the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 12 Jan 2023
Evidence of Nitrogen Loss from Anaerobic Ammonium Oxidation Coupled with Ferric Iron Reduction in the Yellow River Wetland
Abstract. Anaerobic ammonium oxidation coupled with iron(III) reduction (Feammox) is a recently discovered pathway of nitrogen removal. However, little is known about the pathways of N transformation via Feammox in the Yellow River wetland. In this study, the difference between Feammox in a natural wetland (site YJW) and a crop rotation wetland (site TEH) was researched using isotope tracing and metagenome techniques. The results revealed that Feammox occurred in TEH but not in YJW. The Feammox rates in the TEH samples were 0.02–0.13 mg N kg−1 d−1 in different depth intervals (0–5 cm, 5–10 cm, 10–20 cm, and 20–30 cm), and the maximum value for TEH occurred in the 5–10 cm depth interval. Iron reducing bacteria play an essential role in Feammox. Rotational tillage reduced the microbial diversity of the iron-reducing bacteria, but it increased the abundance of iron-reducing bacteria at the genus level, and the dominate iron-reducing bacteria responsible for the Feammox process were Anaeromyxobacter and Geobacter. The Feammox rate was less than the denitrification rate (0.55–1.09 mg N kg−1 d−1), an estimated nitrogen loss of 1.1–7.1 t N km−2 a−1 was associated with the Feammox in the wetland. However, the correlation between the functional genes of the iron-reducing bacteria and the rate remains unclear. Overall, the co-occurrence of ammonium oxidation and iron reduction suggest that Feammox can play an essential role in the pathway of nitrogen removal in the Yellow River wetland.
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Status: closed
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RC1: 'Comment on egusphere-2022-1209', Anonymous Referee #1, 02 Mar 2023
Comments: The authors examined microbial activities and microbes involved in anaerobic ammonium oxidation with iron reduction (Feammox). They performed 15N tracing experiments and metagenomic analysis using samples collected from a Yellow River wetland. Innovatively, this study provides a more in-depth research perspective than earlier Feammox publications. It is suggested that this work be accepted with revisions.
1. Total nitrogen loss = Denitrification+ Anammox + Feammox, how do you calculate each nitrogen removal efficiency? It is not mentioned in the experiment.
2. Line 32: There should be NO2− rather than NO3− in chemical equation of anammox.
3. Line 39: Why is Equation 1 significant in the studied condition? Possibly more clarification is required. For instance, from the standpoint of under neutral pH, more dissolved Fe (II) and/or low organic matter conditions (please ref: Tan, X., Xie, G.-J., Nie, W.-B., Xing, D.-F., Liu, B.-F., Ding, J. and Ren, N.-Q. (2022) Fe(III)-mediated anaerobic ammonium oxidation: A novel microbial nitrogen cycle pathway and potential applications. Crit Rev Env Sci Tec 52, 2962-2994.)
4. Line 40-45: Fe2+ should be Fe2+.
5. Line 63-69: Why is it important to specifically characterize Feammox contribution to nitrogen loss in the Yellow River wetland? The authors should state the motivation for the study.
6. Line 105: Were the residual nitrite and nitrate verified after the 2-day pre-incubation? If residual nitrite and nitrate are present, the isotopic tracer experiment results will be impacted.
7. Line 119: This is not metagenomic analysis, but 16S rRNA diversity analysis.
8. Line 162: 73% of what?
9. Line 164: Sediment is the topic of this paragraph. Please clarify it here. How was TOC determined? It is not addressed in the methodology section.
10. Line 175: Regarding the generation concentration-time of 29N2 and 30N2, does Fig 2 represent sediment from YJW and Fig 3 from TEH? I suppose that the authors should rewrite it.
11. Line 179: “…the samples accumulated 29N2 at a faster rate than…” the samples here refer to these from TEH? I suggest rewriting the context appropriately.
12. Line 191: What does 8.3% in parenthesis represent?
13. Line 196: please clarify that the 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, are the rates of the control of YJW and TEH, respectively.
14. Line 207: It is surprising that no microorganisms in natural wetland samples at this depth. Have the authors used other methods to verify this finding?
15. Line 207-209: "At the phylum level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, Firmicutes, and Cyanobacteria have been classified as FeRB" This statement is not correct. How can such a wide range of microorganisms at phylum be classified as FeRB? It's not serious.
16. Line 224: Please clarify that the Feammox rate of the YJW site was lower than that of the TEH site.
17. Line 272: The genus level should be italicized.
18. Lines 309 – 359: Please note that the format of the reference citation should be uniform.
Full listing of the article's contributors is required (e.g., not et al. in line 309)
Either capitalize or lowercase all initial letters of the article title (except the first word).
Journals either use full spelling or abbreviations.
The page numbers follow a beginning and ending format. etc.
Authors should refer to the journal's recommended format and standardize it.
19. Line 392: Spelling error, change dissimilary to dissimilatory.
20. Lines 396 – 409: For a more intuitive reading, it is recommended to indicate the concentration of the added chemical in the caption of the corresponding graph (if applicable).
21. Lines 411,414, 417 and 420: To make your content accessible, you should have a high contrast between the color of your text and its background. For text box, black font on a white backdrop is recommended.
22. It is necessary to have at least two sampling times in two different seasons with markedly different climatic conditions. Soil microbial diversity changes during the year.
23. Line 210-214: "relative abundance of the microbial diversity" and "dominant genus" are based on all microbial taxa detected in the samples or only within the FeRB group.Citation: https://doi.org/10.5194/egusphere-2022-1209-RC1 -
AC1: 'Reply on RC1', Qingsong Guan, 18 Apr 2023
Dear Professor Mariet Hefting,
Thank you very much for giving us an opportunity to reply. I appreciate the editor and reviewers very much for their constructive comments and suggestions for improving our manuscript.
Below you will find point-by-point responses to the reviewers’ comments.
Best regards,
Qingsong Guan
Corresponding author: Qingsong Guan
Email: guanqingsong163@163.com
- Total nitrogen loss = Denitrification+ Anammox + Feammox, how do you calculate each nitrogen removal efficiency? It is not mentioned in the experiment.
Response: Denitrification and Anammox and Feammox rates can be calculated separately. The part of “Appendix I Denitrification and Annamox Rate” indicted the method of Denitrification and Anammox rate. We will add more descriptions in the text.
- Line 32: There should be NO2− rather than NO3− in chemical equation of anammox.
Response: we will revise NO3− to NO2−.
- Line 39: Why is Equation 1 significant in the studied condition? Possibly more clarification is required. For instance, from the standpoint of under neutral pH, more dissolved Fe (II) and/or low organic matter conditions (please ref: Tan, X., Xie, G.-J., Nie, W.-B., Xing, D.-F., Liu, B.-F., Ding, J. and Ren, N.-Q. (2022) Fe(III)-mediated anaerobic ammonium oxidation: A novel microbial nitrogen cycle pathway and potential applications. Crit Rev Env Sci Tec 52, 2962-2994.)
Response: we will add more information according to the suggestion.
- Line 40-45: Fe2+ should be Fe2+.
Response: it should be Fe2+.
- Line 63-69: Why is it important to specifically characterize Feammox contribution to nitrogen loss in the Yellow River wetland? The authors should state the motivation for the study.
Response: we will further summarize the motivation for the study.
- Line 105: Were the residual nitrite and nitrate verified after the 2-day pre-incubation? If residual nitrite and nitrate are present, the isotopic tracer experiment results will be impacted.
Response: the residual nitrite and nitrate after the 2-day pre-incubation was low, which can be ignored in comparison to the concentration of ammonium.
- Line 119: This is not metagenomic analysis, but 16S rRNA diversity analysis.
Response: we accept it.
- Line 162: 73% of what?
Response: it should be “The Fe(II) content of the THE account for 73% of total iron”
- Line 164: Sediment is the topic of this paragraph. Please clarify it here. How was TOC determined? It is not addressed in the methodology section.
Response: TOC and TN were determined using an elemental analyzer (Thermo Fisher Flash 2000 CHNS/O)
- Line 175: Regarding the generation concentration-time of 29N2 and 30N2, does Fig 2 represent sediment from YJW and Fig 3 from TEH? I suppose that the authors should rewrite it.
Response: it should be “Fig. 2 and 3 show the changes in the 29N2 and 30N2 concentrations with incubation time for the YJW and TEH, respectively”.
- Line 179: “…the samples accumulated 29N2 at a faster rate than…” the samples here refer to these from TEH? I suggest rewriting the context appropriately.
Response: it should be “The 15NH4+ and 15NH4+ + Fe(Ⅲ) treatments of the THE samples accumulated 29N2 at a faster rate than those of the corresponding YJW samples.”
- Line 191: What does 8.3% in parenthesis represent?
Response: delete (8.3%).
- Line 196: please clarify that the 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, are the rates of the control of YJW and TEH, respectively.
Response: it should be “The reduction rates of the 15NH4+ addition and the control groups were 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, respectively (Fig. 5).”
- Line 207: It is surprising that no microorganisms in natural wetland samples at this depth. Have the authors used other methods to verify this finding?
Response: in YJW site, the soil below is sand below 13cm, he macrogenomic analysis showed no obvious DNA bands, and we are sorry that no other methods were used.
- Line 207-209: "At the phylum level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, Firmicutes, and Cyanobacteria have been classified as FeRB" This statement is not correct. How can such a wide range of microorganisms at phylum be classified as FeRB? It's not serious.
Response: Actually, this statement cites from a reference (Peng et al., 2016).
- Line 224: Please clarify that the Feammox rate of the YJW site was lower than that of the TEH site.
Response: it should be “The Feammox rate of the YJW site was lower than that of the TEH site”
- Line 272: The genus level should be italicized.
Response: we will correct it.
- Lines 309 – 359: Please note that the format of the reference citation should be uniform.
l Full listing of the article's contributors is required (e.g., not et al. in line 309)
l Either capitalize or lowercase all initial letters of the article title (except the first word).l Journals either use full spelling or abbreviations.
l The page numbers follow a beginning and ending format. etc.
Authors should refer to the journal's recommended format and standardize it.
Response: we will revise the references format.
- Line 392: Spelling error, change dissimilary to dissimilatory.
Response: we will revise dissimilary to dissimilatory.
- Lines 396 – 409: For a more intuitive reading, it is recommended to indicate the concentration of the added chemical in the caption of the corresponding graph (if applicable).
Response: we will add the concentration of the added chemical.
- Lines 411,414, 417 and 420: To make your content accessible, you should have a high contrast between the color of your text and its background. For text box, black font on a white backdrop is recommended.
Response: we accept this suggestion.
- It is necessary to have at least two sampling times in two different seasons with markedly different climatic conditions. Soil microbial diversity changes during the year.
Response: we will continue to collect seasonal samples.
- Line 210-214: "relative abundance of the microbial diversity" and "dominant genus" are based on all microbial taxa detected in the samples or only within the FeRB group.
Response: only within the FeRB group.
Citation: https://doi.org/10.5194/egusphere-2022-1209-AC1
-
AC1: 'Reply on RC1', Qingsong Guan, 18 Apr 2023
-
RC2: 'Comment on egusphere-2022-1209', Anonymous Referee #2, 03 Apr 2023
The authors investigated ammonium oxidation in sediments, collected at two locations in the vicinity of the Yellow River. They closely followed the approach used in previous studies on the same subject. Sediment material was incubated with 15N NH4 and the formation of 15N containing N2 was taken as an indication for Feammox. Additionally, molecular techniques were used to characterize the microbial community. I have the impression that the quality of the data is good and the isotope incubation experiments has been adequately performed. However, a specific research objective, question or hypothesis is missing so that it remained unclear which knowledge gap this study could fill.
In the past years, the Feammox process has been extensively investigated in many environments. Measurements of the rates of Feammox and the corresponding nitrogen loss in diverse environments is relevant for evaluating the global relevance of this process, although not very innovative. In this type of studies, the selection of field sites should be justified based on the relevance of a specific environment or as part of a systematic study. This is, however, not the case for the study performed by Guan et al. How can the two sites contribute to assessing the relevance of Feammox for the N budget in the Yellow River catchment? The study contrasts a natural wetland with a crop rotation wetland, but it does not become clear whether the differences between the sites are caused by different land use or other factors. Hence, the study is not suitable to elucidate the role of one specific factor on Feammox rates. Consequently, the added value of the presented study is not well established besides reporting Feammox rates for two more locations.
Next to the low relevance of the study the manuscript is not suitable for publication for several reasons.
- The main conclusion is not sufficiently supported by the data. The authors attribute differences in Feammox rates to the rotational planting and harvesting (line 298). The two investigated sites differ regarding many environmental factors, potentially exerting influence on Feammox rates. It is unlikely, that differences, for example in Fe contents, can be solely attributed to the difference in land use. Additionally, the conclusion is not an outcome of the discussion and the major part of the related section in the discussion is devoted to comparing the measured rates to those reported in previous studies. The latter discussion does not lead to conclusions and only lists multiple factors influencing Feammox rates in general.
- The methodology and data processing is often not transparent and adequately described. This includes, for example: a) for the chemical analyses often only the instrument is listed but nothing is said about the methodology, detection limit, reproducibility and no references to the origin of the methods is given; b) in the method section, only Fe(III) measurements for water samples are mentioned but Fe(II) and Fe(III) concentrations are reported; c) it is unclear how nitrogen species have been determined for sediment samples, most probable the sediments have been extracted but no extraction is described; d) it is not explicitly explained how rates have been determined and normalized. The description of the procedure for Feammox rates in line 184 and following is confusing and inconsistent. The authors distinguish potential Feammox rate and Feammox rate but they are not discussed or presented separately. The procedure to determine nitrogen loss is cryptic and it is unclear how larger rates can lead to smaller nitrogen loss (when comparing with literature values); e) explain the choices for depth intervals. This is only a selection to illustrate the problem.
- The methodology and data processing has weaknesses. a) All conclusions are based on 29N2 accumulation, which is a clear indicator for ammonium oxidation, but the data should be presented in the context of a nitrogen mass balance. The accumulation of N2 should have been reported if measured. What are the initial reservoirs in NH4 (labeled and unlabeled). The initial ratio should be reflected in the ratio between 29N2, 30N2 and 28N2 production – or not? In general, concentrations should be reported in mol to facilitate the evaluation of stoichiometries and comparing Fe(II) and N2 production.
b)Fe(II) production rates are reported but only relying on the final Fe(II) contents. Without any knowledge about the temporal evolution no statements about rates can be made. Instead, the extents of Fe(II) and N2 production could be compared for the end of the experiment.
c) Prior to ammonium addition the sediments are incubated under anoxic conditions in order to remove nitrate and oxygen. However, it is not tested whether nitrate and oxygen have been indeed completely consumed after two days.
d) The authors consider multiple pathways for 29N2 and 30N2 production (Table 3) but, eventually, 29N2 production was exclusively attributed to Feammox.
Furthermore, the quality of presentation is low. Several statements appeared pointless to me and I was wondering whether I missed an important aspect due to the phrasing. However, there are also several obvious flaws, which should have been noticed before submission. Here I am only listing a few:
- Table 3 is literally the same as Table 1 in Yang etal (2012), this is plagiarism when no indicated
- Line32: nitrite is used in the annamox reaction or not?
- Line 36: microorganisms are oxidized
- Line 40: DrGm needs to be defined, is it under standard state besides pH 7? For which temperature.
- Line 56 – Line 62: the whole section is repetitive or contains redundant information.
- Line 57: including paddy soil….paddy soil.
- Site description: I do not understand the operation of the dam, is it indeed used to discharge muddy soil in summer? How can it store clear water in winter and spring when the rain is concentrated in July and September?
- Line 90: unclear what the random sampling scheme is. I got the impression that the sampling was not random but two specific sites and distinct depth intervals were selected.
- I suspect that “TEH” has been auto-corrected to “the” a few times and, by this, the sentence lost its meaning (e.g. Line 175 , line 179, line 224 etc.)
Citation: https://doi.org/10.5194/egusphere-2022-1209-RC2 -
AC2: 'Reply on RC2', Qingsong Guan, 18 Apr 2023
Thank you very much for the reviewer comment our manuscript.
The reviewer trusts our research data and process. However, the reviewer points out that a specific research objective, question or hypothesis is missing, and the description of methods is not specific enough. Also, the main conclusion is not sufficiently supported by the data.
We will make modifications according to the comments provided by the reviewer to ensure that they meet your requirements.
Citation: https://doi.org/10.5194/egusphere-2022-1209-AC2
Status: closed
-
RC1: 'Comment on egusphere-2022-1209', Anonymous Referee #1, 02 Mar 2023
Comments: The authors examined microbial activities and microbes involved in anaerobic ammonium oxidation with iron reduction (Feammox). They performed 15N tracing experiments and metagenomic analysis using samples collected from a Yellow River wetland. Innovatively, this study provides a more in-depth research perspective than earlier Feammox publications. It is suggested that this work be accepted with revisions.
1. Total nitrogen loss = Denitrification+ Anammox + Feammox, how do you calculate each nitrogen removal efficiency? It is not mentioned in the experiment.
2. Line 32: There should be NO2− rather than NO3− in chemical equation of anammox.
3. Line 39: Why is Equation 1 significant in the studied condition? Possibly more clarification is required. For instance, from the standpoint of under neutral pH, more dissolved Fe (II) and/or low organic matter conditions (please ref: Tan, X., Xie, G.-J., Nie, W.-B., Xing, D.-F., Liu, B.-F., Ding, J. and Ren, N.-Q. (2022) Fe(III)-mediated anaerobic ammonium oxidation: A novel microbial nitrogen cycle pathway and potential applications. Crit Rev Env Sci Tec 52, 2962-2994.)
4. Line 40-45: Fe2+ should be Fe2+.
5. Line 63-69: Why is it important to specifically characterize Feammox contribution to nitrogen loss in the Yellow River wetland? The authors should state the motivation for the study.
6. Line 105: Were the residual nitrite and nitrate verified after the 2-day pre-incubation? If residual nitrite and nitrate are present, the isotopic tracer experiment results will be impacted.
7. Line 119: This is not metagenomic analysis, but 16S rRNA diversity analysis.
8. Line 162: 73% of what?
9. Line 164: Sediment is the topic of this paragraph. Please clarify it here. How was TOC determined? It is not addressed in the methodology section.
10. Line 175: Regarding the generation concentration-time of 29N2 and 30N2, does Fig 2 represent sediment from YJW and Fig 3 from TEH? I suppose that the authors should rewrite it.
11. Line 179: “…the samples accumulated 29N2 at a faster rate than…” the samples here refer to these from TEH? I suggest rewriting the context appropriately.
12. Line 191: What does 8.3% in parenthesis represent?
13. Line 196: please clarify that the 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, are the rates of the control of YJW and TEH, respectively.
14. Line 207: It is surprising that no microorganisms in natural wetland samples at this depth. Have the authors used other methods to verify this finding?
15. Line 207-209: "At the phylum level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, Firmicutes, and Cyanobacteria have been classified as FeRB" This statement is not correct. How can such a wide range of microorganisms at phylum be classified as FeRB? It's not serious.
16. Line 224: Please clarify that the Feammox rate of the YJW site was lower than that of the TEH site.
17. Line 272: The genus level should be italicized.
18. Lines 309 – 359: Please note that the format of the reference citation should be uniform.
Full listing of the article's contributors is required (e.g., not et al. in line 309)
Either capitalize or lowercase all initial letters of the article title (except the first word).
Journals either use full spelling or abbreviations.
The page numbers follow a beginning and ending format. etc.
Authors should refer to the journal's recommended format and standardize it.
19. Line 392: Spelling error, change dissimilary to dissimilatory.
20. Lines 396 – 409: For a more intuitive reading, it is recommended to indicate the concentration of the added chemical in the caption of the corresponding graph (if applicable).
21. Lines 411,414, 417 and 420: To make your content accessible, you should have a high contrast between the color of your text and its background. For text box, black font on a white backdrop is recommended.
22. It is necessary to have at least two sampling times in two different seasons with markedly different climatic conditions. Soil microbial diversity changes during the year.
23. Line 210-214: "relative abundance of the microbial diversity" and "dominant genus" are based on all microbial taxa detected in the samples or only within the FeRB group.Citation: https://doi.org/10.5194/egusphere-2022-1209-RC1 -
AC1: 'Reply on RC1', Qingsong Guan, 18 Apr 2023
Dear Professor Mariet Hefting,
Thank you very much for giving us an opportunity to reply. I appreciate the editor and reviewers very much for their constructive comments and suggestions for improving our manuscript.
Below you will find point-by-point responses to the reviewers’ comments.
Best regards,
Qingsong Guan
Corresponding author: Qingsong Guan
Email: guanqingsong163@163.com
- Total nitrogen loss = Denitrification+ Anammox + Feammox, how do you calculate each nitrogen removal efficiency? It is not mentioned in the experiment.
Response: Denitrification and Anammox and Feammox rates can be calculated separately. The part of “Appendix I Denitrification and Annamox Rate” indicted the method of Denitrification and Anammox rate. We will add more descriptions in the text.
- Line 32: There should be NO2− rather than NO3− in chemical equation of anammox.
Response: we will revise NO3− to NO2−.
- Line 39: Why is Equation 1 significant in the studied condition? Possibly more clarification is required. For instance, from the standpoint of under neutral pH, more dissolved Fe (II) and/or low organic matter conditions (please ref: Tan, X., Xie, G.-J., Nie, W.-B., Xing, D.-F., Liu, B.-F., Ding, J. and Ren, N.-Q. (2022) Fe(III)-mediated anaerobic ammonium oxidation: A novel microbial nitrogen cycle pathway and potential applications. Crit Rev Env Sci Tec 52, 2962-2994.)
Response: we will add more information according to the suggestion.
- Line 40-45: Fe2+ should be Fe2+.
Response: it should be Fe2+.
- Line 63-69: Why is it important to specifically characterize Feammox contribution to nitrogen loss in the Yellow River wetland? The authors should state the motivation for the study.
Response: we will further summarize the motivation for the study.
- Line 105: Were the residual nitrite and nitrate verified after the 2-day pre-incubation? If residual nitrite and nitrate are present, the isotopic tracer experiment results will be impacted.
Response: the residual nitrite and nitrate after the 2-day pre-incubation was low, which can be ignored in comparison to the concentration of ammonium.
- Line 119: This is not metagenomic analysis, but 16S rRNA diversity analysis.
Response: we accept it.
- Line 162: 73% of what?
Response: it should be “The Fe(II) content of the THE account for 73% of total iron”
- Line 164: Sediment is the topic of this paragraph. Please clarify it here. How was TOC determined? It is not addressed in the methodology section.
Response: TOC and TN were determined using an elemental analyzer (Thermo Fisher Flash 2000 CHNS/O)
- Line 175: Regarding the generation concentration-time of 29N2 and 30N2, does Fig 2 represent sediment from YJW and Fig 3 from TEH? I suppose that the authors should rewrite it.
Response: it should be “Fig. 2 and 3 show the changes in the 29N2 and 30N2 concentrations with incubation time for the YJW and TEH, respectively”.
- Line 179: “…the samples accumulated 29N2 at a faster rate than…” the samples here refer to these from TEH? I suggest rewriting the context appropriately.
Response: it should be “The 15NH4+ and 15NH4+ + Fe(Ⅲ) treatments of the THE samples accumulated 29N2 at a faster rate than those of the corresponding YJW samples.”
- Line 191: What does 8.3% in parenthesis represent?
Response: delete (8.3%).
- Line 196: please clarify that the 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, are the rates of the control of YJW and TEH, respectively.
Response: it should be “The reduction rates of the 15NH4+ addition and the control groups were 0–66 mg Fe kg−1 d−1 and 2–5 mg Fe kg−1 d−1, respectively (Fig. 5).”
- Line 207: It is surprising that no microorganisms in natural wetland samples at this depth. Have the authors used other methods to verify this finding?
Response: in YJW site, the soil below is sand below 13cm, he macrogenomic analysis showed no obvious DNA bands, and we are sorry that no other methods were used.
- Line 207-209: "At the phylum level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, Firmicutes, and Cyanobacteria have been classified as FeRB" This statement is not correct. How can such a wide range of microorganisms at phylum be classified as FeRB? It's not serious.
Response: Actually, this statement cites from a reference (Peng et al., 2016).
- Line 224: Please clarify that the Feammox rate of the YJW site was lower than that of the TEH site.
Response: it should be “The Feammox rate of the YJW site was lower than that of the TEH site”
- Line 272: The genus level should be italicized.
Response: we will correct it.
- Lines 309 – 359: Please note that the format of the reference citation should be uniform.
l Full listing of the article's contributors is required (e.g., not et al. in line 309)
l Either capitalize or lowercase all initial letters of the article title (except the first word).l Journals either use full spelling or abbreviations.
l The page numbers follow a beginning and ending format. etc.
Authors should refer to the journal's recommended format and standardize it.
Response: we will revise the references format.
- Line 392: Spelling error, change dissimilary to dissimilatory.
Response: we will revise dissimilary to dissimilatory.
- Lines 396 – 409: For a more intuitive reading, it is recommended to indicate the concentration of the added chemical in the caption of the corresponding graph (if applicable).
Response: we will add the concentration of the added chemical.
- Lines 411,414, 417 and 420: To make your content accessible, you should have a high contrast between the color of your text and its background. For text box, black font on a white backdrop is recommended.
Response: we accept this suggestion.
- It is necessary to have at least two sampling times in two different seasons with markedly different climatic conditions. Soil microbial diversity changes during the year.
Response: we will continue to collect seasonal samples.
- Line 210-214: "relative abundance of the microbial diversity" and "dominant genus" are based on all microbial taxa detected in the samples or only within the FeRB group.
Response: only within the FeRB group.
Citation: https://doi.org/10.5194/egusphere-2022-1209-AC1
-
AC1: 'Reply on RC1', Qingsong Guan, 18 Apr 2023
-
RC2: 'Comment on egusphere-2022-1209', Anonymous Referee #2, 03 Apr 2023
The authors investigated ammonium oxidation in sediments, collected at two locations in the vicinity of the Yellow River. They closely followed the approach used in previous studies on the same subject. Sediment material was incubated with 15N NH4 and the formation of 15N containing N2 was taken as an indication for Feammox. Additionally, molecular techniques were used to characterize the microbial community. I have the impression that the quality of the data is good and the isotope incubation experiments has been adequately performed. However, a specific research objective, question or hypothesis is missing so that it remained unclear which knowledge gap this study could fill.
In the past years, the Feammox process has been extensively investigated in many environments. Measurements of the rates of Feammox and the corresponding nitrogen loss in diverse environments is relevant for evaluating the global relevance of this process, although not very innovative. In this type of studies, the selection of field sites should be justified based on the relevance of a specific environment or as part of a systematic study. This is, however, not the case for the study performed by Guan et al. How can the two sites contribute to assessing the relevance of Feammox for the N budget in the Yellow River catchment? The study contrasts a natural wetland with a crop rotation wetland, but it does not become clear whether the differences between the sites are caused by different land use or other factors. Hence, the study is not suitable to elucidate the role of one specific factor on Feammox rates. Consequently, the added value of the presented study is not well established besides reporting Feammox rates for two more locations.
Next to the low relevance of the study the manuscript is not suitable for publication for several reasons.
- The main conclusion is not sufficiently supported by the data. The authors attribute differences in Feammox rates to the rotational planting and harvesting (line 298). The two investigated sites differ regarding many environmental factors, potentially exerting influence on Feammox rates. It is unlikely, that differences, for example in Fe contents, can be solely attributed to the difference in land use. Additionally, the conclusion is not an outcome of the discussion and the major part of the related section in the discussion is devoted to comparing the measured rates to those reported in previous studies. The latter discussion does not lead to conclusions and only lists multiple factors influencing Feammox rates in general.
- The methodology and data processing is often not transparent and adequately described. This includes, for example: a) for the chemical analyses often only the instrument is listed but nothing is said about the methodology, detection limit, reproducibility and no references to the origin of the methods is given; b) in the method section, only Fe(III) measurements for water samples are mentioned but Fe(II) and Fe(III) concentrations are reported; c) it is unclear how nitrogen species have been determined for sediment samples, most probable the sediments have been extracted but no extraction is described; d) it is not explicitly explained how rates have been determined and normalized. The description of the procedure for Feammox rates in line 184 and following is confusing and inconsistent. The authors distinguish potential Feammox rate and Feammox rate but they are not discussed or presented separately. The procedure to determine nitrogen loss is cryptic and it is unclear how larger rates can lead to smaller nitrogen loss (when comparing with literature values); e) explain the choices for depth intervals. This is only a selection to illustrate the problem.
- The methodology and data processing has weaknesses. a) All conclusions are based on 29N2 accumulation, which is a clear indicator for ammonium oxidation, but the data should be presented in the context of a nitrogen mass balance. The accumulation of N2 should have been reported if measured. What are the initial reservoirs in NH4 (labeled and unlabeled). The initial ratio should be reflected in the ratio between 29N2, 30N2 and 28N2 production – or not? In general, concentrations should be reported in mol to facilitate the evaluation of stoichiometries and comparing Fe(II) and N2 production.
b)Fe(II) production rates are reported but only relying on the final Fe(II) contents. Without any knowledge about the temporal evolution no statements about rates can be made. Instead, the extents of Fe(II) and N2 production could be compared for the end of the experiment.
c) Prior to ammonium addition the sediments are incubated under anoxic conditions in order to remove nitrate and oxygen. However, it is not tested whether nitrate and oxygen have been indeed completely consumed after two days.
d) The authors consider multiple pathways for 29N2 and 30N2 production (Table 3) but, eventually, 29N2 production was exclusively attributed to Feammox.
Furthermore, the quality of presentation is low. Several statements appeared pointless to me and I was wondering whether I missed an important aspect due to the phrasing. However, there are also several obvious flaws, which should have been noticed before submission. Here I am only listing a few:
- Table 3 is literally the same as Table 1 in Yang etal (2012), this is plagiarism when no indicated
- Line32: nitrite is used in the annamox reaction or not?
- Line 36: microorganisms are oxidized
- Line 40: DrGm needs to be defined, is it under standard state besides pH 7? For which temperature.
- Line 56 – Line 62: the whole section is repetitive or contains redundant information.
- Line 57: including paddy soil….paddy soil.
- Site description: I do not understand the operation of the dam, is it indeed used to discharge muddy soil in summer? How can it store clear water in winter and spring when the rain is concentrated in July and September?
- Line 90: unclear what the random sampling scheme is. I got the impression that the sampling was not random but two specific sites and distinct depth intervals were selected.
- I suspect that “TEH” has been auto-corrected to “the” a few times and, by this, the sentence lost its meaning (e.g. Line 175 , line 179, line 224 etc.)
Citation: https://doi.org/10.5194/egusphere-2022-1209-RC2 -
AC2: 'Reply on RC2', Qingsong Guan, 18 Apr 2023
Thank you very much for the reviewer comment our manuscript.
The reviewer trusts our research data and process. However, the reviewer points out that a specific research objective, question or hypothesis is missing, and the description of methods is not specific enough. Also, the main conclusion is not sufficiently supported by the data.
We will make modifications according to the comments provided by the reviewer to ensure that they meet your requirements.
Citation: https://doi.org/10.5194/egusphere-2022-1209-AC2
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