the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 26 Mar 2024
Interactions of fertilisation and crop productivity on soil nitrogen cycle microbiome and gas emissions
Abstract. Fertilised soils are a significant source of nitrous oxide (N2O), a highly active greenhouse gas and stratospheric ozone depleter. Nitrogen (N) fertilisers, while boosting crop yield, also lead to N2O into the atmosphere, impacting global warming. We investigated relationships between mineral N fertilisation rates and additional manure amendment with different crop types through the analysis of abundances of N cycle functional genes, soil N2O and N2 emissions, nitrogen use efficiency (NUE), soil physicochemical analysis and biomass production. Our study indicates that N2O emissions are predominantly dependent on the mineral N fertilisation rate and enhance with increased mineral N fertilisation rate. Higher N2O emissions were attained with the application of manure. Manure amendment also increased the number of N cycle genes that are significant in the change of N2O. Contrary to our hypothesis, there was no significant influence of crop type on soil N2O emissions. The study indicated dominance of nitrification over denitrification in the soil. Microbial analyses also showed the potential role of comammox and DNRA processes as a source of N2O. Our study did not find soil moisture to be significantly linked to N2O emissions. Results of the study provide evidence that for wheat, a fertilisation rate of 80 kg N ha−1 is closest to the optimal rate for balancing biomass yield, N2O emissions, and achieving high NUE. Sorghum showed potential for cultivation in temperate climate, as sorghum maintained low N2O emissions and N losses on mineral N fertilisation rate of 80 kg N ha−1.
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RC1: 'Comment on egusphere-2024-593', Anonymous Referee #1, 14 Jun 2024
The manuscript discusses the effects of mineral N fertilization rates and manure amendment on soil N2O emissions across barley, wheat, and sorghum. The study leverages soil physicochemical analysis, nitrification- and denitrification-related genes, and gas emissions to assess the impact of fertilization strategies on N use efficiency and N2O emissions. The long-term aspects of the study site, the monthly variations of N2O emissions across crops, and the genetic components of N transforming pathways, provide a rich, publishable study. However, although the data is interesting, I do think this manuscript is not in good shape yet.
The manuscript's writing style, particularly in the Introduction and Discussion sections, is disjointed and verbose. The introduction is wordy with some redundancies. For example, the first and second paragraphs could be consolidated. The factors that influence nitrification, denitrification, comammox, and DNRA should be provided. Background information about effects of different crops on N2O emissions should be provided. Additionally, the manuscript should clarify why nitrification is considered a primary source of N2O fluxes. Though nitrification is the dominant step over denitrification in the soil, N2O is not the major product of nitrification. Hypothesis 5 (do you really need that many hypotheses?) is not a testable/measurable hypothesis, and how would the authors define "prospective"? Adaptability? Yield? N use efficiency? Water use efficiency?
My major concern in M&M is the experimental designs. It’s more like a pseudo-replicated (the three study plots within each crop are not independent) instead of a completed randomized block design by looking at Fig 1. Besides, the authors should consider providing more information about manure amendments, like the major source, the CN ratio of the manure, and whether the manure application is just one-time for this experiment or it’s a part of long-term experiments (if so, the manure application started since which year?) A climate diagram or bi-weekly/monthly precipitation amount should also be provided to align with soil moisture (Fig. S6) and N2O emissions plots (Fig. 5). Other soil properties like pH, texture, and bulk density, which influence nutrient dynamics and gas emissions, are also crucial and should be included in the study. The author should report soil organic C instead of total C.
I don’t understand why the authors use PCA instead of simpler methods like bar charts to present soil C, N, and inorganic N in different sites. I also think the authors should consider using other approaches (like structural equation models or approaches that can consider contributions from multiple factors) in addition to ANOVA and Pearson correlation to analyze their data. N cycling is complicated and has been influenced by many factors including vegetation, texture, soil moisture (precipitation), temperature, soil fertility and C concentration, and management practices like tillage, fertilization, etc. Simple correlation analysis may not always be the best way to capture those complicated interactions. And since the authors measured N2O emissions with time, I think they should analyze the data by different time periods instead of just cumulative fluxes. It’s also odd for me to compare sorghum + manure with barley/wheat without manure application in Fig 6. It should be barley vs sorghum vs wheat as one part, and sorghum vs sorghum w/ manure as another part.
The Discussion sections probably need some major work. There are many repetitive parts (N2O emissions increased with high mineral N application) and many statements are contradicted with each other in the current version. For example, the authors said there is no correlation between soil moisture levels and N2O emissions or functional marker gene abundances. Then the authors note that the lowest levels of N2O emissions and functional marker gene abundances occurred during periods of low soil moisture. Another example is the authors said no significant influence of crop type on N2O emissions, then the authors suggested sorghum as a potential crop in Northern Europe as sorghum maintained low N2O emissions. The authors also said N2O emissions increased with fertilization rates for wheat and barley plots, but the statistical results in Fig 6 showed no significant differences between N0 and N80. I also don’t know how to use the ratio of gene copy numbers to infer the resources of N2O. Both nitrification and denitrification contribute to N2O emissions. "dominance" might overstate the results given the weak strength of the correlation. Nitrification and denitrification are complex processes influenced by a variety of environmental and microbial factors. This correlation alone does not conclusively establish dominance or any cause-effect relationship.
It always needs extra caution on suggestions replacing current crops with sorghum. Assuming that sorghum with enhanced biological nitrification inhibition properties could reduce N2O given the same levels of N as other crops (corn, wheat, barley, etc) is applied, how much grain demand could be met by sorghum when considering large-scale implementation of the practice? Instead, including sorghum in the existing crop rotation and understanding its subsequent effects on N dynamics seems a more practical approach.
Please use upper case L to represent liter.
L52: 70% of N fertilizers were lost due to nitrification and denitrification? You just said about half of applied N to the field is not taken by plants (L44). In addition, how about N leaching and volatilization?
L94: IOSDV: Should put full name first and abbreviation in the parentheses.
L96: on crop type? Did you mean on crop responses of various crops?
Table S1:. Please express the unit of herbicide application as L ha-1. Should be the same order as other figures: barley - Sorghum – wheat
L120: Ø: diameter? is this 65 L the entire volume of PVC collars+lid? Looks like the volume is not consistent across treatments due to chamber extension, which create another sources of variable
L188: What’s biomass yield produced? Biomass production?
Figures: The figures should be labeled in order in the Result section. Fig S5 comes first in this draft so it should be S1. Similarly, Fig S6 -> S2. And Figure S1-4 should be S3-6. And please use the correct format for unit, like using mg kg-1 instead of mg/kg
L221: The unit of Y axis in Fig 3B is not concentration.
Fig 3a: Using ton ha-1 in the y axis may better align with the context
Fig 5: precipitation data should be provided.
L267: The statement that cumulative barley
Fig 6: I don’t understand the reason for estimating N2 emissions in this study.
Table 1: I am not sure if the reader needs to know sum of squares, means square, w2. And it’s odd that manure amendment is significant for N2O emissions in Table 1 but not in Fig 6.
L334: It seems long-term manure application showed no significant difference in NO3, NH4, N2O, and N2? That is odd. In Fig 6A, soils under 231 kg N ha-1 (N0 at sorghum w/ manure) treatment produce lower cumulative N2O compared to those under 80 kg N ha-1 (N80 at sorghum). That required some explanations.
L339: This paragraph needs further expended. We generally expect organic fertilization would increase SOC, total N, yield, and N2O due to direct C & N (both labile and recalcitrant) inputs. Same as L379, what's the potential reason for different results in this study and previous studies?
L381: there are no significant difference between sorghum and sorghum + manure in N0 and N160 (Fig 6A).
L434: if there is a liner response, the authors should provide p-value and r2
Citation: https://doi.org/10.5194/egusphere-2024-593-RC1 - AC1: 'Reply on RC1', Laura Kuusemets, 24 Jul 2024
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RC2: 'Comment on egusphere-2024-593', Anonymous Referee #2, 19 Jun 2024
The article “Interactions of fertilization and crop productivity on soil nitrogen cycle microbiome and gas emissions” studies the relationship between fertilization and crop productivity and how it relates to the N cycle microbiome and gas emissions, including an important greenhouse gas N2O. This is a really interesting topic as the production and usage of chemical fertilizers has increased exponentially since the discovery of the Haber-Bosch process for the production of ammonia. And nitrogen fertilized soils are a source of N2O, a strong greenhouse gas.
The manuscript fits well with the SOIL aims and scope. In general, it is well-organized and presented. And I would recommend this paper for publication. I would just suggest some points that I hope may be useful for the authors.
From the general perspective, I have noticed that the authors have calculated the N2 in addition to N2O in the results. However, they have not mentioned that data on the discussion. I was wondering if the authors could relate the data mentioned to the genes and the completion of the denitrification process, for example.
Some minor specific comments:
L 89: Hypothesis (5) is not clear. Does it mean optimal?
L 120: I understand that the corresponding corrections where made for the calculations when using the chamber extensions.
L 130: It would be important to state how much time lasted from the soil sample collection until analysis.
L 183 – L 185: Here it would be important to explain what data is parametric and what non-parametric as ANOVA is a parametric test and the Spearman’s rank a non-parametric one.
Citation: https://doi.org/10.5194/egusphere-2024-593-RC2 - AC2: 'Reply on RC2', Laura Kuusemets, 24 Jul 2024
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RC3: 'Comment on egusphere-2024-593', Anonymous Referee #3, 22 Jun 2024
This work presents a relevant topic, that is soil N2O emissions management, to understand N fertilization and crop type impact, along with a possible involvment of soil microbiome. Therefore, in my opinion, it is relevant to SOIL aims and scope. Introduction is well constructed, and it highlights the relevance of the study in a broader context. Some modifications in text structure are required. Hypotheses and objectives are clearly stated and coherent with the methodology used, even though some of them might require improvements. The biggest problem of this paper is a major lack in appropriately describing the experimental design. In fact, it is necessary to specify which crops immediately preceded the ones tested in the current experiment and which fertilization treatments they received. The reason why it is so important is that this experimental design would not be valid if this wasn’t a long-term experiment, as there aren’t multiple separate and randomized plots. In fact, I think that what you refer to as “replicates” are only multiple sampling points of one unique plot per treatment. If this lack in methodological information will not be addressed appropriately, I fear that it might seriously undermine the reproducibility of this work. In addition, some information about one of the treatments is lacking (manure amendment). Results have been described quite clearly, although sometimes too much detail is given about findings that don’t have a wide importance. In some graphs I think there are some mistakes in results presentation. In the discussion section there are some problems related to the flowing of the text. In fact, often the description of the same topic is divided into multiple, short paragraphs, thus creating some confusion for the reader. Moreover, some of the speculations are too strong based on the presented results. Overall, the manuscript has a great potential to be improved, but only if the issue with the experimental design is correctly and extensively addressed, as it is the most serious problem of this work.
Hereafter follow the comments I made on the text. Highlighted text indicates citations from the manuscript. When multiple lines are indicated, the comments refer either to a full sentence or to a meaningful part of it. L.(L.s) is used as a short form for “line(s)”.
L. 12: N2O I think the term emission is missing here.
L.s 16-17 You mean higher compared to the application of mineral fertilizers?
L. 19 Microbial analyses Could you be more specific here?
L. 23 sorghum It is not necessary to repeat the term sorghum here again.
L. 33 maise There is a spelling mistake here.
L. 34 in present agricultural regions due to climate system changes I am sorry, but this is not very clear to me. Could you please rephrase it.
L. 38 This sentence does not fit well in this paragraph. I think it is better to move it to the following one, when you introduce the problem of N2O emissions.
L.s 51-62 I think it's better to unite these two paragraphs in one. Otherwise, it results confusing since the topic discussed continues from the first to the second.
L. 63 contributes Probably some words are missing here.
L. 63 for biological production as a N fertiliser This seems confusing to me. Could you rephrase it?
L. 65 both requiring NO3-I don't think it is necessary to repeat this here again.
L. 68 clad There is a spelling mistake here.
L. 71 the Hatch-Slack pathway This phrase requires to be included in commas.
L. 71 maise This is a spelling mistake.
L.s 83-84 Could you rephrase this part? It sounds confusing to me.
L. 86 in arable mineral soil What do you mean by "mineral soil"?
L. 88 decreases This term here is not fitting.
L. 88 affects the soil microbial community This hypothesis seems too general. Could you be more specific?
L. 89-90 This hypothesis does not sound very fitting to me for the purposes of the paper. I think it might be better to focus on the performance of this crop in comparison to the others. Otherwise, please provide further insights.
L. 98 It might be better to move this sentence to the section preceding the description of the crop rotation.
L.s 104-109 In my opinion, this is the biggest problem of the whole manuscript. Although you specify there are three replicates per treatment, only one is present. This would be a major issue, if it wasn’t a long-term field experiment. Therefore, please specify which crops have preceded in the few years before the described experiment started. Also, please specify if fertilization has been applied in the previous years and its entity. In addition, it would be ideal if you could provide further insights about the farmyard manure composition and whether it has been treated somehow (composting or something else).
L. 104 I do not understand where the three replications are. A replicate is a treatment group to which the same levels of factors tested were applied in a way that allows to account for environmental factors’ variability. Based on the experimental design you have provided, it seems that you only have one replicate per treatment group. If this is not the case, I think you only have pseudo-replicates.
L.s 107-108 Manure was applied only to sorghum. I think you should make it clear also from the text.
L.s 113-114 As further specified in my comment on the text, it is better to clarify that additional N was applied with manure in the sorghum plots.
L. 125 electron capture and flame ionisation detectors Probably it is better to provide more details about these two instruments.
L.s 129-130 Please provide more details on the soil sampling process implemented (number of samples, rhizosphere or bulk soil).
L.s 131-132 What are the instruments used for these analyses?
L. 134 Could you provide the reference for this method?L.134 There is no need to use the full term here. It is better to use "C".
L.s 131-132 This paragraph should be united with the preceding one. Separating paragraphs discussing the same topic results in difficulty for the reader to understand their meaning.
L. 146 I have some doubts about the term "total". In fact, you sampled roots up to a depth of 18 cm, but all these species’ roots can easily reach lower depths.
L. 148 maturity phase Could you provide insights about the date when the measurement was done for each species?
L. 150 Frasier et al. (2018) This reference is not reported in the cited literature.
L. 171 extracted DNA What is the DNA concentration used?
L.s 171, 172 ml I really think that here you mean microliter.
L.s 175-176 Could you specify which are the standard curve ranges used?
L.180 analysis Since it is plural, it would be "analyses".
L. 183 Analysis of variance (ANOVA) Probably here it is necessary to specify the type of ANOVA used. Please provide me with your opinion.
L. 185 and This "and" should be substituted by a comma.
L.s 185-187 Later, in the results, you use a different terminology to refer to this term.
L. 193 2017 Parentheses are missing here.
L.s 199-200 in the soil It is redundant to repeat this phrase.
L. 207 Over all Probably a different conjunction here would make the discussion more fluent.
L. 221 increased The form of this verb does not seem correct. Probably it is better to say that they "caused an increase in".
L. 226 p < 0.05 This should be written without spaces.
L.s 226-227 For biomass yield, the hierarchy of uppercase letters seems to be B>C>A while for N content it seems to be C>D>B>A for yellow bars and A>B>C for the other two colours. I am right? If so, please correct the graph.
L. 234 In table S3, N2 emissions are just an estimation based on N2O emission if I'm not wrong. If this is the case, please specify it, otherwise it would be misleading.
L.s 240-246 Could you please specify in the text which of these differences are significant? It would also be ideal if you could specify the significance of the results with different letters in the table.
L. 264-265 Could you rephrase this? It seems quite redundant.
L. 286 The effect of mineral N fertilisation It would be better to write "mineral N fertilization effect".
L. 287 to effects of crop type The correct form would be "to the effects of crop type".
L. 299 Feature selection algorithm Please align the name of this methodology with the one described in the methodology.
L. 301 change I'm not sure about the term "change". Probably "variations" or "alterations" are better.
L.s 316-325 I'm not sure if it is really necessary to describe all the significant correlations. Probably it's better to just choose the relevant ones.
L.s 333-334 Please, make it clear that you are specifying this in support of what previously stated.
L. 342 indicates the dominance of nitrification over denitrification in N2O-producing processes Reference is lacking for the relevance of this ratio between the two genes.
L.s 350-351 What does this mean?
L. 359 important This term is not correct in this context.
L.s 362-363 A reference is lacking.
L. 364 What about the soil water content?
L.s 376-388 There is no need to separate this section in two different paragraphs, as the topic discussed is the same. Furthermore, this recurred also in other parts of the manuscript. It would be better to avoid writing short paragraphs with just a few sentences while separating the same topic in multiple paragraphs.
L. 386 manure enhances the activity of soil microbes It is better to not directly give this conclusion. It would be ideal if you propose this as one of the possible hypotheses.
L.s 389-390 with emissions increasing slower than linearly with the fertilisation rate I don't think that this phrase describes correctly the observed trend.
L.s 390-391 Are you sure this conclusion can be derived from just three points you have?
L. 394 N2O emissions often grow exponentially when the applied N exceeds the necessary amount for crops I don't understand what you mean by "often". I think that whether the growth is exponential or not depends on the number of N doses tested and their entity. I'm not sure about this so please let me know.
L. 397 positive linear response Here the same comment as before applies. Please provide further explanation about the linear or exponential response.
L.s 400-405 I don't think that discussing this part can provide useful insights to this work.
L. 406 indirectly affecting Probably it is better to say: "as it directly affects".
L. 410 Based on my personal experience, this information does not seem very fitting. Could you please check that it is correct?
L.s 411-412 This sentence has no reference, and it is not very clear as it is not specified how water scarcity might enhance N gases emissions.
L. 414 N2O management should align with crop yield It is not very clear here what is intended for "align".
L. 414 Biomass It would be better to say "biomass production".
L. 416 fertilisation rate 160 kg N ha−1 "of" is missing here.
L.s 416-417 but our study shows increasing N2O emissions at higher fertilisation rates (Figure 6A), suggesting potential overfertilisation. I don't understand what the connection with the previous sentence is.
L.s 419-421 If this sentence is added to sustain the previously discussed results, I think it is better to specify it. Otherwise, it does not seem very clear.
L. 422 rate 160 kg N ha−1 "of" is missing here.
L. 422 increase Probably it is better to use the past tense, as you are describing results you observed.
L. 423 The fertilisation rate 80 kg N ha−1 "of" is missing here.
L.s 433-434 There are some typos in this sentence.
L.s 437-438 As you studied the abundance of microbial functional groups based on a DNA approach, I think that this speculation is too strong. What you can say is that the nitrification potential was higher than the denitrification one, but not that one process prevailed over one other.
L. 438 N cycle A hyphen is required.
L.s 442-443 fertilisation rate 80 kg N ha−1 "of" is missing here.
L. 444 positioning This term is not very fitting here. Please, change it.
Citation: https://doi.org/10.5194/egusphere-2024-593-RC3 - AC3: 'Reply on RC3', Laura Kuusemets, 24 Jul 2024
Status: closed
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RC1: 'Comment on egusphere-2024-593', Anonymous Referee #1, 14 Jun 2024
The manuscript discusses the effects of mineral N fertilization rates and manure amendment on soil N2O emissions across barley, wheat, and sorghum. The study leverages soil physicochemical analysis, nitrification- and denitrification-related genes, and gas emissions to assess the impact of fertilization strategies on N use efficiency and N2O emissions. The long-term aspects of the study site, the monthly variations of N2O emissions across crops, and the genetic components of N transforming pathways, provide a rich, publishable study. However, although the data is interesting, I do think this manuscript is not in good shape yet.
The manuscript's writing style, particularly in the Introduction and Discussion sections, is disjointed and verbose. The introduction is wordy with some redundancies. For example, the first and second paragraphs could be consolidated. The factors that influence nitrification, denitrification, comammox, and DNRA should be provided. Background information about effects of different crops on N2O emissions should be provided. Additionally, the manuscript should clarify why nitrification is considered a primary source of N2O fluxes. Though nitrification is the dominant step over denitrification in the soil, N2O is not the major product of nitrification. Hypothesis 5 (do you really need that many hypotheses?) is not a testable/measurable hypothesis, and how would the authors define "prospective"? Adaptability? Yield? N use efficiency? Water use efficiency?
My major concern in M&M is the experimental designs. It’s more like a pseudo-replicated (the three study plots within each crop are not independent) instead of a completed randomized block design by looking at Fig 1. Besides, the authors should consider providing more information about manure amendments, like the major source, the CN ratio of the manure, and whether the manure application is just one-time for this experiment or it’s a part of long-term experiments (if so, the manure application started since which year?) A climate diagram or bi-weekly/monthly precipitation amount should also be provided to align with soil moisture (Fig. S6) and N2O emissions plots (Fig. 5). Other soil properties like pH, texture, and bulk density, which influence nutrient dynamics and gas emissions, are also crucial and should be included in the study. The author should report soil organic C instead of total C.
I don’t understand why the authors use PCA instead of simpler methods like bar charts to present soil C, N, and inorganic N in different sites. I also think the authors should consider using other approaches (like structural equation models or approaches that can consider contributions from multiple factors) in addition to ANOVA and Pearson correlation to analyze their data. N cycling is complicated and has been influenced by many factors including vegetation, texture, soil moisture (precipitation), temperature, soil fertility and C concentration, and management practices like tillage, fertilization, etc. Simple correlation analysis may not always be the best way to capture those complicated interactions. And since the authors measured N2O emissions with time, I think they should analyze the data by different time periods instead of just cumulative fluxes. It’s also odd for me to compare sorghum + manure with barley/wheat without manure application in Fig 6. It should be barley vs sorghum vs wheat as one part, and sorghum vs sorghum w/ manure as another part.
The Discussion sections probably need some major work. There are many repetitive parts (N2O emissions increased with high mineral N application) and many statements are contradicted with each other in the current version. For example, the authors said there is no correlation between soil moisture levels and N2O emissions or functional marker gene abundances. Then the authors note that the lowest levels of N2O emissions and functional marker gene abundances occurred during periods of low soil moisture. Another example is the authors said no significant influence of crop type on N2O emissions, then the authors suggested sorghum as a potential crop in Northern Europe as sorghum maintained low N2O emissions. The authors also said N2O emissions increased with fertilization rates for wheat and barley plots, but the statistical results in Fig 6 showed no significant differences between N0 and N80. I also don’t know how to use the ratio of gene copy numbers to infer the resources of N2O. Both nitrification and denitrification contribute to N2O emissions. "dominance" might overstate the results given the weak strength of the correlation. Nitrification and denitrification are complex processes influenced by a variety of environmental and microbial factors. This correlation alone does not conclusively establish dominance or any cause-effect relationship.
It always needs extra caution on suggestions replacing current crops with sorghum. Assuming that sorghum with enhanced biological nitrification inhibition properties could reduce N2O given the same levels of N as other crops (corn, wheat, barley, etc) is applied, how much grain demand could be met by sorghum when considering large-scale implementation of the practice? Instead, including sorghum in the existing crop rotation and understanding its subsequent effects on N dynamics seems a more practical approach.
Please use upper case L to represent liter.
L52: 70% of N fertilizers were lost due to nitrification and denitrification? You just said about half of applied N to the field is not taken by plants (L44). In addition, how about N leaching and volatilization?
L94: IOSDV: Should put full name first and abbreviation in the parentheses.
L96: on crop type? Did you mean on crop responses of various crops?
Table S1:. Please express the unit of herbicide application as L ha-1. Should be the same order as other figures: barley - Sorghum – wheat
L120: Ø: diameter? is this 65 L the entire volume of PVC collars+lid? Looks like the volume is not consistent across treatments due to chamber extension, which create another sources of variable
L188: What’s biomass yield produced? Biomass production?
Figures: The figures should be labeled in order in the Result section. Fig S5 comes first in this draft so it should be S1. Similarly, Fig S6 -> S2. And Figure S1-4 should be S3-6. And please use the correct format for unit, like using mg kg-1 instead of mg/kg
L221: The unit of Y axis in Fig 3B is not concentration.
Fig 3a: Using ton ha-1 in the y axis may better align with the context
Fig 5: precipitation data should be provided.
L267: The statement that cumulative barley
Fig 6: I don’t understand the reason for estimating N2 emissions in this study.
Table 1: I am not sure if the reader needs to know sum of squares, means square, w2. And it’s odd that manure amendment is significant for N2O emissions in Table 1 but not in Fig 6.
L334: It seems long-term manure application showed no significant difference in NO3, NH4, N2O, and N2? That is odd. In Fig 6A, soils under 231 kg N ha-1 (N0 at sorghum w/ manure) treatment produce lower cumulative N2O compared to those under 80 kg N ha-1 (N80 at sorghum). That required some explanations.
L339: This paragraph needs further expended. We generally expect organic fertilization would increase SOC, total N, yield, and N2O due to direct C & N (both labile and recalcitrant) inputs. Same as L379, what's the potential reason for different results in this study and previous studies?
L381: there are no significant difference between sorghum and sorghum + manure in N0 and N160 (Fig 6A).
L434: if there is a liner response, the authors should provide p-value and r2
Citation: https://doi.org/10.5194/egusphere-2024-593-RC1 - AC1: 'Reply on RC1', Laura Kuusemets, 24 Jul 2024
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RC2: 'Comment on egusphere-2024-593', Anonymous Referee #2, 19 Jun 2024
The article “Interactions of fertilization and crop productivity on soil nitrogen cycle microbiome and gas emissions” studies the relationship between fertilization and crop productivity and how it relates to the N cycle microbiome and gas emissions, including an important greenhouse gas N2O. This is a really interesting topic as the production and usage of chemical fertilizers has increased exponentially since the discovery of the Haber-Bosch process for the production of ammonia. And nitrogen fertilized soils are a source of N2O, a strong greenhouse gas.
The manuscript fits well with the SOIL aims and scope. In general, it is well-organized and presented. And I would recommend this paper for publication. I would just suggest some points that I hope may be useful for the authors.
From the general perspective, I have noticed that the authors have calculated the N2 in addition to N2O in the results. However, they have not mentioned that data on the discussion. I was wondering if the authors could relate the data mentioned to the genes and the completion of the denitrification process, for example.
Some minor specific comments:
L 89: Hypothesis (5) is not clear. Does it mean optimal?
L 120: I understand that the corresponding corrections where made for the calculations when using the chamber extensions.
L 130: It would be important to state how much time lasted from the soil sample collection until analysis.
L 183 – L 185: Here it would be important to explain what data is parametric and what non-parametric as ANOVA is a parametric test and the Spearman’s rank a non-parametric one.
Citation: https://doi.org/10.5194/egusphere-2024-593-RC2 - AC2: 'Reply on RC2', Laura Kuusemets, 24 Jul 2024
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RC3: 'Comment on egusphere-2024-593', Anonymous Referee #3, 22 Jun 2024
This work presents a relevant topic, that is soil N2O emissions management, to understand N fertilization and crop type impact, along with a possible involvment of soil microbiome. Therefore, in my opinion, it is relevant to SOIL aims and scope. Introduction is well constructed, and it highlights the relevance of the study in a broader context. Some modifications in text structure are required. Hypotheses and objectives are clearly stated and coherent with the methodology used, even though some of them might require improvements. The biggest problem of this paper is a major lack in appropriately describing the experimental design. In fact, it is necessary to specify which crops immediately preceded the ones tested in the current experiment and which fertilization treatments they received. The reason why it is so important is that this experimental design would not be valid if this wasn’t a long-term experiment, as there aren’t multiple separate and randomized plots. In fact, I think that what you refer to as “replicates” are only multiple sampling points of one unique plot per treatment. If this lack in methodological information will not be addressed appropriately, I fear that it might seriously undermine the reproducibility of this work. In addition, some information about one of the treatments is lacking (manure amendment). Results have been described quite clearly, although sometimes too much detail is given about findings that don’t have a wide importance. In some graphs I think there are some mistakes in results presentation. In the discussion section there are some problems related to the flowing of the text. In fact, often the description of the same topic is divided into multiple, short paragraphs, thus creating some confusion for the reader. Moreover, some of the speculations are too strong based on the presented results. Overall, the manuscript has a great potential to be improved, but only if the issue with the experimental design is correctly and extensively addressed, as it is the most serious problem of this work.
Hereafter follow the comments I made on the text. Highlighted text indicates citations from the manuscript. When multiple lines are indicated, the comments refer either to a full sentence or to a meaningful part of it. L.(L.s) is used as a short form for “line(s)”.
L. 12: N2O I think the term emission is missing here.
L.s 16-17 You mean higher compared to the application of mineral fertilizers?
L. 19 Microbial analyses Could you be more specific here?
L. 23 sorghum It is not necessary to repeat the term sorghum here again.
L. 33 maise There is a spelling mistake here.
L. 34 in present agricultural regions due to climate system changes I am sorry, but this is not very clear to me. Could you please rephrase it.
L. 38 This sentence does not fit well in this paragraph. I think it is better to move it to the following one, when you introduce the problem of N2O emissions.
L.s 51-62 I think it's better to unite these two paragraphs in one. Otherwise, it results confusing since the topic discussed continues from the first to the second.
L. 63 contributes Probably some words are missing here.
L. 63 for biological production as a N fertiliser This seems confusing to me. Could you rephrase it?
L. 65 both requiring NO3-I don't think it is necessary to repeat this here again.
L. 68 clad There is a spelling mistake here.
L. 71 the Hatch-Slack pathway This phrase requires to be included in commas.
L. 71 maise This is a spelling mistake.
L.s 83-84 Could you rephrase this part? It sounds confusing to me.
L. 86 in arable mineral soil What do you mean by "mineral soil"?
L. 88 decreases This term here is not fitting.
L. 88 affects the soil microbial community This hypothesis seems too general. Could you be more specific?
L. 89-90 This hypothesis does not sound very fitting to me for the purposes of the paper. I think it might be better to focus on the performance of this crop in comparison to the others. Otherwise, please provide further insights.
L. 98 It might be better to move this sentence to the section preceding the description of the crop rotation.
L.s 104-109 In my opinion, this is the biggest problem of the whole manuscript. Although you specify there are three replicates per treatment, only one is present. This would be a major issue, if it wasn’t a long-term field experiment. Therefore, please specify which crops have preceded in the few years before the described experiment started. Also, please specify if fertilization has been applied in the previous years and its entity. In addition, it would be ideal if you could provide further insights about the farmyard manure composition and whether it has been treated somehow (composting or something else).
L. 104 I do not understand where the three replications are. A replicate is a treatment group to which the same levels of factors tested were applied in a way that allows to account for environmental factors’ variability. Based on the experimental design you have provided, it seems that you only have one replicate per treatment group. If this is not the case, I think you only have pseudo-replicates.
L.s 107-108 Manure was applied only to sorghum. I think you should make it clear also from the text.
L.s 113-114 As further specified in my comment on the text, it is better to clarify that additional N was applied with manure in the sorghum plots.
L. 125 electron capture and flame ionisation detectors Probably it is better to provide more details about these two instruments.
L.s 129-130 Please provide more details on the soil sampling process implemented (number of samples, rhizosphere or bulk soil).
L.s 131-132 What are the instruments used for these analyses?
L. 134 Could you provide the reference for this method?L.134 There is no need to use the full term here. It is better to use "C".
L.s 131-132 This paragraph should be united with the preceding one. Separating paragraphs discussing the same topic results in difficulty for the reader to understand their meaning.
L. 146 I have some doubts about the term "total". In fact, you sampled roots up to a depth of 18 cm, but all these species’ roots can easily reach lower depths.
L. 148 maturity phase Could you provide insights about the date when the measurement was done for each species?
L. 150 Frasier et al. (2018) This reference is not reported in the cited literature.
L. 171 extracted DNA What is the DNA concentration used?
L.s 171, 172 ml I really think that here you mean microliter.
L.s 175-176 Could you specify which are the standard curve ranges used?
L.180 analysis Since it is plural, it would be "analyses".
L. 183 Analysis of variance (ANOVA) Probably here it is necessary to specify the type of ANOVA used. Please provide me with your opinion.
L. 185 and This "and" should be substituted by a comma.
L.s 185-187 Later, in the results, you use a different terminology to refer to this term.
L. 193 2017 Parentheses are missing here.
L.s 199-200 in the soil It is redundant to repeat this phrase.
L. 207 Over all Probably a different conjunction here would make the discussion more fluent.
L. 221 increased The form of this verb does not seem correct. Probably it is better to say that they "caused an increase in".
L. 226 p < 0.05 This should be written without spaces.
L.s 226-227 For biomass yield, the hierarchy of uppercase letters seems to be B>C>A while for N content it seems to be C>D>B>A for yellow bars and A>B>C for the other two colours. I am right? If so, please correct the graph.
L. 234 In table S3, N2 emissions are just an estimation based on N2O emission if I'm not wrong. If this is the case, please specify it, otherwise it would be misleading.
L.s 240-246 Could you please specify in the text which of these differences are significant? It would also be ideal if you could specify the significance of the results with different letters in the table.
L. 264-265 Could you rephrase this? It seems quite redundant.
L. 286 The effect of mineral N fertilisation It would be better to write "mineral N fertilization effect".
L. 287 to effects of crop type The correct form would be "to the effects of crop type".
L. 299 Feature selection algorithm Please align the name of this methodology with the one described in the methodology.
L. 301 change I'm not sure about the term "change". Probably "variations" or "alterations" are better.
L.s 316-325 I'm not sure if it is really necessary to describe all the significant correlations. Probably it's better to just choose the relevant ones.
L.s 333-334 Please, make it clear that you are specifying this in support of what previously stated.
L. 342 indicates the dominance of nitrification over denitrification in N2O-producing processes Reference is lacking for the relevance of this ratio between the two genes.
L.s 350-351 What does this mean?
L. 359 important This term is not correct in this context.
L.s 362-363 A reference is lacking.
L. 364 What about the soil water content?
L.s 376-388 There is no need to separate this section in two different paragraphs, as the topic discussed is the same. Furthermore, this recurred also in other parts of the manuscript. It would be better to avoid writing short paragraphs with just a few sentences while separating the same topic in multiple paragraphs.
L. 386 manure enhances the activity of soil microbes It is better to not directly give this conclusion. It would be ideal if you propose this as one of the possible hypotheses.
L.s 389-390 with emissions increasing slower than linearly with the fertilisation rate I don't think that this phrase describes correctly the observed trend.
L.s 390-391 Are you sure this conclusion can be derived from just three points you have?
L. 394 N2O emissions often grow exponentially when the applied N exceeds the necessary amount for crops I don't understand what you mean by "often". I think that whether the growth is exponential or not depends on the number of N doses tested and their entity. I'm not sure about this so please let me know.
L. 397 positive linear response Here the same comment as before applies. Please provide further explanation about the linear or exponential response.
L.s 400-405 I don't think that discussing this part can provide useful insights to this work.
L. 406 indirectly affecting Probably it is better to say: "as it directly affects".
L. 410 Based on my personal experience, this information does not seem very fitting. Could you please check that it is correct?
L.s 411-412 This sentence has no reference, and it is not very clear as it is not specified how water scarcity might enhance N gases emissions.
L. 414 N2O management should align with crop yield It is not very clear here what is intended for "align".
L. 414 Biomass It would be better to say "biomass production".
L. 416 fertilisation rate 160 kg N ha−1 "of" is missing here.
L.s 416-417 but our study shows increasing N2O emissions at higher fertilisation rates (Figure 6A), suggesting potential overfertilisation. I don't understand what the connection with the previous sentence is.
L.s 419-421 If this sentence is added to sustain the previously discussed results, I think it is better to specify it. Otherwise, it does not seem very clear.
L. 422 rate 160 kg N ha−1 "of" is missing here.
L. 422 increase Probably it is better to use the past tense, as you are describing results you observed.
L. 423 The fertilisation rate 80 kg N ha−1 "of" is missing here.
L.s 433-434 There are some typos in this sentence.
L.s 437-438 As you studied the abundance of microbial functional groups based on a DNA approach, I think that this speculation is too strong. What you can say is that the nitrification potential was higher than the denitrification one, but not that one process prevailed over one other.
L. 438 N cycle A hyphen is required.
L.s 442-443 fertilisation rate 80 kg N ha−1 "of" is missing here.
L. 444 positioning This term is not very fitting here. Please, change it.
Citation: https://doi.org/10.5194/egusphere-2024-593-RC3 - AC3: 'Reply on RC3', Laura Kuusemets, 24 Jul 2024
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