Denitrification Mechanism of Heterotrophic Aerobic Denitrifying Pseudomonas hunanensis Strain DC-2 and Its Application in Aquaculture Wastewater

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Current manuscript was well designed and informative, very good for future aquaculturist for the treatment of Aquatic wastes by using denitrifying bacteria like pseudomonas strains,

However it's needs some minor improvements like

1. Please add the refrences in methodologies section that from where you have adopted this all methodology of identifying and sequencing and measuring nitrites and carbon, nitrogen, ammonium removal level by using  bacterial  strains during aquatic wastes treatment of bottom soil sediments.

2. I think if you add also the side affects of these bacterial strains on aquaculture ecosystem, fishes, or their consumers like human populations, then it will be more better before using your ideas in Aquaculture practices. Please reconsidered and  includes the  chances of any negative impact of these strains. if you found these  possibilities in previous studies, then let us know that how they could be balanced or under controlled in Aquaculture practices.

Wish you best of luck 

Regards 

Reviewer 

Author Response

Dear reviewer,

Thanks for your efforts, we very much appreciate the comments and suggestions concerning our manuscript from reviewers.  We feel the comments are very instructive and helpful to further improve the quality of our paper.  We did our best to make a revision for the manuscript.  Revised portions were highlighted in yellow in the revised manuscript, and all responses to these questions are shown in the specific comments on the document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well-written and presented.

Line 76 ‘’Surface sediments were gathered’’, please be specific. Is there any information on the physicochemical characteristics of the sediments where the strain was isolated?

Lines 181-182  ‘’Strain DC-2 demonstrated the capability to remove ammonium-N within aerobic conditions (Figure 2a)’’. In the figure there is no increase in the concentrations of nitrite and nitrate. How is this evaluated by the authors? The explanation given in lines 194-196 is not adequate, given that the initial ammonium nitrogen concentration is high. The concentrations of the carbon sources are not included in the materials and methods section. Nitrogen and carbon sources were used simultaneously? There is no discussion-justification of the results for sections 3.4.3, 3.4.4, and 3.5.

Author Response

Dear reviewer,

Thanks for your efforts, we very much appreciate the comments and suggestions concerning our manuscript from reviewers.  We feel the comments are very instructive and helpful to further improve the quality of our paper.  We did our best to make a revision for the manuscript.  Revised portions were highlighted in yellow in the revised manuscript, and all responses to these questions are shown in the specific comments on the document.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

ln.39 > define HN-AD bacteria

ln.91

"...supplementary materials (see Table S1 in Supplementary materials)"

> I couldn't found this material...

lns.145-6

> ion chromatography for nitrate quantification

Why not also for nitrite?

Fig.3b

> values indicated on the abscissa axis are confusing...

> in Fig.3b why not to represent a XY dispersion (since you have fifferent increments in abscissa...)

Section 3.3. Effects of environmental factors on nitrogen removal...

> you are dealing with at least 4 factors: carbon source type (fig.3a), C/N ration (fig.3b), c) media pH value (fig.3c) and d) temperature (fig.3c)

As depicted from these figures, these 4 factors are affecting the removal efficiency...

How to ensure BEST simultaneous conditions?

Are you varying one factor at each time?  Keeping pseudo-constant conditions?

> maybe you need a full factorial design... (e.g. 3 level design with 4 factors -> 81 experiments)?

> alternatively, use multivariate modeling approach to estimate and evaluate relevant factors and respective interactions...

Table 1

> the meaning for "nt" (not determined?  not quantifiable?)

Figure 5

> add more explanation to this figures legend in order to emphasize the relevance of this figure

Table 1, table 2 and text...

> excess of significant digits!

> accordingly to EURACHEM / CITAC Guide CG 4 (2012) for "Quantifying Uncertainty in

Analytical Measurement" (https://www.eurachem.org/images/stories/Guides/pdf/QUAM2012_P1.pdf)

a) uncertainties should be reported with a maximum of 2 significant digits

b) respective central estimate should be rounded to decimal places of corresponding uncertainty

e.g.: in Table 2, instead of 110214.39 ± 2744.87

use scientific notation and express as...

(1102 ± 27) x 10^2

> same observation to several values reported among text like... e.g. 

"30.42% ± 4.09%" in Ln.403

"9.5 ± 0.52" in Ln.418

"15.2 ± 0.03 mg/L to 2.3 ± 0.4" in Ln.421

4. Conclusions

"The optimal conditions for NH4+-N removal were identified as a temperature of 30°C, an initial pH of 7, and a

C/N ratio of 10..."

> this seems to be a very complex system...  In reported experimental conditions (using pseudo-fixed conditions)...

I do not believe that BEST conditions are already established!

What profs do you have that, in fact, thees conditions meet best nitrogen removal efficiency? 

Author Response

Dear reviewer,

Thanks for your efforts, we very much appreciate the comments and suggestions concerning our manuscript from reviewers.  We feel the comments are very instructive and helpful to further improve the quality of our paper.  We did our best to make a revision for the manuscript.  Revised portions were highlighted in yellow in the revised manuscript, and all responses to these questions are shown in the specific comments on the document.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your responses.

Please "round" the following results accordingly to respective "uncertainties"...

Due to experimental uncertainties...  I believe that, percentages should be only represented with 1 decimal place...

#ln.13 98.84% -> 98.8%   

#ln.14 88.43% -> 88.4%

#ln.22 84.76% -> 84.8%   

#ln.84 97.36 ± 15.66 -> 97 ± 16

#ln.196 98.84% -> 98.8%

#ln.218 61.50% -> 61.5%

#ln.233 88.43% -> 88.4%

#ln.239 93.20% -> 93.2%

#ln.243 95.11% -> 95.1%

#ln.282 96.99 -> 97.0

#ln.291 94.91% -> 94.9%

#lns.309-321 > several cases of ##.##%!

Table 1

100.01 ± 2.85 -> 100.0 ± 2.9

34.43 ± 4.68 -> 34.4 ± 4.7

63.18 ± 4.03 -> 63.2 ± 4.0

30.42 ± 4.09 -> 30.4 ± 4.1

41.53 ± 1.90 -> 41.5 ± 1.9

9.20 ± 2.18 -> 9.2 ± 2.2

98.80 ± 2.95 -> 98.8 ± 3.0

1.51 ± 1.11 -> 1.5 ± 1.1

55.32 ± 3.65 -> 55.3 ± 3.7

55.39 ± 3.65 -> 55.4 ± 3.7

28.57 ± 5.64 -> 28.8 ± 5.6

Table 2

58.08 ± 3.20 -> 58.1 ± 3.2   (uncertainty represented with only 2 significant digits)

#lns.419-420 30.42 ± 4.09% -> 30.4 ± 4.1%

#ln.436

19.14% -> 19.1%

62.63% -> 62.6%

#ln.439

84.76 ± 2.92 -> 84.8 ± 2.9   (uncertainty represented with only 2 significant digits)

#ln.440

84.22% -> 84.2%

Table S1 

> add horizontal lines separating mediums?  (avoid confusion?)

Author Response

Thank you again for your efforts. We greatly appreciate the reviewers' comments and suggestions on our manuscript. We have made corresponding revisions to the manuscript based on your comments. Revised portions were highlighted in blue in the revised manuscript, and all responses to these questions are shown in the specific comments on the document.

Author Response File: Author Response.pdf