Review Reports - Influence of Biogas Slurry and a Nitrification Inhibitor Application in Nitrous Oxide Emissions by Soil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting paper about quantification of nitrous oxide emissions from biogas slurry application and its causes depending on the application conditions. The subject is very interesting, but the text is very confusing, in the ideas and in the language usage – English. Namely the title should be reviewed once the sentence is not well constructed. Several sentences seem that have no sequence… They are highlighted in the document.

The paper can be very important for sharing the knowledge about this subject. Please see the following issues. Also, in the document attached are highlighted some errors that should corrected and sone doubts that I think that should be clarified.

Page 2 – Line 72

“The changed soil properties resulting from the initial application, such as elevated soil pH and NH4+ content, as well as the reduced NO3- levels, mostly contribute to these emissions. Furthermore, the combined application of BS and DMPP at the same time would exert the best inhibitory efficacy in N2O emissions.” This sentence is already a conclusion?

Page 3, line 113: In gas kinetics analysis description, it is not clear: the DMPP was only added after the soil treatment? Only to the 30 g of soil after 30- and 60-days treatment? It should not be done in the beginning of the test with the soil?

Page 4, line 163 “The content of DOC was 108 mg kg-1 in the B0 treatment, decreasing to 62.9 mg kg-1 in treatment B1. After the repeated application of BS, the DOC content in the B2 treatment increased to 104 mg kg-1 and showed no significant difference in comparison with B0.” - Why does the DOC decrease with the BS addition until the 30 days?

Page 8, line 230 – “Soil NH4+ and pH were strong positive predictors of cumulative N2O emissions, CO2 emissions, N2O/(N2O+N2) and O2 consumption (P < 0.05). While these gas emissions were negatively correlated with soil NO3- content (P < 0.01). The emission characteristics of N2 and CO2 were also remarkably explained by soil DOC content (P < 0.05, Figure 5A).” This was not expected?

Page 9, Line 283: Where is this shown? “Soil pH was shown to be amplified with the simple application of BS”. Probably it should be added “after 30 days”. And the figure citation is incorrect.

Page 9, Line 287: “by the rich weakly acidic functional groups such as polysaccharides and humic acid in BS” If this is true it should be noticed in the beginning. Temo zero. But is was only after 30 days. Probably due to the organic matter decomposition?

Age 19, Line 327 – Where is this seen? – “When the BS was initially applied, the soil pH increased significantly.”

Page 10, Line 329 - Where is this seen? – “BS amended soil was generally characterized by an elevated pH”

Paga 10, line 339 – “Combining an application of the nitrification inhibitor DMPP with a single BS application gives best productivity …” where are the results that confirm this statement?

Overall questions:

- Is the DMPP toxic for the environment? Has it side effects?

- Why was the DMPP addition done aonly after the soil assays? It should not be added in the beginning?

- Why was necessary to add BS again after 30 days of assay?

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The subject is very interesting, but the text is very confusing, in the ideas and in the language usage – English. Namely the title should be reviewed once the sentence is not well constructed. Several sentences seem that have no sequence… They are highlighted in the document.

Author Response

Response: Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files. We have already had the language of this paper corrected by an English native speaker who did a proofreading for the entire manuscript, and we will also be submitting the English language editing certificate.

Comments 1: Page 2 Line 72 “The changed soil properties resulting from the initial application, such as elevated soil pH and NH4+ content, as well as the reduced NO3- levels, mostly contribute to these emissions. Furthermore, the combined application of BS and DMPP at the same time would exert the best inhibitory efficacy in N2O emissions.” This sentence is already a conclusion?

Response 1: Thank you for pointing this out. We appreciate your observation that the original hypothesis seemed to align closely with the study’s conclusions. To address this concern, we have revised the hypothesis in Page 2, Lines 82-89 in the revised manuscript to better reflect the exploratory nature of the study and its preliminary assumptions.

Page 2, Lines 82-89: Based on the above information, it was hypothesized that a single application of BS would initially lead to higher N₂O emissions from the soil compared to multiple BS applications. We anticipated that this initial emission spike could be attributed to changes in soil properties resulting from the single application, such as increased soil pH and NH₄⁺ content, along with decreased NO₃^- levels. Additionally, the simultaneous application of BS with DMPP would exert the best inhibitory efficacy in N₂O emissions.

Comments 2: Page 3, line 113: In gas kinetics analysis description, it is not clear: the DMPP was only added after the soil treatment? Only to the 30 g of soil after 30- and 60-days treatment? It should not be done in the beginning of the test with the soil?

Response 2: Thank you for your comment. We have revised the description in the gas kinetics analysis to make it clearer in Page 3, Lines 130-145 in the revised manuscript. Prior to the 108-h robotized incubation-monitoring (Robot) system being employed, in the treatments B0-DMPP, B1-DMPP, and B2-DMPP, the DMPP solution was evenly dripped onto the 30 g of soil after 0-, 30- and 60-days treatment from the 2.2.1 incubation experiment. Importantly, the addition of DMPP was carried out at the beginning of the 108-h gas kinetics monitoring incubation. Additionally, we have provided a schematic representation of the experimental design in Figure S1 for better understanding.

Page 3, Lines 130-145: A 108-h robotized incubation-monitoring (Robot) system was conducted to observe the characteristics of soil N₂O, nitrogen (N₂), oxygen (O₂) and CO₂emissions. This investigation employed 30 g of soil after 0-, 30- and 60-days treatment as outlined in 2.2.1 experiment. To specifically assess the efficacy of DMPP addition, an additional 30 g of soil from treatments B0, B1, and B2 underwent parallel monitoring of gas emissions (as depicted in Figure S1). The incubation was established in 120 mL serum bottles, featuring nine specific treatments: CK0, CK1, CK2, B0, B0+DMPP, B1, B1+DMPP, B2, and B2+DMPP, with each treatment replicated three times. Prior to incubation, for treatments involving DMPP addition, the DMPP solution was evenly dripped onto the surface of the soil at a rate of 1% of N contained in BS (i.e. the commercially recommended rate). The volume of DMPP solution and deionized water addition was determined for adjusting the soil water content of all treatments to 70% WHC. Once prepared, all bottles were immediately sealed with a rubber plug and an aluminum cap (Macherey-Nagel, Germany).

Comments 3: Page 4, line 163 “The content of DOC was 108 mg kg-1 in the B0 treatment, decreasing to 62.9 mg kg-1 in treatment B1. After the repeated application of BS, the DOC content in the B2 treatment increased to 104 mg kg-1 and showed no significant difference in comparison with B0.” - Why does the DOC decrease with the BS addition until the 30 days?

Response 3: Thank you for your comment. One potential reason for the decrease in DOC content in the B1 treatment is the microbial degradation of organic matter present in the BS. When BS is applied to the soil, it introduces a significant amount of organic material that serves as a nutrient source for soil microorganisms [1]. These microorganisms consume the available organic carbon for energy and growth, resulting in a decrease in the DOC content of the soil. Additionally, the composition of the BS itself may contain organic compounds that, upon application, temporarily alter the balance of soil organic carbon dynamics [2]. It is also possible that the adsorption of organic carbon to soil particles may occur, leading to a decrease in dissolved organic forms [3]. The increase in DOC in B2 treatment, following the repeated application of biogas slurry, is due to the replenishment of fresh organic matter from the additional slurry. This new organic matter undergoes microbial decomposition, releasing more dissolved organic carbon into the soil, thus elevating the DOC content.

References

1. Giles, M.E.; Daniell, T.J.; Baggs, E.M. Compound driven differences in N₂ and N₂O emission from soil; the role of substrate use efficiency and the microbial community. Soil Biology and Biochemistry 2017, 106, 90-98, doi: https://doi.org/10.1016/j.soilbio.2016.11.028.

2. Chen, Q.; Liu, T. Biogas system in rural China: upgrading from decentralized to centralized? Renewable and Sustainable Energy Reviews 2017, 78, 933-944, doi: https://doi.org/1016/j.rser.2017.04.113.

3. Tang, J., Pan, F., Davy, A.J., Yin, J., Wu, D., Yang Q. Responses of water‐stable aggregates, their associated organic carbon and crop yield to the application of biogas slurry in a fluvo‐aquic soil of the North China plain. Soil Use and Management 2024, 40, e12969. doi: https://doi.org/10.1111/sum.12969

Comments 4: Page 8, line 230 – “Soil NH4+ and pH were strong positive predictors of cumulative N2O emissions, CO2 emissions, N2O/(N2O+N2) and O2 consumption (P < 0.05). While these gas emissions were negatively correlated with soil NO3- content (P < 0.01). The emission characteristics of N2 and CO2 were also remarkably explained by soil DOC content (P < 0.05, Figure 5A).” This was not expected?

Response 4: Thank you for your comment. The correlation between gas emission characteristics and soil variables in soils with different BS application frequency was determined based on Pearson’s correlation coefficients, which are widely recognized for their reliability in measuring linear relationships between variables. Higher NH₄⁺ levels can stimulate nitrification and denitrification processes, which are known to produce N₂O as a byproduct [4]. Additionally, an increase in soil pH can influence microbial activity by creating a more favorable environment for certain microbial groups involved in the N cycle [5-6]. On the other hand, the negative correlation with soil NO₃^- content indicates that when levels of nitrate are high, it may lead to increased N₂ formation rather than N₂O, suggesting a shift in microbial processes possibly driven by the availability of electron acceptors [7]. The influence of soil DOC on N₂ and CO₂ emissions aligns with previous studies indicating that DOC serves as a carbon source for heterotrophic microorganisms, thereby promoting microbial respiration and potentially leading to increased emissions of these gases [8-9]. We have supplied the related discussion of these results you pointed out in Page 8 Lines 252-255, Page 9 Lines 293-295, 307-311 in the revised manuscript.

Page 8 Lines 252-255: The emission characteristics of N₂ and CO₂ were significantly associated with soil DOC content (P < 0.05, Figure 5A), where N₂ emissions exhibited a negative correlation with DOC content, while CO₂emissions showed a positive correlation with DOC content.

Page 9 Lines 293-295: Well et al. [33] also concluded that N₂O emissions were governed by NH₄⁺ availability, which stimulate nitrification and denitrification processes.

Page 9 Lines 307-311: The higher N₂O emission intensity in the B0 treatment would also be attributed to higher soil pH, which can influence microbial activity by creating a more favorable environment for certain microbial groups involved in nitrification and is generally more conducive to related N₂O emissions [5].

References

4. Well, R.; Flessa, H.; Xing, L.; Xiaotang, J.; Römheld, V. Isotopologue ratios of N₂O emitted from microcosms with NH4+ fertilized arable soils under conditions favoring nitrification. Soil Biology and Biochemistry 2008, 40, 2416-2426, doi: https://doi.org/10.1016/j.soilbio.2008.06.003.

5. Kong, F.; Li, Q.; Yang, Z.; Chen, Y. Does the application of biogas slurry reduce soil N₂O emissions and increase crop yield? –A systematic review. J. Environ. Manage. 2023, 342, 118339, doi: https://doi.org/10.1016/j.jenvman.2023.118339.

6. Xu, H; Wang, X; Li, H ; Yao, H; Su, J; Zhu, Y. Biochar Impacts Soil Microbial Community Composition and Nitrogen Cycling in an Acidic Soil Planted with Rape. Environ. Sci. Technol. 2014, 48, 9391-9399, doi: https://doi.org/10.1021/es5021058.

7. Harter, J., Krause, H. M., Schuettler, S., Ruser, R., Fromme, M., Scholten, T., Kappler, A., Behrens, S. Linking N₂O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community. The ISME journal, 2014, 8(3), 660-674. doi: https://doi.org/10.1038/ismej.2013.160.

8. Azam, F.; Müller, C.; Weiske, A.; Benckiser, G.; Ottow, J. Nitrification and denitrification as sources of atmospheric nitrous oxide – role of oxidizable carbon and applied nitrogen. Biol. Fertil. Soils 2002, 35, 54-61, doi: https://doi.org/1007/s00374-001-0441-5.

9.Jirout, J. Nitrous oxide productivity of soil fungi along a gradient of cattle impact. Fungal Ecol. 2015, 17, 155-163. doi: https://doi.org/10.1016/j.funeco.2015.07.003.

Comments 5: Page 9, Line 283: Where is this shown? “Soil pH was shown to be amplified with the simple application of BS”. Probably it should be added “after 30 days”. And the figure citation is incorrect.

Response 5: Thank you for your comment. We have corrected the figure citation in Page 9 Line 307 in the revised manuscript from “Figure 1B” to “Figure 1A”. B0 stimulates the practice where BS was applied once and to examine the immediate effect of BS application on soil properties and N₂O emissions. From the present results in Figure 1A, the soil pH of B0 treatment was significantly higher than that of the B1 and B2 treatment, which demonstrated that soil pH was shown to be amplified with the simple application of BS.

Page 9 Lines 306-307: Soil pH was shown to be amplified with the simple application of BS (Figure 1A).

Comments 6: Page 9, Line 287: “by the rich weakly acidic functional groups such as polysaccharides and humic acid in BS” If this is true it should be noticed in the beginning. Temo zero. But is was only after 30 days. Probably due to the organic matter decomposition?

Response 6: Thank you for your comment. We acknowledge that the rich weakly acidic functional groups such as polysaccharides and humic acid in BS may initially exert a slight influence on the observed pH levels. However, it is likely that their impact became more pronounced over time, particularly after 30 days. The accumulation of such functional groups could not only reduce soil pH but improve the buffering performance against changes in pH. Upon initial application to the soil, the alkaline nature of BS is the main reason for the immediate increase in soil pH, which may temporarily counteract the effects of the weakly acidic functional groups present in the BS. Furthermore, as you pointed out, this delay could probably be attributed to the ongoing decomposition of organic matter within the biogas slurry. As biochemical processes mature, the breakdown of organic materials may lead to the gradual release of these weakly acidic compounds, thereby affecting pH levels more significantly later in the observation period.

Comments 7: Page 19, Line 327 – Where is this seen? – “When the BS was initially applied, the soil pH increased significantly.”

Response 7: Thank you for your comment. The statement "When the BS was initially applied, the soil pH increased significantly" is supported by the results shown in Figure 1A. Specifically, the pH level of the soil treated with single biogas slurry application (B0) was significantly higher than that of the corresponding control group (CK0), which only received deionized water. This indicates that the initial application of the BS led to a significant increase in soil pH.

Comments 8: Page 10, Line 329 - Where is this seen? – “BS amended soil was generally characterized by an elevated pH”

Response 8: Thank you for your comment. You are correct in noting that the statement "BS amended soil was generally characterized by an elevated pH" is based on previous literature, rather than a direct observation from our current study's results. I apologize for any confusion this may have caused. To clarify, our study did observe that when BS was initially applied to the soil, the pH increased significantly due to the alkaline nature of the BS used. This increase in soil pH was consistent with our expectations and is supported by our experimental results. To address your concern, I propose modifying the relevant sentence in the discussion to clarify that the statement about BS-amended soil generally having an elevated pH is based on previous studies, while our own results focus on the temporal changes in soil pH observed in our experimental conditions. We have revised the sentence in the Page 10, Lines 355-361 to "Previous studies have reported that BS-amended soil is generally characterized by an elevated pH [50]. In the present study, it was observed that when the BS was initially applied, the soil pH increased significantly due to the alkaline nature of the BS. However, with the incubation and the repeated application of BS …" in the revised manuscript.

Page 10, Lines 355-361: Previous studies have reported that BS-amended soil is generally characterized by an elevated pH [50]. In the present study, it was observed that when the BS was initially applied, the soil pH increased significantly due to the alkaline nature of the BS. However, with the incubation and the repeated application of BS, the soil pH decreased, contributing to the low DMPP efficacy in the B2-DMPP.

Comments 9: Page 10, line 339 – “Combining an application of the nitrification inhibitor DMPP with a single BS application gives best productivity …” where are the results that confirm this statement?

Response 9: Thank you for your comment. Upon reviewing our data and results, we recognize that we do not have direct evidence to conclusively support this specific statement. This was an oversight on our part, and we apologize for any confusion it may have caused. We have revised this statement to “Combining an application of the nitrification inhibitor DMPP with a single BS application gives best environmental outcomes” in Page 10, Lines 370-372 in the revised manuscript. We will ensure all statements are consistent with our data.

Page 10, Lines 370-372: Combining an application of the nitrification inhibitor DMPP with a single BS application gives best environmental outcomes.

Overall questions:

Comments 10: Is the DMPP toxic for the environment? Has it side effects?

Response 10: Thank you for your comment. DMPP, a nitrification inhibitor, has been extensively studied and used in agriculture to improve nitrogen use efficiency. It specifically targets the nitrification process, reducing nitrogen losses and mitigating negative environmental impacts. DMPP exhibits low toxicity to non-target organisms, biodegrades in soil, and does not accumulate over time. Its use is associated with reduced nitrogen pollution and greenhouse gas emissions. While caution and monitoring are advised, current evidence suggests that DMPP has minimal negative environmental effects when applied appropriately.

Comments 11: Why was the DMPP addition done aonly after the soil assays? It should not be added in the beginning?

Response 11: Thank you for your comment. We have revised the description in the gas kinetics analysis to make it clearer in Page 3, Lines 130-145 in the revised manuscript. Prior to the 108-h robotized incubation-monitoring (Robot) system being employed, in the treatments B0-DMPP, B1-DMPP, and B2-DMPP, the DMPP solution was evenly dripped onto the 30 g of soil after 0-, 30- and 60-days treatment from the 2.2.1 incubation experiment. Importantly, the addition of DMPP was carried out at the beginning of the 108-h gas kinetics monitoring incubation. Additionally, we have provided a schematic representation of the experimental design in Figure S1 for better understanding.

Page 3, Lines 130-145: A 108-h robotized incubation-monitoring (Robot) system was conducted to observe the characteristics of soil N₂O, nitrogen (N₂), oxygen (O₂) and CO₂emissions. This investigation employed 30 g of soil after 0-, 30- and 60-days treatment as outlined in 2.2.1 experiment. To specifically assess the efficacy of DMPP addition, an additional 30 g of soil from treatments B0, B1, and B2 underwent parallel monitoring of gas emissions (as depicted in Figure S1). The incubation was established in 120 mL serum bottles, featuring nine specific treatments: CK0, CK1, CK2, B0, B0+DMPP, B1, B1+DMPP, B2, and B2+DMPP, with each treatment replicated three times. Prior to incubation, for treatments involving DMPP addition, the DMPP solution was evenly dripped onto the surface of the soil at a rate of 1% of N contained in BS (i.e. the commercially recommended rate). The volume of DMPP solution and deionized water addition was determined for adjusting the soil water content of all treatments to 70% WHC. Once prepared, all bottles were immediately sealed with a rubber plug and an aluminum cap (Macherey-Nagel, Germany).

Comments 12: Why was necessary to add BS again after 30 days of assay?

Response 12: Thank you for your comment. After 30 days of assay, the incorporation of multiple applications of biogas slurry allows us to further investigate the cumulative effects of repeated BS application on soil N₂O emissions. By comparing the results of single application versus multiple applications, we can assess how the soil responds to continuous BS input. This includes examining adjustments in soil physicochemical properties and the changes in N₂O emission patterns over time. Moreover, repeated applications of BS are essential as they realistically simulate agricultural practices, where farmers often apply BS multiple times based on crop growth requirements. This approach not only reflects practical agricultural scenarios but also provides a more comprehensive evaluation of the long-term and cumulative impacts of biogas slurry on soil N₂O emissions. In summary, this study aims to offer valuable insights for developing scientifically-based fertilization strategies by understanding the effects of BS application over time.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present the results of work related to the dynamics of soil nitrogen transformations due to the addition of biogas slurry. Below are comments to which the authors should refer.
In the text, the style of expressing units, chemical formulas (superscripts, subscripts) should be improved.
In Section 2.2.1 there is a reference to Figure S1, which is not in the manuscript.
In line 101 the authors give the value of BS application " rate of 1.25 m3 ha-1", on what basis the authors determined this value, it is an unrealistic value when it comes to recommended BS dosage.
There is no diagram of the experiment cited as Figure S1 by the authors.
The title of the manuscript is inadequate for the results presented, as the results section includes data on carbon dioxide emissions, nitrogen N2, oxygen consumption.

Author Response

The authors present the results of work related to the dynamics of soil nitrogen transformations due to the addition of biogas slurry. Below are comments to which the authors should refer.

Response: Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

Comments 1: In the text, the style of expressing units, chemical formulas (superscripts, subscripts) should be improved.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have thoroughly reviewed and revised the text to ensure consistent and accurate expression of units and chemical formulas, including proper use of superscripts and subscripts.

Comments 2: In Section 2.2.1 there is a reference to Figure S1, which is not in the manuscript.

Response 2: Thank you for your comment. We apologize for the inconvenience. During the manuscript submission phase, we did submit supplementary materials, including Figure S1. However, it appears that these materials may not have been available during the peer review process. We will re-upload the supplementary materials to ensure that Figure S1 and any other relevant content are properly included.

Comments 3: In line 101 the authors give the value of BS application " rate of 1.25 m3 ha-1", on what basis the authors determined this value, it is an unrealistic value when it comes to recommended BS dosage.

Response 3: Thank you for pointing this out. In response to your comment regarding the description of BS volume, we have carefully reviewed our text and made the necessary corrections. Previously, we wrote incorrectly the amount of BS used in our experiments as "1.25 m³ ha^-1", whereas the correct value should be "300 m³ ha^-1". This rate is within the recommended range in greenhouse soil to enhance soil fertility without causing environmental harm [1-2]. This correction has been implemented in Page 3, Line 114. We believe this clarification will enhance the accuracy and clarity of our research findings. The calculations of the BS application value in our experiment are as follows:

First, we calculate the weight of surface soil per hectare using Equation 1:

Weight=Volume×Density (1)

where:

Volume is the product of the area and the depth of the topsoil layer: 10000 m²×0.2 m = 2000 m³

Density of the collected soil used for the experiment is 1.20 g cm^-3, which converts to 1200 kg m³

Thus, the weight of surface soil per hectare is 10000 m²×0.2 m×1200 kg m^-3=2400 t

The recommended application rate of BS is 300 m³ ha^-1, which is equivalent to 125 mL kg^-1of soil.

References

1. Guo Q, Gong X, Liu H. Study on Effect of Long-term Application of Biogas Manure on Soil Nutrients and Salt in Protected-land Vegetable Field. Acta Agriculturae Boreali-occidentalis Sinica, 2020, 29,127-134. doi:10. 7606/j. issn.1004-1389. 2020.01. 015.

2. Guo Q, Ge Y, Gong X, Wang Q. Effect of Amount of Biogas Slurry on Nutrients, Salt Accumulation and Migration in Greenhouse Soil. Shaanxi Journal of Agricultural Sciences, 2022, 68, 56-61.

Comments 4: There is no diagram of the experiment cited as Figure S1 by the authors.

Response 4: Thank you for your comment. We will re-upload the supplementary materials to ensure that Figure S1 and any other relevant content are properly included.

Comments 5: The title of the manuscript is inadequate for the results presented, as the results section includes data on carbon dioxide emissions, nitrogen N2, oxygen consumption.

Response 5: Thank you for your comment. We have revised the title of the manuscript to “The gas emission characteristics from biogas slurry application as influenced by application frequency and co-application with a nitrification inhibitor” based on your suggestion.

Author Response File: Author Response.pdf