Bacteriophage and Their Potential Use in Bioaugmentation of Biological Wastewater Treatment Processes

Round 1

Reviewer 1 Report

This paper illustrated the usage of phage cocktails for improving the performance of biological wastewater treatment. The manuscript needs substantial enhancements in terms of the discussion  and basic format/design, but these points does not invalid this work. I suggest a meticulous revision of both the manuscript and the associated figures.

1.     Abstract:

Please revise the abstract and highlight some important and quantitative results you get. Some statement are not very clear. For example:

Line 12: “improve functional microbiota”: improve what aspect? Stability?

Line 14: “the entire microbiome including microeukaryotes was modified by the bacteriophages” is very unclear. Modify what exactly? How much? What’s it for?

Line 16: how is adding antibiotic relevant to the main claim? The logical connection in the abstract needs to be improved. 

2.     Introduction:

Please briefly introduce the mechanism of BWT approach. How do bacteria regulate the removal of pollutants?

Please briefly introduce what MBR exactly is, before explaining the advantages. 

Line 68: “optimization of the microbial strains and modifications”: optimize what exactly? “Optimize strains” is a confusing statement. Do you mean “optimize their performance of wastewater treatment”?

3.     In the manuscript, could you provide more information about the phage cocktail you use? For example, how many types of phages, names, sources.

4.     Please justify the novelty of this work with your previous research on using phage cocktails for membrane bioreactors. For example, 

https://doi.org/10.1016/j.scitotenv.2022.154716

https://doi.org/10.1080/08927014.2022.2045484

You cited some of these papers, but the comparison/improvement of this paper can be added. Or what is the problem your previous papers didn’t cover while this paper solved.

5.     Shotgun metagenomic sequencing has already been used for analyzing microbial communities (for at least 11 years according to your reference). Why is it worthy to highlight this established characterization method in your abstract, introduction and conclusion?

6.     Design of some figures can be improved. For example: 

Figure 2, 6: Some notes are too small to read. 

Figure 3: What’s the meaning of the arrow behind “Ascomycota”?

Font is not consistent among figures or even in the same figure. 

7.     Conclusion should have quantified statement.

Author Response

Thanks to Reviewer 1 for the comments. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Thanks for reviewing the manuscript: "Bacteriophage and their potential use in bioaugmentation of biological wastewater treatment processes". Some revisions are necessary before acceptance into Sustainability.

1) keywords are long, please review.

2) Line 136 after the period (.) appears the percentage symbol (%1 ....), review this writing.

3) improve the caption of figure 2, it is too small.

4) Figure 3 is very interesting and necessary for the manuscript, but needs to be improved.

5) unfortunately figure 4 has poor visualization quality, redo it.

6) suggestion Figure 5 is great for viewing. do the same for the others.

7) check the references with the entire text.

After these considerations, the article is ready to be approved in the Journal.

Author Response

Thanks to Reviewer 2 for the comments. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

This research project holds immense potential. The application of bacteriophages for wastewater treatment is innovative and could revolutionize the sector. This approach not only addresses the environmental issue but also brings to light an effective and sustainable alternative to traditional methods. The project's success could result in significant improvements in water treatment, potentially reducing public health risks associated with waterborne diseases.

I look forward to seeing how the study progresses and the impact it could make in the field of wastewater treatment. The manuscript is organized about materials and methods; results are clearly presented however

I have the following comments.

1. Can you provide more detail on the exact sources from which the bacteriophages will be isolated, and how they were selected to ensure a diverse representation?
2. What specific metrics or indicators will be used to assess the effectiveness of bacteriophages in wastewater treatment? How will you quantify the bacteriophage's ability to eliminate bacteria?
3. Considering potential challenges, how will you ensure that the selected bacteriophages will be capable of surviving and maintaining their bacterial lysis ability in different real-world wastewater conditions?
4. Could you elaborate on the relevance of your study in the context of current wastewater treatment practices? How does bacteriophage treatment compare to existing treatments in terms of efficiency, cost, and environmental impact?
5. What is the anticipated timeline for the full implementation of the bacteriophage treatment process, including isolation, characterization, in vitro and in vivo testing, and evaluation?
6. Could you specify the statistical models or analysis methods that will be employed to analyze your results, and how they will contribute to proving your hypothesis within the projected timeline?
7. Can you provide more specific details on how the presence of antibiotics enriches gram-negative Proteobacteria in the BP reactor?
8. How significant is the impact of the mtrA gene in Proteobacteria's resistance to antibiotics, and how was this measured in your study?
9. Considering the results of your study, what strategies would be most achievable in controlling the proliferation of Proteobacteria in the BP reactor?
10. Given the prominence of RND-type Drug Efflux Pumps in antibiotic resistance, what relevant interventions could be used to counteract this resistance mechanism?
11. In future research, what is the projected timeline for implementing additional bioaugmentation agents to mitigate ARG formation, particularly by targeting Proteobacteria, in the aerobic membrane bioreactor?

carefully editing of English language required

Author Response

Thanks to Reviewer 3 for the comments. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

It is OK.

Reviewer 3 Report

This time authors answered to all my questions well. Thank for your hard working.