Responses of N2O Production and Abundances of Associated Microorganisms to Soil Profiles and Water Regime in Two Paddy Soils
, Qingling Fu 2,*, Jun Zhu 2, Heng Zheng 2, Minghao Mo 1 and Anguo Tu 1
Round 1
Reviewer 1 Report
This manuscript provided interesting information on the effects of different soil layers and soil moisture on N2O emissions as well as related microbiological mechanisms. The authors interestingly present the influence of the physical and chemical properties of soil on the number of soil nitrifier and denitrifier function genes with soil depth. The subject of the manuscript is in line with the scope of the journal. The authors used tests commonly used in this type of research. The authors received many interesting results that were correctly interpreted and statistically analyzed. They discussed the results in an interesting way against the background of world literature. Therefore, the presented study is original. I recommend this manuscript for publication in this journal, but with some corrections. Detailed comments are provided below.
Line 5: shouldn't there be a capital letter here? (Tu Anguo)
Line 20: please use superscript correctly throughout the manuscript e.g. NH4+
Line 28, 336: please change to nirK
Line 29, 273, 277, 323, 338: please standardize throughout the manuscript: AOA amoA or AOA
Line 47-53: in the introduction, please describe microorganisms that carry out processes nitrification and denitrification as well as this processes
Line 71-73: please expand this thought more; what individual authors write about
Line 104, 115: please change the symbol of the degree throughout the manuscript, e.g. 16.1°C
Line 113: All figures and tables should be placed next to the first citation throughout the text and
numbered according to their occurrence number.
Line 164, 167, 191: why ha-2 and not ha-1?
Line 178-179: are you sure in all treatments? was it the same in treatments related to 60%WHC?
Line 182-183: are you sure for all treatments? or only for 100% and 200% WHC?
Line 192: why there are no cumulation diagrams for NO3 and NH4 analogous to NO2?
Line 199: where these values come from? no means on the chart
Line 194: (Fig.3) should (Fig. 3)
Line 204-205: was it really similar to the NO3 200% WHC trend?
Line 213-216: what about 60% WHC in the case of Fe3 +?
Line 220, 226: (Fig.6) should (Fig. 6)
Line 224, 229: Table S2- please add the line with the calculated mean for individual treatments in table S2
Line 225-229: How did humidity affect the nosZ and nirK?
Line 236-237: no reference to a table or figure
Line 241: Please reduce and standardize the font in all figures. Please make color charts.
Line 249: Figure 2. it should be bold Figure 2
Line 276: in figure 7 there is no N2O
Line 280: please format all tables, e.g. reduce font and line spacing
Line 307: are you sure the same trend for NH4?
Line 321-322: why? explain and develop the influence of pH on nitrification and denitrification genes
Line 324-325: how Fe2 and Fe3 influenced nitrifying and denitrifying microorganisms? look for references in the literature
Line 364: Conclusions- What is the conclusion from these studies for agricultural practice?
Line 391: References- please format as required by the journal
Author Response
Please see the attachment
Reviewer 2 Report
In my understanding, the text needs a language review to clarify a few points. For example, in the abstract, the sentence "The abundance of function microbes involved in nitrification and denitrification were determined by quantitative PCR", does not make much sense, and is likely meant to be "functional microbes" or even better just "microbes involved in". This is not the single point in which language review would enhance the manuscript.
In the material and methods section, the soils should be better described, including their taxonomic status, preferrably under the FAO WRB system. The WHC is a central point of this study, but neither the values, nor the method and definition used are presented. Why was WHC chosen to represent water status instead of pore-volumes for example?
In my understanding, the microcosm experiment is not sufficiently well described. For example, what do you mean by "activated soils"? Just the soils that were kept at 25C and 20% WHC for a week ? Can a 25 g sieved soil sample be considered sufficiently representative to discuss results in a 10 or 20 cm depth layer? Does it make sense to translate kg N.ha-1.year-1 into N rates for different soil layers independently, not considering that in a field condition (as per the field rate used as a basis), this N would be applied to one of the layers, likely the 0-10 cm, and then somehow affect the other layers?
I have serious objections to the Duncan test, since it protects the error in a comparison-wide basis. Thus the overall experiment-wide cumulative error is higher than the predicted by the test. Although with a relatively small experiment such as this the deviance is likely not major, I strongly recommend using an experiment-wide type of test, such as Tukey.
You affirm there is a trend of increasing N2O emission up to a peak in all treatments, but it is simply not true, since (as expected) it does not happen to those soils under 60 % WHC. Although you later mention the difference between this and the remaining treatments, your very first sentence of the results is not based on the actual data.
I have very strong objections on making field predictions based on an experiment like this, since you are basing them on an experimental setup in which N was equally applied to each soil layer, which most definitly would not be the case under field conditions, and all soil layers are equally exposed to the ambient atmosphere and at the same soil saturation, which again would not likely occur under field conditions. This is not meant to say the experiment does not have value, but that the extrapolation from it to the field conditions as in the second paragraph of section 3.1 is highly dubius, in my opinion at least. Besides this, Figure 2 indicates 0 emissions for the 20-40 cm layer. You also did not present the difference found between the soil behaviors, since while in the hydromorphic there is a very slight difference between 100 and 200% WHC, this difference is much more pronounced for the gleyed soil.
Although Table 2 indicates a significant interaction between soil layer and WHC, this is not presented in Figure 6, in which the means comparison test was made considering equally all nine combinations of a soil layer and a WHC, when it should be made separately for the soil layers for each WHC and vice-versa. From the data presentation in Table 2 it seems that the statistical analysis was conducted separately for each soil type, which was not my understanding from the statistical analysis section of the Material and Methods section of the manuscript.
I recommend always considering the size of the correlation (r value), rather than just if it is significant or not. For example, although there is indeed a significant correlation between Fe2+ content and N2O flux, it is a rather weak one, with r=0.280, and as such it is difficult to be confident on its implications to understand the system. Also there is simply no reason to include self-correlations (such as pH with pH) in this kind of table.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Round 2
Reviewer 2 Report
Although most of the previous comments were adequately covered, the manuscript still does nod define WHC as applied to this research, nor indicate why this was chosen,
Although the authors have corrected some of the data analysis problems previously mentioned, and indicate using a two-way factorial, the Tukey tests as presented, for example, in Figures 2, 5 or 6 and Table 1 do not indicate that the interaction was adequately evaluated by the Tukey test, since there is a single line of comparison of all soil x layer combinations, and not a comparison between soils for a given layer, or between layers for a given soil, as should be the case if the interaction was significant. On the other hand, if the interaction was not significant, then the comparison should be only between layers (disregarding soils) or soil (disregarding layers) according to the ANOVA.
As I said earlier, most of points were correctly addressed, but these are remaining issues with the manuscript at this point.
Author Response
Please see the attachment
Author Response File: Author Response.docx
