Review Reports

Round 1

Reviewer 1 Report

In this study, the authors applied a NBRC 0190 immobilized alginate hydrogel to remove nitrate-nitrogen from synthetic contaminated groundwater and real contaminated groundwater. this application holds great potential to be applied for groundwater remediation. However, I found this paper has several flaws that shall be addressed before being re-considered for publication: 

1. experimental replicates are missing for most of the comparison studies. and the R. gra growth data should be provided along with the nitrogen species data.
2. I did not see much of discussion involved in the Result section. this makes the paper read more like a report. The authors could include some interpretation of the data, and comparison with similar studies.
3. It seems glucose (carbon source) need to be added when the R. gra immobilized bead is used for groundwater treatment. it may increase the overall cost.

specific comments are below:

In the introduction section, the author introduced the "heterotrophic nitrification-aerobic denitrification". this is not the typical nitrogen transformation pathway used for domestic wastewater treatment or nitrate removal from groundwater. It would be better if the authors could explain more details regarding the electron donor  (such as organic carbon?) used for aerobic denitrification. in the experimental design, the authors used glucose as the electron donor/carbon source. However, the natural organic matter level in groundwater may be limited.

line 56-58, it would be helpful to provide the influent water nitrogen level and justify why immobilization of R. gra is necessary, and for what practical application?

line 105-106, belongs to materials and methods.

Figure 1, it is hard to interpret the "zoom-in" data, the x-axis info is missing. it would be better to add the legend explained in the figures. 

Figure 2, replicate is missing for each experimental group.

Figure 3, The alginate bead and R. gra concentration shall be provided in the figure caption. Legend should be added to the figure, the detection limit of nitrate and ammonium shall be explained in the figure caption, rather than in the figure. replicate is missing for each experimental group.

Figure 4. Glucose concentration change shall also be provided, so we could understand how much organic carbon is needed to remove 10 ppm of nitrate-nitrogen.

line 194, for figure 5, if statistical analysis was conducted, the authors should conduct replicate experiments for each experimental group. 

 

Author Response

I would like to thank you for your careful review and appropriate comments. I have revised the manuscript and rewrote all the figures according to your suggestions.

1. The experiment was conducted multiple times, and errors were added to the figure to make it clearer. The growth rate of R.gra in immobilized alginate beads was not measured, but there was a color change after the experiment due to the growth of R.gra. 326-327 lines added.

 

2. Following your suggestion I've added a few more considerations.

 

3. Added description of glucose to the 92-94, 333-338, and 514-517 lines.

 

Line 56-58: Following your suggestion, I added the nitrogen level in 74-77 line.

 

Line 105-106: The sentence has been moved to 2.1 Materials (101-102 lines).

 

All figures have been rewritten, and experimental conditions and other information have been added to the figure captions.

Author Response File: Author Response.pdf

Reviewer 2 Report

My suggestions, comments and recommendations can be found below in bullet points.

- The title of the manuscript sounds informative and attractive.

- The introduction, which serves as the theoretical background to the paper, is nicely done but little literature in terms of application, R. plays in immobilized calcium alginate hydrogel beads.

- The methodology of the experiment seems to be good developed.

The experiment is simple but therefore clear and you can see the relationships.

 

However, Figure 2 is unclear, please describe the markings. Or explain the introduction of the brown dots. 

It is not clear which alginate beads were used for the ground water experiment. Please explain.

 

Please standardise in the article the units either ppm or mg/L for concentrations, I propose mg/L.

Please make editorial corrections in the lines: 94, 114, 123,

here is an example : reaction was evaluated

 

Author Response

I would like to express my deepest gratitude for your accurate remarks. We have revised the manuscript and added detailed explanations as you suggested.

Figure 2: To clarify the meaning of the symbols in Figure 2, the following text has been added to the figure caption and 316-319 lines have been written in the manuscript.

 

“Red symbols, Volume of R.gra suspension was varied from 1, 3, 5 and 10 mL to 1 mL of sodium alginate of 1.5% con-centration. Circular symbols, Volume of sodium alginate at a concentration of 1.5% was varied from 3, 5, 10 and 15 mL to 1 mL of R.gra suspension.”

 

Figure 5 (ground water experiments): The alginate beads were prepared by adding 0.5 mL of R. gra suspension to 5 mL of 1.5% concentration sodium alginate solution. I added this sentence to the figure capyion.

 

In accordance with your suggestion, we have standardized the unit to mg/L.

 

The English manuscript has been revised.

Author Response File: Author Response.docx

Reviewer 3 Report

Please see comments in the attached file.

Comments for author File: Comments.pdf

Author Response

Thank you very much for your careful review and detailed comments on the grammatical errors. We have revised the text and added detailed explanations as you suggested.

43: Yes, I changed the 43-63 lines.

 

54: Replace "safe" with "environment-friendly".

 

71&103: My mistakes. I have rewritten it.

 

90: We added the experimental conditions as shown in the following sentence. These waters had been sterilized in an autoclave at 110℃ for 10 minutes for use in the experiments.

 

101: The detection limits for nitrate-nitrogen, ammonia-nitrogen, and glucose concentrations were added.

 

104: I added the following sentence. It was confirmed that there was a direct proportional relationship between the value of turbidity and the number of R. gra yeast counted by the microscopic method.

 

154: I rewrote the sentence as follows. Nitrate-nitrogen removal and ammonia-nitrogen removal experiments were performed independently using R. gra-immobilized alginate beads prepared under standard conditions (10 mL of 1.5% concentration sodium alginate solution and 1 mL of R. gra suspension).

 

170: The experiment was conducted multiple times, and errors were added to the figure to make it clearer. The data is added in Figure 3.

 

183: We were not able to conduct our experiments at night.

 

221: “about 24 hours” has been rewritten to “within 24 hours” (line 552).

 

245: “at a culture time of 120 hours” was added.

 

248: “In order to apply the removal of nitrate-nitrogen by R. gra immobilized alginate beads in a drinking water treatment plant, the removal rate of nitrate-nitrogen in a packed bed bioreactor must be considered. The post-treatment of treated water containing residual R. gra, etc. by filtration, adsorption or disinfection should be investigated in the future.” Was added in conclusions.

 

The English manuscript has been revised.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The quality of the manuscript has been significantly improved after revision.

In the figures, the detection limit shall be explained in the figure caption, not in the figure. specifically for figure 3 and 4, legend should be added instead of using arrows indicating the data.

 

One additional concern is the glucose concentration used in this study was way much higher than the organic carbon concentration in the groundwater. Therefore, how does the results translate to the groundwater remediation? In addition,  there is a lack of microbial density assessment. how does the immobilized R. gra respond to environmental conditions change and the relevant cell growth shall be considered before its application in the field.

Author Response

(1) The detection limits of nitrate-nitrogen and ammonia-nitrogen have been added to the figure captions in Figs. 3 and 4.

(2) The post treatment of glucose and R. Gra need to be investigate in the future and is described in lines 593-594.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors,

Thank you for faithfully considering all of my previous comments and making the numerous changes. I feel that both the major comments and the "finish" of the article have now been well addressed. The only lacking comment is the control experiment and replicates which would have strengthened the work. I assume this would not be easy to provide at this point, and I do not feel it should be required for publication.

One minor revision in the methods section, line 106. I requested clarification of the term "turbidity". However, your addition of "with no optical absorption" has only worsened the explanation (probably no evidence for this) and I would hate to be the cause of making your paper less clear.

Although it is not my area, I assume you are measuring light transmission - and that you do not know how much light actually is scattered (turbidity) and how much is absorbed. Therefore, I suggest you revert to your original text on this point.

Or perhaps you could provide a third explanation. I assume that 660 nm is so long a wavelength, that light absorption by dissolved compounds is expected to be low and that the method can therefore be used to indicate presence of yeast cells which scatter light and dominate at this wavelength. Is this correct? My main complaint was simply that I do not think you should measure transmission (scattering and absorption) and then call it turbidity (scattering only). I trust you will find a simple and short solution to this minor comment. 

    

Author Response

I am very grateful for the careful peer review. As the reviewer's suggestion, "with no optical absorption" has been deleted. The following sentence was added to explain the accuracy of the turbidity. Not all samples were measured, but the ones that were measured gave good results.

"It was confirmed that there was a direct proportional relationship between the value of turbidity and the number of R. gra counted by the microscopic method."

Turbidity: I added the following sentence. It was confirmed that there was a direct proportional relationship between the value of turbidity and the number of R. gra yeast counted by the microscopic method.

 

Author Response File: Author Response.docx