Nitrite-Oxidizing Bacterial Strains Isolated from Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction

Round 1

Reviewer 1 Report (New Reviewer)

The work is relevant, and the results are good if it really reduces 40% and 94%. There is a note:

- The name of the culture is written in italics;

- line 102-106 refers to the introductory part;

- It is necessary to change the map in the 2nd drawing, because it is not clear, not clear, and not clear;

- Why does the article use blue text color on many pages? Does it mean something?

- Review all tables so that all borders are clear, you don't look like a table.

- the аigure 5 should be replaced, not informative and not a clear picture;

- аigure 6 needs to be changed, add a color scheme.

- in the 6th figure, the statistics are not correct, for everyone, how can this be?

- part of the methodology must be correctly formulated and redone, write what you are doing there. Remove the word "we". And you need to write how the technique is performed, the sequence of work;

- You write "All strains and their replicas were tested from day 15 with a time interval of 5 days until reaching 40 days." Why only for 15 days? What are the primary (1 day) data?

- on the 3rd figure of the reactors it is not visible, as you write in line 189;

- you need to change Figure 1, because the text is hard to read;

- the methods in the article are simple and do not use the latest innovations.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

NITRITE OXIDIZING BACTERIAL STRAINS ISOLATED 2 FROM SOILS OF ANDEAN ECOSYSTEMS AND THEIR PO- 3 TENTIAL USE IN NITROGEN REDUCTION

Please check plagiarism 

Comments

Abstract

Line 10 rewrite this used in our favor in different processes such as bioremediation………..add more process

Line 13, 14   Name of following microbes should be italic  Aeromonas, Bacillus, Buttiauxella, Mycobacterium, Paeni-bacillus, Serratia, and Yersenia  

Line 13,    which are part of the cultivable microbial from soil int a native  correct the sentence

It should be italic  Serratia and Yersinia

Lime 16 spelling check  litoautotrophic or lithotrotrophi

Line 20 It should be italic Serratia and Yersinia

Introduction

Line 56  spelling checking  chemiolitoautotrophos,

Line 82 checking both in water and in sediments,

Line 85 , public Effect of land use ………….. replace effect

Line 89   It should be italic Nitrosomonas

Line 95 spelling check disponibility

 

2. Materials and Methods

Line 115      10 gr means……….gram

Line  120- 121   write in same format    ph  ph-meter pH of 5.6 

Line 127  for bacteria we use 24hours why you use  48 hours any region please specify

Line 156  please specify two seedings

Line 168   why use 150 rpm for 3 to 6 weeks. Long duration is required for bacteria?

Line 174 2.7.  Spelling is incomplete   ……pplication of nitrifying

Line204  Why you are using  cellular retention time (TRH) of 40 days?

Line 211, 213 . check this  y Yersinia

Line 215, 219   spelling check litoautotrophic

Line 222  table and figure 4 use same format in all manuscript

Line 246     It should be italic Yersenia enterocolitica

Line 268  Figure 1. Oxidation phase of nitrite by bacteria (NOB), the object of this study. no need to write in figure caption

Line 272     Mention the location details in this  Figure 3. Location of plots for soil sampling

Line 280    MgSO4·7H20, KH2PO4 it should be written like this KH₂PO₄  number should be subscript

Line 283      Table 3. Behavioral dynamics of applied bacteria strains ,,,,,,explain in which behavioral dynamic

References

Some References are not complete and not in same format

It should be written properly

 

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

The manuscript shows relevant information on heterotrophic and litho-autotrophic strains with nitrifying potential. This research reveals the uniqueness of the microbiological diversity of Andes’ soils and gives an interesting perspective on the applications these microorganisms can have in environmental remediation. The information shown is of great value. However, the following recommendations are suggested to improve the quality of the manuscript.

1. Write impersonally, avoiding personal form (we, our…).

2. Put keywords in alphabetical order

4. Line 31. Modify “Nitrogen starts its cycle” by “The nitrogen cycle starts in…”

5. Line 35. Change “Ammonia” to “ammonia”

6. Line 81. Modify “carry out..” by “carrying out bacterium identification”

7. Line 82. Modify “both in water and in sediments, they” by “in water and sediments and finding…”

8. Improve the wording of lines 87-90.

9. Line 119. Modify “gr” by “g”

10. Line 120. Modify “ph” by “pH”

11. Lines 132-133. Complete the phrase. The sentence is incomplete.

12. Lines 136 and 144. Separate the units from the numeric value.

13. Line 174. Modify “pplication” by “Application of nitrifying bacteria in a synthetic medium..”

14. Line 196. Change “prior to” by “before”

15. Line 201. Modify “The use of” by “Using…”

16. Improve the resolution and quality of Figure 1.

17. Line 216. Change “nót” to “not”

18. Line 240. Modify “by anoxic way” by “in an anoxic way”. “allows to establish” by “allows establishing”

19. Line 255. Change “other” to “Other”

20. Standardize the word “lithoautotrophic” in the whole manuscript since sometimes it is mentioned as litoautotrophic.

21. Line 344. Modify “The” by “They”

22. Review the format of reference number 30.

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Reviewer 4 Report (New Reviewer)

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Reviewer 5 Report (New Reviewer)

The manuscript emphasized nitrification bacteria and their important application in bioremediation. This study’s topic is an essential issue in agricultural pollution and is of broad interest. However, the methods and results in this manuscript are very common and basic. Numerous studies have been published regarding the isolation and characterization of nitrification bacteria with even more advanced technologies, such as microbiome/metagenomic approaches, from batch to semi-continuous experiments, and application in the actual polluted soil/wastewater. It is difficult to highlight the excellent novelty of this manuscript. To further point out the critical issues of this paper, several comments are provided as follows:

1.       Abstract. Ln 10. the sentence is a general term that is not important to explain. It shall not be found anywhere in the abstract, where the information must be straightforward.

2.       The structure of the abstract is not constructed properly. The introduction is very long before the aim is mentioned. Moreover, the result and final statement in the abstract are also very limited. The application in wastewater and its potency for bioremediation discussed early in the abstract is also not mentioned further.

3.       In the introduction, the authors shall focus on the previous study where the nitrification bacteria shall be more emphasized in the mechanisms and diversity in the soil. The application of bioremediation is also essential to mention and add by citing previous studies that isolate, characterize, identify, and apply these strains to polluted soil, wastewater, or other bioremediation application.

4.       The figures are poorly arranged and misplaced. The contents are also questionable. Why did the authors prefer to use two lines of standard errors/deviation in figure 6? It is rare to have this style nowadays.

5.       Why did the authors prefer to use the MALDI-TOF technique? It is common to use morphological observation and physiological approaches to identify isolated bacteria. Moreover, molecular techniques are also available to obtain detailed strain genus and species results.

6.       Ln 185, the process is called batch system. This term is a general term.

7.       Giving the codes and strain names help explain the results and discussion.

8.       The halo zone shall be measured triplicate and mentioned by mean ± SD.

9.       The reason and characteristics of the sampling site are essential to show and discuss. It may be attached in the supplementary materials.

10.   The potency of bioremediation is not clearly explained. It is also necessary to address this issue since the title mentions this potency. Isolating and nitrogen-related activity are essential to be discussed before the ability and application of this step.

11.   Is the occurrence of the specific strains related to the condition of the sampling site? Are these strains commonly found in forest soil?

12.   What is the difference between R1 and R2 in figure 6? The method is not clearly stated in the material and method sections.

13.   Photos in figure 5 shall be replaced with more proper documentation.

14.   The limitation and further possibilities shall be stated and discussed as well.

15.   Bioremediation focuses on a broad spectrum of wastes and pollutants. In which category of bioremediation may these bacteria be applied?

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Reviewer 6 Report (New Reviewer)

The MS discusses the potential use of bacterial strains in bioremediation processes, specifically in wastewater treatment, through the activation of nitrification and denitrification processes. The study evaluated the nitrification potential of bacteria strains belonging to different genera. Specific culture media for ammonium oxidative and nitrate oxidation bacteria were used to identify heterotrophic and litoautotrophic strains. The study found strains with nitrifying potential, with Serratia and Yersinia genera showing a high NO2- oxidation capacity. These strains were inoculated in synthetic water rich in nitrogenous products, which resulted in a significant reduction in nitrite and high cell retention times. The study concludes that these results are promising for the possible use of these strains in environmental bioremediation processes, specifically in the biological removal of nitrogenous compounds from wastewater.

Overall, the abstract provides a clear and concise summary of the study's objectives, methodology, and findings. However, it lacks information on the study's limitations and implications, which could have enhanced its impact.

Top of Form

-Correct” It is crucial to determine the bacteria strains with nitrification potential and subsequently use these in terrestrial and aquatic ecosystem bioremediation processes [11],[12],[13],[14], contributing in this way to the disponibility of nitrogen used in the plant development “ to  “It is crucial to identify bacteria strains with nitrification potential and subsequently use them in terrestrial and aquatic ecosystem bioremediation processes [11-14], thus contributing to the availability of nitrogen for plant development."

- "Each quadrant collected 1 kg of soil sample at a depth of 10 cm (after removing the organic layer). For the microbiological analysis, we used 10 kg of sample per plot, which was composed of 10 subsamples of soil [17]. We also measured pH and temperature in the soils. We took 10 g of soil from each plot and dissolved it in 990 ml of distilled water. The pH was measured using a HACH® model HQ40D pH-meter. In the native forest, we recorded a pH of 5.6 and a temperature of 15 °C; in the pine forest, a pH of 3.5 and a temperature of 13 °C."

The corrected version makes the text grammatically correct and more accurate. The changes include fixing capitalization, punctuation, and spelling errors, as well as rephrasing some sentences to improve clarity.

-"I found many grammar errors in the article; please correct them before publication."

- what the importance of that "2.4. Bacterial Density by Most Probable Number (MPN)"

• - "The discussion is of poor quality and needs improvement."
• - "The conclusion is poorly written and requires rewriting and summarization."

 

Author Response

Dear Reviewer,

Thank you for taking the time to look at our article and I am sure your suggestions will improve it significantly. Corrections have been made according to your observations.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Thanks for the work you've done!

Author Response

Thank you for your time and comments, which have improved the article.

Reviewer 4 Report (New Reviewer)

I find the authors have made extensive revisions based on the reviewers' comments, and the manuscript has been improved. However, there are still some contents needing further improvement before it can be finally published.

Frist, in L. 102, the authors mentioned "For this reason". I understand this statement was based on the literature review of Ls. 88-101. However, these contents were just simple list (who have conducted a experiment in somewhere), but no comprehensive "reason" were summarized. This should be supplemented. In addition, there are text mistakes in this paragraph. For example, in Ls. 88-89, what is the subject of "carrying out ... and finding"? It can not be "the United kingdom", but should be the authors of relevant publications (or just "researchers"). Based on these contents, I wonder whether the authors were too hastily in preparing this manuscript or they may need some language edition help.

Second, in Ls. 108-117, The authors well explained the reason why they selected the two forest soil types, which can lead to very attractive scientific questions for relevant readers. However, there are no "scientific question" in the section of Introduction. What is more important is, these statements are missing in the section of Conclusions. What are the differences of results between the two soil types? Did you find one soil contained more strains with stronger fuctions than the other? Therefore, I also hope to see a relevant statements in the sections of Results and Discussions.

Other minor issues:

L. 67. Delete "." before "[9]".

L. 157. by Papen and Von Berg [28].

L. 262. Where is 3.3.2?

Ls.263-283. Please provide some specific data throughout the text.

Fig. 6. R1 and R2 refer to "reactor" or "analysis" or "measurement"? Please use only one of them.

Author Response

Thank you for your time and comments, which have improved the article.

Author Response File: Author Response.pdf

Reviewer 5 Report (New Reviewer)

Several issues have been addressed in this manuscript. However, there are still some concerns that need to be addressed before it can meet the excellent quality standards of the Sustainability journal. For instance, the use of duplicate experiments and outdated techniques does not align with current research practices. To address this, it is recommended that the research methods be updated to include more sophisticated techniques commonly used in the field of bioremediation and environment. 

Furthermore, the presented data and figures are limited in their scope and do not provide enough insights for readers interested in the topic. As such, additional relevant data and figures should be included to provide a more comprehensive picture of the research findings. Additionally, the quality of the figures and tables needs to be improved to make them more visually appealing and informative. It is worth noting that the manuscript has undergone a review process in the past, but the quality of the figures and tables have not been enhanced despite being replaced. 

In light of these concerns, it is suggested that the manuscript be rejected until these issues are addressed.

Author Response

Dear Reviewer,

 

 

Thank for you time.

 

You are undoubtedly right, but it is necessary to mention that although the research was not carried out with the sophisticated methods you recommend, we used those reported in scientific articles dealing with the same subject. It is necessary to mention that all the data have been placed and are very valuable for the working team to take the next step in the research.

 

As for the tables and figures, they have been improved thanks to the suggestions of the previous reviewers.

 

This research is of interest for our country where little or nothing has been done on the potential use of microorganisms, these are certainly the first steps.

 

Thank you for your time and comments, which have certainly contributed to this manuscript and will certainly be taken into account in our future research.

Reviewer 6 Report (New Reviewer)

The manuscript is fine now and could be accepted for publication

 

Author Response

Thank you for your time and comments, which have improved the article.

Round 3

Reviewer 4 Report (New Reviewer)

I think the authors have dealed with the reviewers' comments well. Now I only have a few comments, which are easy to solve. 

1. I think the difference between pine and native forests should be presented in the conclusion section.

2. Line 256. pH "of" 5. Please double check the spelling and grammar of the entire text

Author Response

Dear reviewer,

Thanks for your time and comments.

Author Response File: Author Response.docx

Reviewer 5 Report (New Reviewer)

The reviewer would like to recommend the rejection based on the quality of the experiments and overall merit of the manuscript.

Author Response

Dear reviewer,

 

Thank you for each of your comments

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.

Round 1

Reviewer 1 Report

- Abbreviations have to be spelled out when mentioned for the first time in the text.

- Spell chek is required

- Figure 5: Please identify R1 and R2

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript-sustainability-2060289

 

Review comments:

In this manuscript a Nitrite Oxidizing Bacterial Strains Isolated From Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction was studied.  This type of bacteria play an important role in nitrogen elimination from different liquid waste.

In its present form, the manuscript is not suitable for publication in Sustainability Journal. A major revision is required before publication. The following comments support the decision:

 

Comments concerning the backgrounds of the manuscript:

 

The manuscript entitled” Nitrite Oxidizing Bacterial Strains Isolated From Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction » is relevant in the thematic of sustainability Journal.

1.      The abstract is too general, please improve it. Also, include background statement into the abstract.

2.      Please reveal the results in term of digital value in the abstract.

3.      What is the novelty of the study in comparison with the reference given? State the novelty of this work clearly at the end of introduction.

4.      In section 2. Materials, section 2.2 Culture, isolation, and identification of bacterial strains 2.2 Reactor operation, page 3, this section is note clear, please improve it (as an example :samples preparation , pH measurement….).

5.      Section 2.4 Evaluation of the nitrifying potential/ Nitrifying potential, is not clear try to improve it.

6.      In all figures the errors bars are not provided.

7.      Section 2.6.1 Experimental setup, is an important part in your research, try to give more importance to this part.

8.      In the experimental section, concerning the measurement of different parameters, the used methods must be referenced and added to references part.

9.      The obtained results require a more accurate discussion.

10.  Conclusions:  need improvement regarding the new achievements obtained and in accordance with the abstract description.

Comments for author File: Comments.pdf

Reviewer 3 Report

General comments:

The manuscript “Nitrite Oxidizing Bacterial Strains Isolated From Soils of Andean Ecosystems and Their Potential Use in Nitrogen Reduction” identified and investigated the nitrification potential of strains of soil-cultivable bacteria from Andean forest soils. The topic of this research would be of general interest to the audience of Sustainability. However, the organization of this manuscript is fragmented. Additionally, the English writing of this manuscript needs to be significantly improved to ensure accessibility to the audience. Therefore, my suggestion is rejection. Here are some general comments:

1. Please use subscripts and superscripts when appropriate. It is not standard to write NH4+, NO2-, NO3-, etc., without using subscripts and superscripts.

2. The Introduction needs to be set up better. The transition from the general knowledge of N cycling to the design of the experiment of this study is missing. I think adding hypotheses might work better. Also, consider adding a figure regarding the N process described in the first few paragraphs and then label out the ones closely associated with this study. That way the audience might be able to understand the unique contribution of this work better.

3. There isn’t enough discussion about previous work or state-of-art of this research topic, particularly regarding soil-cultivable bacteria in the Andean region, in the introduction section. Therefore, it is hard to judge the innovation level of this work.

4. Please use a period rather than a comma when stating a number with digits (e.g. pH = 7.4 rather than pH = 7,4).

5. Most figures/ tables are not self-explanatory. Consider modifying legends to make them more accessible.

6. Consider expanding the Discussion section to include future work and broader research implications (e.g. potential applications) of this work.

Specific comments:

L33. N in soils can also come from synthetic fertilizers, manures, and cover crop inputs.

L34. Change “free” into “freely”

L35. No need to capitalize on “Ammonia”

L49. Change “in” into “to”

L84. Consider adding texts about the previous findings related to bacteria cultivation and identification that are relevant to this research topic.

L141. It doesn’t seem to be very meaningful to mention the “10-1 to 10-9” detail.

L142. Use the correct format for degree Celsius

L163. Sentence is missing an object after “incubated”

L217. delete the redundant “C”

L229-231, 245-246, 261-263. Awkward sentences

Figure 5. It’s not clear what R1 and R2 represent in this figure.

Table 1. Should “Pyruvate de Sodio” be translated into English?

L376. Discussion should be section 4, not 3.

L377-384. This paragraph repeated lots of ideas in Introduction. A more in-depth discussion is needed about the implications of this study. Try emphasizing the potential use in nitrogen reduction as indicated in the title.

L397-398. Awkward sentence.

L420. “explained due to” is incorrect usage.

L426. Conclusion should be section 5, not 4. Try combining different paragraphs together.

L428. “ a debate” is confusing in this sentence

L435. Double check if “singularity” is the correct word here.