 8: According to the comment, we have included the following text in the article: ‘’Besides, it can be seen that the similarity of the bacterial community of different composting variants decreased with the increase of time, and that the highest similarity was observed during the thermophilic phase of the composting (days 3 and 7)’’.

 

 

Point 9: The discussion part of this article could be divided into different sections. It not only makes the article more organized but also convenient for readers to read.

Response 9: We agree with the comment and divided the discussion part of the article into sections.

Author Response File: Author Response.docx

Reviewer 2 Report

agriculture-1806087

This is an interesting paper and suitable for Agriculture journal. These issues must be revised.

1) Introduction part: Although it is well written, it seems difficult for the readers who have less knowledge about antibiotics and compost. I strongly recommend adding some more literature review or research papers related to the relationship of compost, antibiotics, crop yield, crop growth, so on. Please see these papers. [ 2020. Soil Organic Carbon in Sandy Paddy Fields of Northeast Thailand: A Review. Agronomy, 10(8), 1061.] [2021. Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments From Conventional and Organic Livestock Farms. Front Vet Sci. 2021; 8: 633858.] [2022. Veterinary antibiotics can reduce crop yields by modifying soil bacterial community and earthworm population in agro-ecosystems. Science of The Total Environment. 808, 152056]

2) Materials and Methods: Some questions must be explained in details for more understanding.

Why “Chelny-Broiler” poultry farm was selected for taking the samples?

How many bedding chicken manure were taken?

How many replication of samples were analyzed?

How many sequence/sample were ran?

3) Figure 1: All texts in the figure must be black color. Also, the meaning of K, B, O, BO, M, BM,MO, BMO should be mentioned at the figure caption.

4) Table 1: Data should be presented in Table as the text, not photo.

5) Figure 5: The texts are too small.

6) Figure 6: It is very difficult to read the text.

7) Table 2: Data should be presented in Table as the text, not photo.

8) Table 3: Data should be presented in Table as the text, not photo.

9) Discussion: It will be more useful if the author (s) try to link compost, crop yield, soil organic carbon and soil fertility.

Author Response

Response to Reviewer 2 Comments

 

Point 1: Introduction part: Although it is well written, it seems difficult for the readers who have less knowledge about antibiotics and compost. I strongly recommend adding some more literature review or research papers related to the relationship of compost, antibiotics, crop yield, crop growth, so on. Please see these papers. [ 2020. Soil Organic Carbon in Sandy Paddy Fields of Northeast Thailand: A Review. Agronomy, 10(8), 1061.] [2021. Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments From Conventional and Organic Livestock Farms. Front Vet Sci. 2021; 8: 633858.] [2022. Veterinary antibiotics can reduce crop yields by modifying soil bacterial community and earthworm population in agro-ecosystems. Science of The Total Environment. 808, 152056]

Response 1: We corrected the Introduction section according to this comment and added more literature review from the suggested papers.

 

 

Point 2: Materials and Methods: Some questions must be explained in details for more understanding.

 

Why “Chelny-Broiler” poultry farm was selected for taking the samples?

 

How many bedding chicken manure were taken?

 

How many replication of samples were analyzed?

 

How many sequence/sample were ran?

Response 2: We have added more details to the Materials and Methods section foo better understanding the design of the experiment according to this comment.

 

 

Point 3: Figure 1: All texts in the figure must be black color. Also, the meaning of K, B, O, BO, M, BM,MO, BMO should be mentioned at the figure caption.

Response 3: We have made changes to the Figure 1 according to the comment.

 

 

Point 4: Table 1: Data should be presented in Table as the text, not photo.

Response 4: According to the comment, we presented the data in the Table 1 as the text.

 

 

Point 5: Figure 5: The texts are too small.

Response 5: According to the comment, the text size in Figure 5 has been increased.

 

 

Point 6: Figure 6: It is very difficult to read the text.

Response 6: According to the comment we have improved the quality of the Figure 6.

 

 

Point 7: Table 2: Data should be presented in Table as the text, not photo.

Response 7: According to the comment, we presented the data in the Table 2 as the text.

 

 

Point 8: Table 3: Data should be presented in Table as the text, not photo.

Response 8: According to the comment, we presented the data in the Table 3 as the text.

 

 

Point 9: Discussion: It will be more useful if the author (s) try to link compost, crop yield, soil organic carbon and soil fertility.

Response 9: According to the comment, we have included some discussion about the connection between compost, crop yield, soil organic carbon and soil fertility in the section.

Author Response File: Author Response.docx

Reviewer 3 Report

 

1. Abstract:

·       The first sentence is incomprehensible, please edit it.

·       I suggest explaining all abbreviations the first time you use them. There is no OTU explanation in the abstract.

·       The abstract should be more precise, detailing the purpose and concept of the research being conducted. The abstract of a good journal article always ends with an outline of the benefits of the results obtained and recommendations as a solution to the presented problem. There is no such information in the presented abstract.

2. Keywords should not repeat phrases contained in the title of the manuscript: „antibiotic resistance genes; biochar; manure; composting”.
3. Introduction: The chapter is well designed. The purpose of the research was precisely presented.
4. Materials and Methods: The research was designed and carried out properly. Well-designed chapter.
5. The results of the conducted research have been prepared very carefully. All figures and tables are legible and carefully interpreted. The discussion of the obtained results was carried out very diligently, and the authors considered the results of the research very carefully to the information contained in the available bibliography. I have no objections.
6. Conclusions need to be improved. They must be convincing statements about what was found to be innovative, contributing to a strong support of the results and discussions. They should highlight the main results of the work.

 

Author Response

Response to Reviewer 3 Comments

 

Point 1: Abstract:

• The first sentence is incomprehensible, please edit it.
• I suggest explaining all abbreviations the first time you use them. There is no OTU explanation in the abstract.
• The abstract should be more precise, detailing the purpose and concept of the research being conducted. The abstract of a good journal article always ends with an outline of the benefits of the results obtained and recommendations as a solution to the presented problem. There is no such information in the presented abstract.

Response 1: We added OTU explantion in the abstract and the abstract was corrected according the comment and comments by other reviewers.

 

 

Point 2: Keywords should not repeat phrases contained in the title of the manuscript: „antibiotic resistance genes; biochar; manure; composting”.

Response 2: We have corrected Keywords as follows: “oxytetracycline; heavy metals; respiration activity; horizontal transfer; microbial community”.

 

 

Point 3: Introduction: The chapter is well designed. The purpose of the research was precisely presented.

 

Point 4: Materials and Methods: The research was designed and carried out properly. Well-designed chapter.

 

Point 5: The results of the conducted research have been prepared very carefully. All figures and tables are legible and carefully interpreted. The discussion of the obtained results was carried out very diligently, and the authors considered the results of the research very carefully to the information contained in the available bibliography. I have no objections.

 

 

Point 6: Conclusions need to be improved. They must be convincing statements about what was found to be innovative, contributing to a strong support of the results and discussions. They should highlight the main results of the work.

Response 6: We have corrected Conclusions according the comment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have well addressed the previous issues, before publication the following questions should be further carefully solved.

Detailed comments:

1. Line19-21: “The bacterial communities in the composting mixtures had a similar structure, and the shifts were mainly determined by the duration of the process.” The sentence seems to be contradictory.

2. Line44-45: Any reference for the sentence?

3. Line51-53: Any reference for the sentence?

4. “Rare reports have even demonstrated the enrichment of compost by ARG [12,13].”. Please make the sentence clear.

5. Line 78-82: There is no reference for the increase or decrease of the ARG copy numbers.

6. Line 97: “its” should be “the”.

7. Line 100: “Moreover” should be “Previously”.

8. Line 101-104: Any reference for the properties of chicken manure biochar?

9. Line 110: Are “genes tet(A) and tet(X)” representative antibiotic genes?

10. Line 133: What tools are used to stir compost.

11. Line 188: Please add relevant references.

12. Line 217-221: Please add relevant references.

13. Line 227: “the character of its change was different” is vague.

14. In Figures 2 and 3, the images are not the same size.

15. Line 267: “and” should be “, and”

16. Line 272: Please add relevant references.

17. Line 410: “however, in some way they affected the level of respiration activity” should be “in addition, they affect the level of respiratory activity to some extent”.

18. Line 462: Please add relevant references.

Author Response

1. Line19-21: “The bacterial communities in the composting mixtures had a similar structure, and the shifts were mainly determined by the duration of the process.” The sentence seems to be contradictory.

Response: according to the comment, we have corrected this sentence as follows: The bacterial communities in different composting mixtures had a similar structure and dynamics. It was revealed that the shifts of the bacterial composition of the composting mixtures were mainly determined by the duration of the process.

 

 

2. Line44-45: Any reference for the sentence?

Response: according to the comment, we added the relevant reference to the text.

 

 

3. Line51-53: Any reference for the sentence?

Response: according to the comment, we added the relevant reference to the text.

 

 

4. “Rare reports have even demonstrated the enrichment of compost by ARG [12,13].”. Please make the sentence clear.

Response: according to the comment, we have corrected this sentence as follows: Rare reports have even demonstrated the increase of ARG copy numbers in the process of composting.

 

 

5. Line 78-82: There is no reference for the increase or decrease of the ARG copy numbers.

Response: according to the comment, we added the relevant reference to the text.

 

 

6. Line 97: “its” should be “the”.

Response: according to the comment, we have corrected this sentence as follows: However, the mechanisms of biochar influence on the compost bacterial community, including the ARG carriers, are not fully clear yet.

 

 

7. Line 100: “Moreover” should be “Previously”.

Response: according to the comment, we have corrected this sentence as follows: Previously, the best results are obtained for biochar made of plant biomass such as straw or wood.

 

 

8. Line 101-104: Any reference for the properties of chicken manure biochar?

Response: according to the comment, we added the relevant reference to the text.

 

 

9. Line 110: Are “genes tet(A) and tet(X)” representative antibiotic genes?

Response: according to the comment, we added the following few sentences to the text to explain why we chose these genes:

“There are several mechanisms by which microorganisms have developed resistance to antibiotics. Those mechanisms can be encoded by one or several specific genes located in chromosomes or more often in plasmids [9]. In case of tetracyclines that are the most widespread antibiotics in poultry husbandry, about 40 different genes encoding resistance have been revealed. For example, genes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J) and tet(Z) encode active antibiotic efflux. Genes tet(M), tet(O), tet(S), tet(W), tet(Q) and tet(T) encode ribosome protection and tet(X) gene encodes antibiotic detoxification [10]. Bacteria may possess several ARG encoding different ways to resist one and the same antibiotic compound, or to resist different antibiotics [11]” (Lines 62-70).

 

“The genes tet(A) and tet(X) encode two different mechanisms of resistance of bacteria to tetracyclines, which are the most widespread group of antibiotics in poultry husbandry” (Lines 111-113).

 

Lines 217-221 also contain a small explanation of why we chose genes tet(A) and tet(X): “About 40 such genes are known from the literature; however, we have chosen two of them for a number of reasons: firstly, they are responsible for two different mechanisms of anti-biotic resistance; secondly, they are among the most frequently analyzed by researchers; and thirdly, as a result of our previous research (data not shown), the results obtained for these two genes were the most reproducible”

 

 

 

10. Line 133: What tools are used to stir compost.

Response: We conducted manure composting process using rotary drum composters. Stirring of compost mixtures was carried out mechanically using the electric motor, which was located on each composter. For clarity, we have added several photos of our composters to the Supplementary material.

Supplement Figure S7. Rotary drum composters used for chicken manure composting.

 

11. Line 188: Please add relevant references.

Response: According to the comment, we added the relevant reference to the text.

 

 

12. Line 217-221: Please add relevant references.

Response: According to the comment, we added the relevant reference to the text.

 

 

13. Line 227: “the character of its change was different” is vague.

Response: Аaccording to the comment, we have corrected this sentence as follows: “the shape of the curve reflecting the gene copy numbers was different”.

 

 

14. In Figures 2 and 3, the images are not the same size.

Response: We corrected the size of images in Figures 2 and 3 according the comment.

 

 

15. Line 267: “and” should be “, and”

Response: We corrected our mistake in the text according the comment.

 

 

16. Line 272: Please add relevant references.

Response: According to the comment, we added the relevant reference to the text.

 

 

17. Line 410: “however, in some way they affected the level of respiration activity” should be “in addition, they affect the level of respiratory activity to some extent”.

Response: According to the comment, we corrected this sentence in the text.

 

 

18. Line 462: Please add relevant references.

Response: According to the comment, we added the relevant reference to the text.

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Accept in present form!

Author Response

Please see the attachment of revised version according to Reviewer 1 comments

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.