Effects of Simulated Nitrogen Deposition on the Bacterial Community of Urban Green Spaces
, Augusto Zanella 2, Xiaohua Chen 1, Bin Peng 1, Jiahui Lin 1, Jiaxuan Su 1, Xinghao Luo 1, Guoliang Xu 1,3,* and Andrea Squartini 4,*Round 1
Reviewer 1 Report
This article well indicates the relationship between N deposition and soil bacterial population structure in urban turf. I recommend the publication in Applied Sciences. Please check the following comments.
35. Which was atmospheric N indicated NOx or total N?
124. Please show an amount of 2M KCl.
141. Please show the mathematical condition of filtering and processing of MiSeq data.
153 Could you show the citation of "Anderson's PERMDISP procedure?
!62. Would you show the reference and version of the R package "betadisper"?
167. Nitrogen (N) deposition -> Inorganic nitrogen dosage
Increasing nitrogen deposition should be the result of N application.
168. Gi1 -> Table 1
175 total nitrogen and total carbon (TN and TC) -> total carbon (TC)
Fig. 4
Please show the contribution ratio of variation of each PCOA 1 and PCoA 2.
Was the PCoAs of each graph the same?
Could you show the meaning of "method=****"
244. 3.4
The soil chemical properties, except TC, might be interacting with each other strongly (Table 1). Could you calculate the correlation between the parameters of soil bacteria abundance and N dosage and between the soil properties? For example, the N dosage of N1 plots at 12months may be equal to N3 at three months.
Author Response
Response to Reviewer 1 Comments
POINT 1: 35. Which was atmospheric N indicated NOx or total N?
ANSWER 1: Both natural and anthropogenic sources contribute to atmospheric N deposition, but anthropogenic reactive N (Nr) emissions dominate N deposition at both continental and regional scales. Anthropogenic Nr is released into the atmosphere either as N oxides (NOx = NO + NO2), primarily from fossil fuel combustion, or as ammonia (NH3), primarily from agriculture. After a series of chemical conversions and physical transport, the ultimate fate of NOx and NH3 is removal by wet and dry deposition on terrestrial ecosystems.
POINT 2: 124. Please show an amount of 2M KCl.
ANSWER 2: Thank you for your suggestion. We have corrected it.
POINT 3: 141. Please show the mathematical condition of filtering and processing of MiSeq data.
ANSWER 3: Thank you for your suggestion. We have modified paragraph 2.3 and showed the mathematical condition of filtering and processing of raw data. The text introduced is the following: “Based on the raw data, pair-end reads were spliced using the principle of 98% overlap of 19 bases using the Connecting Overlapped Pair-End software 36. Barcode and primer sequences were then filtered to obtain the clean data. Operational Taxonomic Units (OTUs) were individuated as those clustering at shared nucleotide identity equal or higher than 97 %. The relative abundance of soil bacteria was calculated according to the species annotation and reads number.”
POINT 4: 153 Could you show the citation of "Anderson's PERMDISP procedure?
ANSWER 4: The link to calculation (with reference) has been added
http://scikit-bio.org/docs/0.5.4/generated/generated/skbio.stats.distance.permdisp.html
POINT 5: 162. Would you show the reference and version of the R package "betadisper"?
ANSWER 5: The description in in a web resource (https://www.rdocumentation.org/packages/vegan/versions/2.4-2/topics/betadisper). The version has been added (version 2.4-2, License: GPL-2)
POINT 6: 167. Nitrogen (N) deposition -> Inorganic nitrogen dosage. Increasing nitrogen deposition should be the result of N application.
ANSWER 6: Correction made.
POINT 7: 168. Gi1 -> Table 1
ANSWER 7: Thank you for your suggestion, we have corrected it.
POINT 8: 175 total nitrogen and total carbon (TN and TC) -> total carbon (TC)
ANSWER 8: Thank you for your suggestion, we have corrected it.
POINT 9: Fig. 4 Please show the contribution ratio of variation of each PCOA 1 and PCoA 2. Was the PCoAs of each graph the same? Could you show the meaning of "method=****"
ANSWER 9: The percentages of explained variation contribution have been added. The “method=**” line was a graphical text leftover error and has been removed.
POINT 10: 244. 3.4 The soil chemical properties, except TC, might be interacting with each other strongly (Table 1). Could you calculate the correlation between the parameters of soil bacteria abundance and N dosage and between the soil properties? For example, the N dosage of N1 plots at 12months may be equal to N3 at three months.
ANSWER 10: This is actually not a linear calculation that can be attempted. Soil has buffering capacity and resilience capability whose coefficients are not straightforwardly extrapolated; Moreover small doses were added along a large span of time. For those reasons, to compare N1 plots at 12 months (50) to N3 (150/12) at three months would be prone to an error which could be larger than the differences.
Hoping to have fulfilled all issues we thank you for your kind attention.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This study assessing the addition of N to simulate atmospheric N deposition is of relevant hypothesis and fits with the scope of the journal. The authors evaluated increasing N rates from 50 to 150 kg N ha-1 on soil chemical transformations and bacterial community changes. My main concern is with the N rates evaluated. For example, on lines 103 and 104, you said that the N deposition ranges from 30.1 to 43.3. In my view, this is the amount of N that N0 received by annual N deposition. If you are focused on the atmospheric N deposition effects, you should have compared with treatment without receiving any N deposition. Otherwise, we can not say that the current atmospheric N deposition is detrimental to the urban bacterial community once the rates you evaluated is not realistic with the annual N inputs. Other than that, I have specific comments that you may address before further consideration. See below:
L19: Which unit is that ha-2 a-1? Is it kg N per hectare per year? If so, please use ha-1 y-1.
L29: there are many keywords that was mentioned in the title already. I suggest you substitute or remove some of them. You can put something like, “ soil DNA extraction”, and also put the names of 2 major group of bacteria assessed here (i.e., Proteobacteria; Acidobacteria).
L36: nitrogen to N. Once you already mentioned what N means, please check throughout the text, there are many sentences where you can replace nitrogen per N.
L100 and 102: What you mean with 20 ~ 22 and 9 ~ 16?
L102 – 104: Are these rates per year? Why did you evaluate rates higher than those? It is not realistic to the current deposition.
L120: Whose procedure you followed? As far as I know, you should have stored it in a -80C fridge.
L168: What is Gi 1?
L168-170: why are you discussing your results here? It is out of place. It should be in the discussion section.
Table 1: It is very strange that NO3-N had no significant difference among N rates at 3 months sampling time.
L201-203: Here is not a place for the conclusion.
L222: which differences were those?
L300-304: does soil bacterial community is affected by the addition of N rates lower than 50 kg N ha-1?
L325-327: please transform units to Kg N ha-1.
L341: It is not realistic with the background you put in the introduction and M&M. What about the effect of N rates consistent with what is deposited annually?
Author Response
Response to Reviewer 2 Comments
This study assessing the addition of N to simulate atmospheric N deposition is of relevant hypothesis and fits with the scope of the journal. The authors evaluated increasing N rates from 50 to 150 kg N ha-1 on soil chemical transformations and bacterial community changes. My main concern is with the N rates evaluated. For example, on lines 103 and 104, you said that the N deposition ranges from 30.1 to 43.3. In my view, this is the amount of N that N0 received by annual N deposition. If you are focused on the atmospheric N deposition effects, you should have compared with treatment without receiving any N deposition. Otherwise, we can not say that the current atmospheric N deposition is detrimental to the urban bacterial community once the rates you evaluated is not realistic with the annual N inputs. Other than that, I have specific comments that you may address before further consideration. See below:
ANSWER: For the same exact reason it was not possible to find in China a soil that had not been already exposed to the background deposition. Given the increased industrial development in China and the prediction of its acceleration, the rates we have chosen to add are actually the ones that are expected to correspond to the customary inputs in the future.
POINT 1: L19: Which unit is that ha-2 a-1? Is it kg N per hectare per year? If so, please use ha-1 y-1.
ANSWER 1: Sorry for the confusion, ha-2 a-1 means kg N per hectare per year, we have corrected it.
POINT 2: L29: there are many keywords that was mentioned in the title already. I suggest you substitute or remove some of them. You can put something like, “soil DNA extraction”, and also put the names of 2 major group of bacteria assessed here (i.e., Proteobacteria; Acidobacteria).
ANSWER 2: Thank you for your suggestion, we have revised the keywords.
POINT 3: L36: nitrogen to N. Once you already mentioned what N means, please check throughout the text, there are many sentences where you can replace nitrogen per N.
ANSWER 3: Thank you for your suggestion, some of 'nitrogen' has been replaced to N.
POINT 4: L100 and 102: What you mean with 20 ~ 22 and 9 ~ 16?
ANSWER 4: Sorry for the confusion, we have checked the annual average temperature and average temperature in January of Guangzhou that are 21.5 °C and 13.3 °C, respectively. We have revised the issue in the manuscript.
POINT 5: L102 – 104: Are these rates per year? Why did you evaluate rates higher than those? It is not realistic to the current deposition.
ANSWER 5: Yes, these rates are per year. Nitrogen (N) deposition has rapidly increased in recent decades due to industrialization and anthropogenic activities. The mean rate of wet N deposition in China increased by about 25% from the 1990s to the 2000s and reached an average rate of 19 kg N ha−1 yr−1 from 2010 to 2014. Therefore, this study set a higher nitrogen deposition which tries to explain the potential risk of increasing nitrogen N deposition in the urban area.
The experimental design refers to this article: Guo-liang, XU, and, Jiang-ming, MO. (2007). Response of soil fauna to simulated nitrogen deposition: a nursery experiment in subtropical China. Journal of Environmental Sciences. https://doi.org/10.1016/S1001-0742(07)60100-4
POINT 6: L120: Whose procedure you followed? As far as I know, you should have stored it in a -80C fridge.
ANSWER 6: Sorry for the wrong figure, samples were stored at −80 °C until use. We have corrected it.
POINT 7: L168: What is Gi 1?
ANSWER 7: Sorry for the wrong word, ‘Gi1’ should have been ‘Table 1’, we have corrected it.
POINT 8: L168-170: why are you discussing your results here? It is out of place. It should be in the discussion section.
ANSWER 8: Thank you for your suggestion. The text has been moved to the discussion section.
POINT 9: Table 1: It is very strange that NO3-N had no significant difference among N rates at 3 months sampling time.
ANSWER 9: Nitrate, being an anion is a very mobile form in the soil, and in part it is bound to be leaching down and run off. Moreover soil has a buffering capacity which can mask changes in soil chemical properties within short term. Only gradually, when the homeostatic resilience is overcome, changes could become noticeble.
POINT 10: L201-203: Here is not a place for the conclusion.
ANSWER 10: Thank you for your suggestion. It has been moved to the discussion section.
POINT 11: L222: which differences were those?
ANSWER 11: Sorry for the confusion. Soil bacterial abundance after 12 months N addition were analyzed by one-way analysis of variance (ANOVA) to determine significant differences among the treatments. The relative abundance of Proteobacteria significantly differed among different treatments (P < 0.01). This figure will attach in the appendix.
Table S1. Effects of N deposition on the relative abundance of dominant phyla after 12 months. Different letters indicate significant differences among treatments within the same sampling time (P < 0.05, ANOVA)
POINT 12: L300-304: does soil bacterial community is affected by the addition of N rates lower than 50 kg N ha-1?
ANSWER 12: In the literature that we cite in that part (Ref. 54) authors showed that the addition of 35 kg N ha-1 was enough to cause changes. However, their control N background was already 33.5 kg N ha-1.
POINT 13: L325-327: please transform units to Kg N ha-1.
ANSWER 13: Thank you for your suggestion. The units have been transformed to Kg N ha-1.
POINT 14: L341: It is not realistic with the background you put in the introduction and M&M. What about the effect of N rates consistent with what is deposited annually?
ANSWER 14: This question can be referred to the answer anticipated for point 5. The industrialization lookout for China is showing a rapid increase and predicting an even faster development; thus the rates of anthropogenic N deposition and other environmental parameters are already showing a trend of intensification. In this sense the present study wishes to emphasize the alert for consequences of an emerging problem, which we are still on time to counteract.
Hoping to have fulfilled all issues we thank you for your kind attention.
Author Response File: Author Response.pdf
Reviewer 3 Report
The article by Mo et al. uses manipulation experiments to explore the effects of nitrogen deposition on microbial communities in grassland soils. They conclude that bacterial diversity and pH decline under high levels of N addition. I really like this paper, as the study of how environmental factors, especially those enhanced by humans, affect microbial communities in soils, and how that affects vegetation, is a cutting edge subject. Many modeling groups are now incorporating these effects into their models, so we need more studies of this kind. I do have some relatively minor suggestions. In particular, the authors need to make sure their description of results is consistent with what is being shown in the figures.
- Introduction can expand upon N deposition beyond just nitrous oxides. Some explanation of the NOy and NOx and wet vs dry deposition would be useful.
- Section 2.4: Explain in words how the Chao1 index describes richness and the Shannon Index describe diversity.
- Lines 175-177: The trends in TC are not really steady, and with both TN and TC not significant, not sure it makes sense to report them here. Also should refer to Table 1 somewhere in that paragraph.
- Paragraph lines 196-203 is repeated.
- Line 207: Define OUT
- Line 223: say Proteobacteria increased not differed.
- Line 251: Bacteroides does show significant correlation with pH.
- Line 253: Progeobacteria does NOT have a significant positive correlation with NH4+
- Line 264: pH, NH4_ and TN are positive correlations, while the others are negative
- Line 281: Capitalize “t” in “these”
- Line 290-292: Figure 5 shows correlations in different directions, depending on the phylum, for NH4+, NO3-, and pH. Table 2 shows positive correlation with pH. Really it is Table 1 that shows that relationships highlighted in this sentence. Please reconcile the positive correlation with pH in Table 2 with the overall effect of decreased pH.
- Line 299: Why highlighting these particular three phylum? Not true for Proteobacteria and NH4+
- Lines 313-314: Perhaps a little more discussion on whether pH affects the bacterial communities or the bacterial communities affect the pH, or whether it is N deposition making things more acidic, which is what then affects the bacterial communities.
- Last paragraph of the discussion: I am interested in what are the functional roles of each of the phylum in this study, or does functional role differ amongst orders within each phylum? This is important as one way to model the effects of microbial communities is to model them by functional role, so is it possible to classify each of the phyla in this study by function? Might also be nice to add a small discussion about how this study can be useful to ongoing modeling efforts.
- Is it possible that the lesser effect on NO3- is because nitrate is being leached, so that it just doesn’t show up as readily as NH4+? And, if that is so, is it something that could be getting into the runoff?
Author Response
Response to Reviewer 3 Comments
POINT 1: Introduction can expand upon N deposition beyond just nitrous oxides. Some explanation of the NOy and NOx and wet vs dry deposition would be useful.
ANSWER 1: Thank you for your suggestion. We have revised the first paragraph.
POINT 2: Section 2.4: Explain in words how the Chao1 index describes richness and the Shannon Index describe diversity.
ANSWER 2: Thank you for your suggestion. The definitions have been added.
POINT 3: Lines 175-177: The trends in TC are not really steady, and with both TN and TC not significant, not sure it makes sense to report them here. Also should refer to Table 1 somewhere in that paragraph.
ANSWER 3: Changes in TC and TN do occur, but they are both time-related and not treatment-related. The text there now specifies it “…total carbon (TN and TC) did not show significant differences (P > 0.05) within the N gradient of the same time point, changes were nevertheless occurring in a time-dependent fashion with a steady decrease of TC and an increase of TN…”. The referral to Table 1 was added at the beginning of the paragraph.
POINT 4: Paragraph lines 196-203 is repeated.
ANSWER 4: Very sorry for the wrong text, we have deleted it.
POINT 5: Line 207: Define OUT
ANSWER 5: Thank you for your suggestion. We have modified the 2.3 and define OTU there (Operational Technical Units).
POINT 6: Line 223: say Proteobacteria increased not differed.
ANSWER 6: Sorry for the confusion. Soil bacterial abundance after 12 months of N addition were analyzed by one-way analysis of variance (ANOVA) to determine significant differences among the treatments. The relative abundance of Proteobacteria significantly differed among different treatments (P < 0.01). This figure will be added attach in appendix.
Table S1. Effects of N deposition on the relative abundance of dominant phyla after 12 months. Different letters indicate significant differences among treatments within the same sampling time (P < 0.05, ANOVA)
POINT 7: Line 251: Bacteroides does show significant correlation with pH.
ANSWER 7: Very sorry for the wrong text, we have deleted it.
POINT 8: Line 253: Progeobacteria does NOT have a significant positive correlation with NH4+
ANSWER 8: Very sorry for the wrong text, we have revised it.
POINT 9: Line 264: pH, NH4_ and TN are positive correlations, while the others are negative
ANSWER 9: Sorry for the confusion, we have corrected it. The correlation coefficients, in decreasing order of absolute values, were, for positive correlations: pH, NH4+-N and TN; while for negative correlations: TC, and NO3—N.
POINT 10: Line 281: Capitalize “t” in “these”
ANSWER 10: Thank you for your suggestion, we have corrected it.
POINT 11: Line 290-292: Figure 5 shows correlations in different directions, depending on the phylum, for NH4+, NO3-, and pH. Table 2 shows positive correlation with pH. Really it is Table 1 that shows that relationships highlighted in this sentence. Please reconcile the positive correlation with pH in Table 2 with the overall effect of decreased pH.
ANSWER 11: the non-opposite trend for pH and ammonium appearing from Table.2 is due to the fact that, although ecological diversity indexes are abated by increasing ammonium and decreasing pH, nevertheless a number of numerically-relevant phyla are enhanced, and thus the overall value of community structure is not as impaired by such substitutions in relative abundance as the Simpson and evenness parameters are.
It must be observed however that in Table 2, while for the pH parameter, the p value is very significant, for the ammonium nitrogen, significance is borderline and the r value is very low.
POINT 12: Line 299: Why highlighting these particular three phylum? Not true for Proteobacteria and NH4+
ANSWER 12: Sorry for the confusion, it has been revised. We want to express that most of the phyla showed the correlations of abundances with soil pH and NH4+-N.
POINT 13: Lines 313-314: Perhaps a little more discussion on whether pH affects the bacterial communities, or the bacterial communities affect the pH, or whether it is N deposition making things more acidic, which is what then affects the bacterial communities.
ANSWER 13: The direct action of bacteria as causes of acidification is limited to environments where lactic fermentation can take place (food contexts) or sulphide oxidation (mine ores) while in soils bacteria have passive roles with respect to pH. We have implemented this point by adding some general chemistry and microbiology comments as follows. “The reason for a pH decrease as a consequence of ammonium-based fertilization is mainly to be seen in the fact that when ammonium undergoes nitrification it leaves net H+ ions into the circulating solution. Bacterial sensitivity to acidic conditions is widespread and mainly due to the scarce attitude to maintain neutrality within the cytoplasm to avoid amino acid charged groups depolarization. “
POINT 14: Last paragraph of the discussion: I am interested in what are the functional roles of each of the phylum in this study, or does functional role differ amongst orders within each phylum? This is important as one way to model the effects of microbial communities is to model them by functional role, so is it possible to classify each of the phyla in this study by function? Might also be nice to add a small discussion about how this study can be useful to ongoing modeling efforts.
ANSWER 14: Beside the groups and phyla for which, throughout the discussion, we have cited some knowledgeably prevailing ‘average’ attitudes, it is not possible to generalize such aspect. The reason is the illegitimate phylogeny that characterizes prokaryotes where horizontal gene exchange is often occurring (unlike the case that regards sexually-reproducing organisms, as plants and animals). While bacterial taxonomy (and its resulting phyla) is constructed on the basis of ribosomal RNA sequences alignment, the actual physiology of each species is often the result of lateral gene transfer of plasmids or chromosome islands that can completely scramble the physiology of the recipient taxon with respect to the rest of its vertical phylogeny clade allies.
POINT 15: Is it possible that the lesser effect on NO3- is because nitrate is being leached, so that it just doesn’t show up as readily as NH4+? And, if that is so, is it something that could be getting into the runoff?
ANSWER 15: If you refer to Table 2, the point is actually relevant as anions are surely much more mobile than cations and their fate is indeed bound to a runoff leaching. Anyhow, judging from Table 1, both for ammonium and for nitrate we observe similarly occurring significant differences linked to the time and concentrations, notwithstanding the differences in quantity which are in agreement with your comment.
Hoping to have fulfilled all issues we thank you for your kind attention.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The paper can be accepted. I just have a few minor suggestions below:
L-29: “Bacterial bacteria” or just “bacteria”?
L-101: you wrote “21.5” with a different font.
L207 and 208: You used “for this reason” twice very shortly. I suggest you change “for this reason” in L208 per “Therefore.”
God job
Author Response
Point 1: L-29: “Bacterial bacteria” or just “bacteria”?
Answer 1: Sorry for the confusion. It should be 'Bacteria'.
Point 2: L-101: you wrote “21.5” with a different font.
Answer 2: It has been formatted and styled.
Point 3: L207 and 208: You used “for this reason” twice very shortly. I suggest you change “for this reason” in L208 per “Therefore.”
Answer 3: Thank you for your suggestion. It has been changed to 'Therefore'.
