Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil
, Jin-Young Lee 1,†, Heejung Kim 3,*, Jae-E Yang 4,*, Hyunji Lee 1,5, Jaewon Jung 1,6 and Ha-Rang Kim 1,7
Round 1
Reviewer 1 Report
Authors claim to have monitored nitrogen cycling by studying microbial genes involved in the different processes.
Major concerns:
1. There is no next-generation sequencing data in the manuscript as claimed in the abstract.
2. Out of the 14 primer pairs selected in Table 3; 7 still have non-specific bands. qPCR provided better detection.
3. The overall writing needs improvement. References have not been used properly. Having 40 references for a general sentence is not the way. If you are determined not to add an experiment like qPCR, I would still suggest a rewrite and consider your results carefully before making any conclusion.
Other comments are attached.
Comments for author File: 
Comments.pdf
Author Response
[Oct. 03, 2022]
Prof. Dr. Takayoshi Kobayashi
Editor-in-Chief
Applied Sciences
Dear Prof. Dr. Takayoshi Kobayashi:
We wish to re-submit the manuscript titled “Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil”. The manuscript ID is applsci-1877754-R1.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We would like to thank you for giving us the opportunity to submit our manuscript with major revisions and the 5 reviewers for their helpful suggestions and comments. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. The changes in the revised manuscript are given in red color.
Thank you again for your decision.
Sincerely,
Corresponding authors Prof. Heejung Kim and Prof. Jay E Yang
Department of Geology, College of Natural Sciences
Kangwon National University, Chuncheon 24341, Republic of Korea
Tel.: +82-33-250-8560
Fax.: +82-33-259-8559
[email protected]
Reviewer #1: Anonymous
Authors claim to have monitored nitrogen cycling by studying microbial genes involved in the different processes.
RESPONSE: AGREEMENT AND EXPLANATION
We would like to thank the reviewer for helping us to improve our manuscript. Many studies on microbial nitrogen cycling in the environment have already been reported. These are difficult to culture, so non-cultured based methods are used to study the diversity of many related genes such as amo, nosZ, nirK, etc. However, various PCR methods have been developed by many researchers to amplify the genes in each gene related to nitrogen cycling. Therefore, some of the problems below may be raised.
1) First of all, the reported nitrogen cycle-related PCR methods are mostly used for research such as non-cultured based diversity, not for the development of excellent diagnostic methods. Therefore, comparative studies between reported PCR methods are insufficient.
2) When you want to identify microorganisms based on the nucleotide sequence, it is theoretically advantageous when you have a long nucleotide sequence. Generally, 400-800 base pair (bp) are suitable, and comparisons should be made with a length of at least 300 bp. This is because most microorganisms have not been reported yet, and they are identified as having the highest similarity to the corresponding nucleotide sequence among the microbial data pools registered on the server. Meanwhile, NGS is used in recent research on non-cultured based diversity of microorganisms, and various platforms are used considering the number of samples, the length of the amplified sequence of the target microorganism, and the number of reads to be analyzed. Therefore, these factors must be considered, so a length of about 400 bp suitable for diversity studies is widely used. However, many studies do not reflect these aspects, such as amplifying short sequences.
3) The PCR primer set is greatly affected by the inhibitor contained in the nucleic acid extracted from the sample. Therefore, suitable PCR primer sets with excellent applicability may exist depending on sample types such as soil and water. Also, the specific primer set used for PCR amplification may not amplify all related microbial genes.
We conducted this study to compare the optimal PCR methods for studying microorganisms related to nitrogen cycling in soil by referring to the above problems and to examine their applicability in soil. Meanwhile, in response to the reviewer's comments, we tried to improve the reader's understanding by modifying the response and manuscript as follows.
1) There is no next-generation sequencing data in the manuscript as claimed in the abstract.
RESPONSE: EXPLANATION AND CHANGES MADE
This study was intended to compare PCR methods for each gene that are suitable for follow-up studies on nitrogen cycle in soil (gene diversity studies using methods such as NGS). We have revised the text in the manuscript accordingly to help readers understand better.
2) Out of the 14 primer pairs selected in Table 3; 7 still have non-specific bands. qPCR provided better detection.
RESPONSE: EXPLANATION AND CHANGES MADE
When a specific nucleic acid is amplified by PCR from total DNA extracted from environmental sample types, unknown non-specific amplification may appear. Quantitative PCR has the advantage of rapidly monitoring short fragments, but it may be difficult to assay for relatively non-specific amplification. On the other hand, in conventional PCR, non-specific amplification of different sizes is visually confirmed. This does not affect gene diversity research as it is possible to remove non-specific bands and recover target bands using gel-purification. This content was also added to the manuscript.
3) The overall writing needs improvement. References have not been used properly. Having 40 references for a general sentence is not the way. If you are determined not to add an experiment like qPCR, I would still suggest a rewrite and consider your results carefully before making any conclusion.
RESPONSE: AGREEMENT AND CHANGES MADE
English proofreading was performed again, and the manuscript was revised as a whole. We have reduced the number of references in that section as well. In addition, we have revised the manuscript as suggested and carefully considered the results and conclusions.
4) Other comments are attached.
RESPONSE: AGREEMENT AND CHANGES MADE
Other comments in the PDF file have been addressed.
Author Response File: Author Response.pdf
Reviewer 2 Report
The authors should describe the climate conditions more extensively in the research area, because this aspect influence the content of nitrogen in soils and also influence the microorganisms activity.
The authors do not present the level of soil supply with total nitrogen, a parameter that influences the results obtained, especially since this study also mentiones the relationship between carbon and nitrogen in the soil.
Author Response
[Oct. 03, 2022]
Prof. Dr. Takayoshi Kobayashi
Editor-in-Chief
Applied Sciences
Dear Prof. Dr. Takayoshi Kobayashi:
We wish to re-submit the manuscript entitled “Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil” The manuscript ID is applsci-1877754-R1.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We would like to thank you for giving us the opportunity to submit our manuscript with major revisions and the 5 reviewers for their helpful suggestions and comments. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. The changes in the revised manuscript are given in red color.
Thank you again for your decision.
Sincerely,
Corresponding authors Prof. Heejung Kim and Prof. Jay E Yang
Department of Geology, College of Natural Sciences
Kangwon National University, Chuncheon 24341, Republic of Korea
Tel.: +82-33-250-8560
Fax.: +82-33-259-8559
[email protected]
Reviewer #2: Anonymous
1) The authors should describe the climate conditions more extensively in the research area, because this aspect influence the content of nitrogen in soils and also influence the microorganisms activity.
RESPONSE: AGREEMENT AND CHANGES MADE
We would like to thank the reviewer for helping us improve our manuscript. The climatic conditions of the sampling area were further described in the discussion section.
2) The authors do not present the level of soil supply with total nitrogen, a parameter that influences the results obtained, especially since this study also mentiones the relationship between carbon and nitrogen in the soil.
RESPONSE: AGREEMENT AND CHANGES MADE
The mentioned section was deleted and the description of the manuscript was comprehensively revised.
Author Response File: Author Response.pdf
Reviewer 3 Report
Major comments:
This study compared the performance of several PCR primer sets in related to microbial nitrogen cycling. Besides, seasonal detection of the nitrogen cycle-related microorganism genes was performed with soil samples from different land-use types in temperate climate regions. The results of this study can provide a reference for choosing PCR primer sets for detecting some nitrogen cycle-related genes. However, the manuscript did not mention the limitation of the methodology. For example, since the soil microbe community is naturally unevenly distributed, and only a tiny amount of soil was selected for soil DNA extraction, increasing the sampling volume or making more sample replicates might help reduce detection errors. The necessity of the study could be stated more clearly.
I will propose that the manuscript could be accepted to be published as a research paper after a language check by a native English speaker and after thorough revision.
Specific comments:
Title
Unnecessary hyphen mark between the word “Surface”.
Abstract
The abstract should include and summarise the essential parts of the paper.
Line 35: “grasslands are crucial for ensuring the continual recurrence of the nitrogen cycle in soil.” the conclusion can not made by such a small sample size. Please draw conclusion carefully.
Introduction
There is only one paragraph for the whole introduction part, and please consider splitting this long paragraph.
This part offered background information on this study to the readers. However, the linkage between background information and the study aims is not close enough. The provided information should lead to the study aim.
Line 86-91: Possibly, you may also find some studies regarding the nitrogen cycle research on surface soil in temperate climate zone, e.g. European region, which could be worth checking and adding as a reference
Line 95-97: This sentence is not easy to follow, please consider re-structure the sentence and check the grammar.
Figure 1
Please consider noting down the full name of the functional genes and the abbreviations in the description part of the figure.
Materials and Methods:
Line 111-113: Please consider re-structure this sentence.
Line 116: Sulface-> Surface
Line 119-120: did you collected totally 25 samples or 125 samples? are the 25 samples for one season or for total five seasons?
Line 121-122: Please consider adding some summarized sentences of the sampling method in the text, which is helpful to give the reader an overview of the representative of the soil samples.
Line 147: “The PCR analysis was performed in 20 μL reaction volume: …..”
Line 303: whats the meaning of “R” in the table?
Results
Please consider replacing the unit nucleotides (nt) with base pairs (bp) since the latter is more commonly used.
Discussion: The discussion was poorly written, the study aim that was subjected in the introduction should be clearly, thoroughly answered, and discussed in the discussion part.
Lines 315-318: please consider re-structure this sentence.
Line 319: please consider re-structure this sentence
Lines 327-330: please consider splitting the sentence.
Line 333:” The nitrogen cycle is more intimately connected…” more initimately connected than what? there should be a subject here
Line335-338: do you mean that nitrogen cycle is more important than carbon cycle? the fact that„approximately 95% of nitrogen in soil existing as SOM” is not enough to support this conclusion.
Author Response
[Oct. 03, 2022]
Prof. Dr. Takayoshi Kobayashi
Editor-in-Chief
Applied Sciences
Dear Prof. Dr. Takayoshi Kobayashi:
We wish to re-submit the manuscript titled “Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil”. The manuscript ID is applsci-1877754-R1.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We would like to thank you for giving us the opportunity to submit our manuscript with major revisions and the 5 reviewers for their helpful suggestions and comments. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. The changes in the revised manuscript are given in red color.
Thank you again for your decision.
Sincerely,
Corresponding authors Prof. Heejung Kim and Prof. Jay E Yang
Department of Geology, College of Natural Sciences
Kangwon National University, Chuncheon 24341, Republic of Korea
Tel.: +82-33-250-8560
Fax.: +82-33-259-8559
[email protected]
Reviewer #3: Anonymous
Major comments
1) This study compared the performance of several PCR primer sets in related to microbial nitrogen cycling. Besides, seasonal detection of the nitrogen cycle-related microorganism genes was performed with soil samples from different land-use types in temperate climate regions. The results of this study can provide a reference for choosing PCR primer sets for detecting some nitrogen cycle-related genes. However, the manuscript did not mention the limitation of the methodology. For example, since the soil microbe community is naturally unevenly distributed, and only a tiny amount of soil was selected for soil DNA extraction, increasing the sampling volume or making more sample replicates might help reduce detection errors. The necessity of the study could be stated more clearly.
RESPONSE: AGREEMENT AND CHANGES MADE
We would like to thank the reviewer for helping us improve our manuscript. The reviewer's comments were reflected in the manuscript, and in particular, the manuscript was revised so that the need for research could be clearly understood.
2) I will propose that the manuscript could be accepted to be published as a research paper after a language check by a native English speaker and after thorough revision.
RESPONSE: AGREE AND CHANGES MADE
The manuscript was edited with the help of a native English speaker.
Specific comments
[Title]
3) Unnecessary hyphen mark between the word “Surface”.
RESPONSE: AGREEMENT AND CHANGES MADE
We corrected the manuscript according to the reviewer’s comment.
[Abstract]
4) The abstract should include and summarise the essential parts of the paper.
RESPONSE: AGREEMENT AND CHANGES MADE
The abstract has been revised, such as summarizing it as a key part of the paper.
5) Line 35: “grasslands are crucial for ensuring the continual recurrence of the nitrogen cycle in soil.” the conclusion can not made by such a small sample size. Please draw conclusion carefully.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
[Introduction]
6) There is only one paragraph for the whole introduction part, and please consider splitting this long paragraph.
RESPONSE: AGREEMENT AND CHANGES MADE
The Introduction section has been completely revised.
7) This part offered background information on this study to the readers. However, the linkage between background information and the study aims is not close enough. The provided information should lead to the study aim.
RESPONSE: AGREEMENT AND CHANGES MADE
The background information of the study was revised in consideration of the research purpose and relevance.
8) Line 86-91: Possibly, you may also find some studies regarding the nitrogen cycle research on surface soil in temperate climate zone, e.g. European region, which could be worth checking and adding as a reference
RESPONSE: AGREEMENT AND CHANGES MADE
In the discussion part, we included a study on the nitrogen cycle in Korea, which is the same as the study according to soil land use types.
9) Line 95-97: This sentence is not easy to follow, please consider re-structure the sentence and check the grammar.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
10) Figure 1: Please consider noting down the full name of the functional genes and the abbreviations in the description part of the figure.
RESPONSE: AGREEMENT AND CHANGES MADE
Please see the added explanation in figure 1.
[Materials and Methods]
11) Line 111-113: Please consider re-structure this sentence.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
12) Line 116: Sulface-> Surface
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
13) Line 119-120: did you collected totally 25 samples or 125 samples? are the 25 samples for one season or for total five seasons?
RESPONSE: AGREEMENT AND CHANGES MADE
The total is 25 samples. We have clarified the content to make it easier for readers to understand.
14) Line 121-122: Please consider adding some summarized sentences of the sampling method in the text, which is helpful to give the reader an overview of the representative of the soil samples.
RESPONSE: AGREEMENT AND CHANGES MADE
’For clarification, we have added sentences related to sampling.
15) Line 147: “The PCR analysis was performed in 20 μL reaction volume: …..”
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
16) Line 303: whats the meaning of “R” in the table?
RESPONSE: AGREEMENT AND CHANGES MADE
The caption for Table 3 has been ’revised accordingly.
[Results]
17) Please consider replacing the unit nucleotides (nt) with base pairs (bp) since the latter is more commonly used.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript accordingly.
[Discussion]
18) The discussion was poorly written, the study aim that was subjected in the introduction should be clearly, thoroughly answered, and discussed in the discussion part.
RESPONSE: AGREEMENT AND CHANGES MADE
The discussion part has been completely revised.
19) Lines 315-318: please consider re-structure this sentence.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript according‘ly.
20) Line 319: please consider re-structure this sentence
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript according‘ly.
21) Lines 327-330: please consider splitting the sentence.
RESPONSE: AGREEMENT AND CHANGES MADE
We have revised the manuscript according‘ly.
22) Line 333:” The nitrogen cycle is more intimately connected…” more initimately connected than what? there should be a subject here
RESPONSE: AGREEMENT AND CHANGES MADE
This section has been appropriately revised.
23) Line335-338: do you mean that nitrogen cycle is more important than carbon cycle? the fact that„approximately 95% of nitrogen in soil existing as SOM” is not enough to support this conclusion.
RESPONSE: AGREEMENT AND CHANGES MADE
The content of the manuscript has been deleted.
Author Response File: Author Response.pdf
Reviewer 4 Report
Siwon Lee and colleagues are are submitting a manuscript on accessing the complete nitrogen cycle with a set of PCR primers each covering individual steps of the N cycle. The novelty seems mostly the approach to cover the whole cycle, as previous work exists on individual or several steps. While this is in principle an interesting study, there are serious methodological flaws with the testing of the PCR primers, which also render the otherwise sound field application part uninterpretable. One of my main points of critique is that the six "positive control" strains are all denitrifying bacteria. Therefore they cannot be used as positive control for genes which are restricted to e.g. nitrifiers or anammox, yet this was done here. Thus it is not surprising that no positive control was achieved for several such genes, such as amoC, hao, hzo and nxrB. Furthermore, there is no indication on whether PCR products were sequenced to actually prove target-specific amplification. I suspect that several primers are not target-specific and amplified other products from the six "positive control" strains. This would explain why some genes such as e.g. amoA and amoB that should not be present in the six control strains still yielded amplicons. As only primers taken from previous works were tested, this might perhaps be of interest to the respective authors and others trying to use and perhaps improve their primer sets. Due to the mentioned methodological flaws I cannot recommend publication of this manuscript in Applied Science.
Author Response
[Oct. 03, 2022]
Prof. Dr. Takayoshi Kobayashi
Editor-in-Chief
Applied Sciences
Dear Prof. Dr. Takayoshi Kobayashi:
We wish to re-submit the manuscript titled “Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil” The manuscript ID is applsci-1877754-R1.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We would like to thank you for giving us the opportunity to submit our manuscript with major revisions and the 5 reviewers for their helpful suggestions and comments. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. The changes in the revised manuscript are given in red color.
Thank you for your decision again.
Sincerely,
Corresponding authors Prof. Heejung Kim and Prof. Jay E Yang
Department of Geology, College of Natural Sciences
Kangwon National University, Chuncheon 24341, Republic of Korea
Tel.: +82-33-250-8560
Fax.: +82-33-259-8559
[email protected]
Reviewer #4: Anonymous
Major comments
Siwon Lee and colleagues are are submitting a manuscript on accessing the complete nitrogen cycle with a set of PCR primers each covering individual steps of the N cycle. The novelty seems mostly the approach to cover the whole cycle, as previous work exists on individual or several steps. While this is in principle an interesting study, there are serious methodological flaws with the testing of the PCR primers, which also render the otherwise sound field application part uninterpretable. One of my main points of critique is that the six "positive control" strains are all denitrifying bacteria. Therefore they cannot be used as positive control for genes which are restricted to e.g. nitrifiers or anammox, yet this was done here. Thus it is not surprising that no positive control was achieved for several such genes, such as amoC, hao, hzo and nxrB. Furthermore, there is no indication on whether PCR products were sequenced to actually prove target-specific amplification. I suspect that several primers are not target-specific and amplified other products from the six "positive control" strains. This would explain why some genes such as e.g. amoA and amoB that should not be present in the six control strains still yielded amplicons. As only primers taken from previous works were tested, this might perhaps be of interest to the respective authors and others trying to use and perhaps improve their primer sets. Due to the mentioned methodological flaws I cannot recommend publication of this manuscript in Applied Science.
RESPONSE: EXPLAINS AND CHANGES MADE
We would like to thank the reviewer for helping us improve our manuscript. Many studies on microbial nitrogen cycling in the environment have already been reported. These are difficult to culture, so non-cultured based methods are used to study the diversity of many related genes such as amo, nosZ, nirK, and etc. However, various PCR methods have been developed by many researchers to amplify the genes in each gene related to nitrogen cycling. Therefore, some of the problems below may be raised.
1) First of all, the reported nitrogen cycle-related PCR methods are mostly used for research such as non-cultured based diversity, not for the development of excellent diagnostic methods. Therefore, comparative studies between reported PCR methods are insufficient.
2) When you want to identify microorganisms based on the nucleotide sequence, it is theoretically advantageous when you have a long nucleotide sequence. Generally, 400-800 base pair (bp) are suitable, and comparisons should be made with a length of at least 300 bp. This is because most microorganisms have not been reported yet, and they are identified as having the highest similarity to the corresponding nucleotide sequence among the microbial data pools registered on the server. Meanwhile, NGS is used in recent research on non-cultured based diversity of microorganisms, and various platforms are used considering the number of samples, the length of the amplified sequence of the target microorganism, and the number of reads to be analyzed. Therefore, these factors must be considered, so a length of about 400 bp suitable for diversity studies is widely used. However, many studies do not reflect these aspects, such as amplifying short sequences.
3) The PCR primer set is greatly affected by the inhibitor contained in the nucleic acid extracted from the sample. Therefore, suitable PCR primer sets with excellent applicability may exist depending on sample types such as soil and water. Moreover, the specific primer set used for PCR amplification may not amplify all related microbial genes.
In related research, comparative experiments with PCR methods in previous reports were found to be sufficient. We conducted this study to compare the optimal PCR methods for studying microorganisms related to nitrogen cycling in soil by referring to the above problems and examining their applicability in soil. Meanwhile, in response to the reviewer's comments, we tried to improve the ’paper’s clarity by modifying the manuscript as follows. In particular, only the genes related to the denitrification process dealt with denitrification bacteria, and the manuscript was changed to secure positives in the samples for other genes. Also, the monitoring part was moved to the discussion section and the contents were reviewed. However, sequencing for proof of amplicon was excluded. A mix peak is expected by Sanger sequencing. A short revision period was given for NGS. Most importantly,the purpose of this study is to suggest highly applicable PCR methods for subsequent studies related to the nitrogen cycle in the soil. Accordingly, we request a re-examination of the revised manuscript.
Author Response File: Author Response.pdf
Reviewer 5 Report
Interesting topic for an article, but in a very narrow discipline.
Abstract written correctly, introduction sufficient. But I have comments to present the research results. The author should once again consider whether in this chapter to introduce a division into such detailed sections describing individual research results. Maybe it's better to do one chapter. Another issue is discussion. In my opinion, it is too short, as is the detailed and comprehensive research results, it should be expanded. The division of conclusions is missing - is it a deliberate omission by the author or does it not present any conclusions?
Author Response
[Oct. 03, 2022]
Prof. Dr. Takayoshi Kobayashi
Editor-in-Chief
Applied Sciences
Dear Prof. Dr. Takayoshi Kobayashi:
We wish to re-submit the manuscript titled “Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil” The manuscript ID is applsci-1877754-R1.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We would like to thank you for giving us the opportunity to submit our manuscript with major revisions and the 5 reviewers for their helpful suggestions and comments. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. The changes in the revised manuscript are given in red color.
Thank you again for your decision.
Sincerely,
Corresponding authors Prof. Heejung Kim and Prof. Jay E Yang
Department of Geology, College of Natural Sciences
Kangwon National University, Chuncheon 24341, Republic of Korea
Tel.: +82-33-250-8560
Fax.: +82-33-259-8559
[email protected]
Reviewer #5: Anonymous
Interesting topic for an article, but in a very narrow discipline. Abstract written correctly, introduction sufficient. But I have comments to present the research results. The author should once again consider whether in this chapter to introduce a division into such detailed sections describing individual research results. Maybe it's better to do one chapter. Another issue is discussion. In my opinion, it is too short, as is the detailed and comprehensive research results, it should be expanded. The division of conclusions is missing - is it a deliberate omission by the author or does it not present any conclusions?
RESPONSE: AGREEMENT AND CHANGES MADE
We would like to thank the reviewer for helping us improve our manuscript. The manuscript content has been revised accordingly. The results that were organized into detailed sections have been modified as one chapter. The review was revised according to the reviewer's comments, and a conclusion was added.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Minor comments:
1. I wonder why you removed the field microbe test part from your abstract. That is important part of your result. Please include it in abstract, it will likely increase the reading of your manuscript too.
2. The second paragraph of discussion is very long. Maybe you can have separate paragraph for each season.
Author Response
Reviewer #1: Anonymous
Minor comments:
- I wonder why you removed the field microbe test part from your abstract. That is important part of your result. Please include it in abstract, it will likely increase the reading of your manuscript too.
RESPONSE: AGREE AND CHANGES MADE
We would like to thank the reviewer for helping us to improve our manuscript. We corrected the manuscript according to the reviewer’s comment.
- The second paragraph of discussion is very long. Maybe you can have separate paragraph for each season.
RESPONSE: AGREE AND CHANGES MADE
The second paragraph of discussion has been properly separated.
Author Response File: 
Author Response.doc
Reviewer 4 Report
I am reviewing this manuscript now for the second time, and there is some improvement. The positive control strains, which all are denitrifiers, are now only used for denitrification genes. I am still missing some information on how the authors are certain that the amplicons are the genes they actually designed the primers for. I am aware that sequencing of the qPCR amplicons is not suitable for species identification, but gene identification should be possible. But, as the amplicon size seems to yielt one major produt that also corresponds in size among samples (nirS set #3 being an exception), I am willing to accept that, keeping also the interesting field study in mind. I would still like to suggest some additional english editing, in particular for the newly added paragraphs.
Author Response
Reviewer #4: Anonymous
I am reviewing this manuscript now for the second time, and there is some improvement. The positive control strains, which all are denitrifiers, are now only used for denitrification genes. I am still missing some information on how the authors are certain that the amplicons are the genes they actually designed the primers for. I am aware that sequencing of the qPCR amplicons is not suitable for species identification, but gene identification should be possible. But, as the amplicon size seems to yielt one major produt that also corresponds in size among samples (nirS set #3 being an exception), I am willing to accept that, keeping also the interesting field study in mind. I would still like to suggest some additional english editing, in particular for the newly added paragraphs.
RESPONSE: EXPLANATION
We would like to thank the reviewer for helping us improve our manuscript. We reflected the reviewer's 1st comment and revised possible parts within the revision period (e.g., use of appropriate positive controls for each gene, changing the direction of the manuscript by reviewing methods rather than monitoring and analysis, etc.).
However, sequencing for the identification of PCR amplicons was excluded. The reasons are as follows: 1) The total DNA used as a positive control is expected to have a mix peak during Sanger’s sequencing. 2) A separate research study is required to review NGS, which is outside the scope of this study, because the purpose of this study is to propose a PCR method that is highly applicable to subsequent studies related to soil nitrogen cycle. 3) In the case of the denitrification strain used as a positive control, it is a strain tested from an authorized culture collection. In addition, the PCR methods used in this study were not developed in this study, but various strains and samples for amplicon in each previous report were sufficiently reviewed to amplify the target gene. Additional English editing including the revised paragraphs has been carried out, and a certificate of English editing is attached below this revised note.
Author Response File: Author Response.doc
