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Article
Peer-Review Record

Effect of the Aerobic Denitrifying Bacterium Pseudomonas furukawaii ZS1 on Microbiota Compositions in Grass Carp Culture Water

Water 2021, 13(10), 1329; https://doi.org/10.3390/w13101329
by 1,2, 1, 1, 1,2, 1,2, 1,2, 1,2, 1,2, 1,2, 1,2,* and 3,4,*
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Water 2021, 13(10), 1329; https://doi.org/10.3390/w13101329
Received: 8 April 2021 / Revised: 1 May 2021 / Accepted: 7 May 2021 / Published: 11 May 2021

Round 1

Reviewer 1 Report

The paper entitled "Effect of the aerobic denitrifying bacterium Pseudomonas furukawaii ZS1 on microbiota compositions in grass carp culture water" reveals some information that may be interesting to readers in the fields of agriculture, water and environmental sciences. The manuscript is well-written and is easy to understand, while still have many points that need to be addressed. Don’t use the notion like ‘we’ or ‘our’ etc., as these are the redundant words (not the research words) for the standard journal manuscripts. Also check and correct grammatical and space errors throughout the article.

 

# An abstract should have the following in this order: Introductory/rationale statement that leads to an objective statement (should start like this "Therefore, the objective (s) was...."), then a description of the experimental design/treatments measurements and ending with the largest amount of the abstract text as results/major conclusions. The description of the experimental design/treatments measurements is Ok, but the authors need to improve the other points indicated. In the conclusion section, authors should emphasize the novelty of the research rather than summarizing findings. Please rewrite it.

# The change in microbial composition analysis via high-throughput sequencing should be written in brief in the abstract section.

# Justify the novelty in introduction and discussion, as well.

# The introduction does not provide sufficient background and does not include all relevant references. It needs the addition of some recent new research progress in this field. Present a hypothesis in introduction. Justify, how does your research contribute to the microbiota compositions in grass carp culture? Also add some points related to the significance of aerobic denitrifying bacterium P. furukawaii ZS1.

# The introduction of the paper must be extended and reformulated in order to provide a more comprehensive approach.

# In materials and method section, any background information why authors used aerobic denitrifying bacterium P. furukawaii ZS1bacterial strain? Need to mention the reason for choosing.

# Line no 70; What do you mean by “during which gas was continuously imported”??

# Line no 72; Assessment of water quality should be described properly with references.

# Give the details of DNA extraction from the sample. Exactly how much sample was taken for DNA extraction?

# For DNA extraction, Was it dried sample or fresh sample?

# Results and discussion: Provide a better explanation for your data. Interpret and discuss the meaning of your results more deeply.  Discussions need to be supported by the latest references and need to be explained in depth. The authors should highlight the reason of their result findings in the light of available literature.

# Authors are suggested to add discussion by explaining trends in the obtained results along with the possible mechanisms behind the trends.

# It is strongly recommended to add a subsection, 'practical implications of this study,' outlining the challenges in the current research, future work, and recommendations, before the conclusion.

# Conclusion: Conclusions is not just about summarizing the key results of the study, it should highlight the insights and the applicability of your findings/results for further work.  Please enrich your conclusion section.

Author Response

Responses to the Comments and Suggestions of the Reviewer #1

Reviewer #1 comment

The paper entitled "Effect of the aerobic denitrifying bacterium Pseudomonas furukawaii ZS1 on microbiota compositions in grass carp culture water" reveals some information that may be interesting to readers in the fields of agriculture, water and environmental sciences. The manuscript is well-written and is easy to understand, while still have many points that need to be addressed. Don’t use the notion like ‘we’ or ‘our’ etc., as these are the redundant words (not the research words) for the standard journal manuscripts. Also check and correct grammatical and space errors throughout the article.

Response

Thank you very much for your comments. The comments are valuable for us to improve our manuscript. We have revised our manuscript according to your and another reviewer’s comments. The have deleted the notion like “we” or “our” and corrected grammatical and space errors according to your comment.

 

Reviewer #1 comment

# An abstract should have the following in this order: Introductory/rationale statement that leads to an objective statement (should start like this "Therefore, the objective (s) was...."), then a description of the experimental design/treatments measurements and ending with the largest amount of the abstract text as results/major conclusions. The description of the experimental design/treatments measurements is Ok, but the authors need to improve the other points indicated. In the conclusion section, authors should emphasize the novelty of the research rather than summarizing findings. Please rewrite it.

Response

Thank you for your comments. We have changed the abstract according to your comments.

 

Reviewer #1 comment

# The change in microbial composition analysis via high-throughput sequencing should be written in brief in the abstract section.

Response

Thank you very much for your comment. We have written the sentence in the abstract section according our comment.

 

Reviewer #1 comment

# Justify the novelty in introduction and discussion, as well.

Response

Thank you very much for your comment. We have revised the introduction and discussion sections of our manuscript according to your comment.

 

Reviewer #1 comment

# The introduction does not provide sufficient background and does not include all relevant references. It needs the addition of some recent new research progress in this field. Present a hypothesis in introduction. Justify, how does your research contribute to the microbiota compositions in grass carp culture? Also add some points related to the significance of aerobic denitrifying bacterium P. furukawaii ZS1.

Response

Thank you for your comments. We have strengthened the introduction according to your comments.

 

Reviewer #1 comment

# The introduction of the paper must be extended and reformulated in order to provide a more comprehensive approach.

Response

The introduction of our manuscript were revised according to your comment.

 

Reviewer #1 comment

# In materials and method section, any background information why authors used aerobic denitrifying bacterium P. furukawaii ZS1bacterial strain? Need to mention the reason for choosing.

Response

We added the description of the aerobic denitrifying bacterium P. furukawaii ZS1 in the introduction section of our revised manuscript.

 

Reviewer #1 comment

# Line no 70; What do you mean by “during which gas was continuously imported”??

Response

All experimental tanks were continuously aerated air gas during the experiment. We have changed the sentence.

 

Reviewer #1 comment

# Line no 72; Assessment of water quality should be described properly with references.

Response

We have added the description of water quality assessment.

 

Reviewer #1 comment

# Give the details of DNA extraction from the sample. Exactly how much sample was taken for DNA extraction?

Response

Approximately 500 mL of water was simultaneously collected from middle position of each tank and filtered through glass-fiber membranes (GF/C) with 0.22-μm apertures to collect microbiota for DNA extraction. The recovered filter membranes were stored at −80 °C for subsequent DNA extraction. Genomic DNA of the water microbiota was extracted from the filter membranes using a PowerSoil DNA isolation kit (QIAGEN, Germany). These have been described in the methods section of our manuscript.

 

Reviewer #1 comment

# For DNA extraction, Was it dried sample or fresh sample?

Response

Approximately 500 mL of water was simultaneously collected from middle position of each tank and filtered through glass-fiber membranes (GF/C) with 0.22-μm apertures to collect microbiota and then the recovered filter membranes were used to DNA extraction by a PowerSoil DNA isolation kit. The recovered filter membranes were fresh sample stored at −80 °C.

 

Reviewer #1 comment

# Results and discussion: Provide a better explanation for your data. Interpret and discuss the meaning of your results more deeply.  Discussions need to be supported by the latest references and need to be explained in depth. The authors should highlight the reason of their result findings in the light of available literature.

Response

Thank you very much for your comments. We have revised our manuscript according to your comments.

 

Reviewer #1 comment

# Authors are suggested to add discussion by explaining trends in the obtained results along with the possible mechanisms behind the trends.

Response

Thank you for your comment. We have added discussion according to your comment.

 

Reviewer #1 comment

# It is strongly recommended to add a subsection, 'practical implications of this study,' outlining the challenges in the current research, future work, and recommendations, before the conclusion.

Response

Thank you for your comment. We have added a subsection according to your comment.

 

Reviewer #1 comment

# Conclusion: Conclusions is not just about summarizing the key results of the study, it should highlight the insights and the applicability of your findings/results for further work.  Please enrich your conclusion section.

Response

Thank you for your comment. We have revised the conclusions according to your comment.

Reviewer 2 Report

The manuscript focussed on denitrification effect of Pseudomonas furukawaii in nitrogen-rich aquatic system. The study is interesting. However, need some revisions.
Line 43........Please add the formula of Ammonia-, nitrite-, nitrate-nitrogen, and organic
nitrogen......as stated in Line 73.
Line 44........Delete comma after showed that
Line 64........Write full name of KNO 3 and place the chemical formula in bracket.
Line 66.......Did you measure C/N ratio? How?
How Pseudomonas furukawaii was cultured?
Line 217....... addition “of ” P. furukawaii ZS1
The initial chemical and biological characteristics of water must be presented in materials
section.
Improve the materials and methods section as it is confusing. Many things have not been
discussed. Detail the experimental setup. Did you apply aeration during the experiment? Why 8
days was chosen?
Figure 1 (D) Write the scientific name in italics
Figure 3 is not visible. Please improve the quality.
Explain the increment in pH.
Line 209......What is COD? Please specify water temperature, nitrate concentration, and pH
ranges.....suitable for denitrification or microbial changes
Use international literature for the introduction and discussion parts.
Strengthen the discussion part.
The changes in microbiota composition in water should be highlighted in the abstract and
conclusion.
The organic matter and C/N ratio not highlighted in discussion as expected.....

Author Response

Responses to Comments and Suggestions of the Reviewer #2

Reviewer #2 comment

The manuscript focused on denitrification effect of Pseudomonas furukawaii in nitrogen-rich aquatic system. The study is interesting. However, need some revisions.

Response

Thank you very much for your comments and suggestions. The comments and suggestions are very useful for us to improve our manuscript and guide our follow-up research. We have revised our manuscript according to your and another reviewer’s comments and suggestions.

 

Reviewer #2 comment

Line 43........Please add the formula of Ammonia-, nitrite-, nitrate-nitrogen, and organic nitrogen......as stated in Line 73.

Response

We have added the formula of Ammonia-, nitrite-, and nitrate-nitrogen. However, due to the variety of organic nitrogen, there is no unified formula for organic nitrogen, so we did not provided the formula of organic nitrogen.

 

Reviewer #2 comment

Line 44........Delete comma after showed that

Response

We have deleted the comma according to your comment.

 

Reviewer #2 comment

Line 64........Write full name of KNO 3 and place the chemical formula in bracket.

Response

We have added the full name of KNO3 and placed the chemical formula in bracket according to your comment.

 

Reviewer #2 comment

Line 66.......Did you measure C/N ratio? How?

Response

We are very sorry for our error that the C/N ratio should be 5:1. We did not measure the C/N ratio directly. The C/N ratio (5:1) was calculated. At the beginning of the experiment, we measured the NH4+-N, NO3--N, NO2--N, and COD concentrations of the culture water from a grass carp culture pond. We assumed that the NH4+-N, NO2--N, and COD concentrations remained unchanged, then calculated the total nitrogen concentration of the culture water when the concentration of NO3--N reached to 50 mg L-1 by adding potassium nitrate. Then we calculated the weight of sodium acetate to be added according to the C/N ratio of 5:1 and COD concentration. By adding sodium acetate, the C/N ratio of the culture water at the beginning of the experiment was 5:1 in theory.

 

Reviewer #2 comment

How Pseudomonas furukawaii was cultured?

Response

Before the experiment, P. furukawaii ZS1 preserved in glycerin at -80 °C were cultured for 24 hours using denitrifying enrichment medium (containing 3.0 g yeast extract, 5.0 g peptone, 1.0 g KNO3, and 1000 mL distilled water, ph = 7.6) in a constant temperature shaker (180 r/min) at 30 °C. Then, the bacterial culture was divided into 10 conical flasks with 5 L capacity, and 2 L denitrifying enrichment medium was added to each flask. The bacterial cells were cultured in a constant temperature shaker (180 r/min) for 24 hours at 30 °C. We have added the description in the methods section of our revised manuscript.

 

Reviewer #2 comment

Line 217....... addition “of ” P. furukawaii ZS1

Response

We have added the word according to your comment.

 

Reviewer #2 comment

The initial chemical and biological characteristics of water must be presented in materials section.

Response

As we described, water temperature was maintained at 30 °C. We did not measure the C/N ratio directly. The C/N ratio (5:1) was calculated. At the beginning of the experiment, we measured the NH4+-N, NO3--N, NO2--N, and COD concentrations of the culture water from a grass carp culture pond. We assumed that the NH4+-N, NO2--N, and COD concentrations remained unchanged, then calculated the total nitrogen concentration of the culture water when the concentration of NO3--N reached to 50 mg L-1 by adding potassium nitrate. Then we calculated the weight of sodium acetate to be added according to the C/N ratio of 5:1 and COD concentration. By adding sodium acetate, the C/N ratio of the culture water at the beginning of the experiment was 5:1 in theory.

 

Reviewer #2 comment

Improve the materials and methods section as it is confusing. Many things have not been discussed. Detail the experimental setup. Did you apply aeration during the experiment? Why 8 days was chosen?

Response

Thank you for your comments. We have added some details of the experiments. All experimental tanks were continuously aerated air gas during the experiment. According to our previous study in another aquaculture water treatment system (Li et al., 2017), the microbial community structure of aquaculture water recovered after approximately 1 week of artificial disturbance. Therefore, the experiment lasted 8 days.

 

Reviewer #2 comment

Figure 1 (D) Write the scientific name in italics

Response

We have changed the scientific name according to your comment.

 

Reviewer #2 comment

Figure 3 is not visible. Please improve the quality.

Response

Thank you for your comment. We have deleted the PCA profiles to improve the visualization of Figure 3.

 

Reviewer #2 comment

Explain the increment in pH.

Response

Thank you for your comment. Even though we observed the increment in pH, we are very sorry we don’t have a reasonable explanation for the increment in pH.

 

Reviewer #2 comment

Line 209......What is COD? Please specify water temperature, nitrate concentration, and pH ranges.....suitable for denitrification or microbial changes

Response

COD is chemical oxygen demand. We have revised the word. We have added the discussion about the influence of the environmental parameters on the denitrification efficiency. Thank you for your comment.

 

Reviewer #2 comment

Use international literature for the introduction and discussion parts.

Response

We have added many international literatures in the introduction and discussion sections.

 

Reviewer #2 comment

Strengthen the discussion part.

Response

We have strengthened the discussion part according to your comment.

 

Reviewer #2 comment

The changes in microbiota composition in water should be highlighted in the abstract and conclusion.

Response

We have highlighted the changes in microbiota composition in water in conclusion section. However, due to the limited number of words, we can not elaborate on the changes of microbiota composition in the abstract.

 

Reviewer #2 comment

The organic matter and C/N ratio not highlighted in discussion as expected.....

Response

We have added the discussion about the organic matter and C/N ratio in our revised manuscript.

Round 2

Reviewer 1 Report

The authors have addressed all the comments, therefore this manuscript may be accepted in the present form. 

Reviewer 2 Report

Auther has successfully replied to all the comments. I recommend accepting the manuscript in the present form.

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