Different Quality Classes of Decomposing Plant Residues Influence Dissolved Organic Matter Stoichiometry Which Results in Different Soil Microbial Processing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is interesting in elucidating the effects of various chemical quality of plant residues on soil organic matter stoichiometry.  The result is novel and can be referenced. However, there are some flaws to be corrected.

1. In the title: the word “of” was lost. Contrasting quality of...

2. The abstract was not clearly demonstrated. Materials and methods should be described in brief.

3. The field and lab experiments were not clear.

4. Soil samples were collected from the year of 2007-2008, how the soil samples were preserved ?

5. Figure 1 should be put ahead of the table 2. You should describe the data before the data analysis. In fig. 1 (b), what was the organic residue used ? where is the other 4 kinds ?

6. Fig. 2 is not clear. You can change the scatter to column.

Author Response

Rvw 1 comments with responses from authors:

Suggestions for Authors

The manuscript is interesting in elucidating the effects of various chemical quality of plant residues on soil organic matter stoichiometry. The result is novel and can be referenced. However, there are some flaws to be corrected.

1.1. In the title: the word “of” was lost. Contrasting quality of...

Response: It has been corrected and the title has been modified.

1.2. The abstract was not clearly demonstrated. Materials and methods should be described in brief.

Response: The abstract has been revised. The brief methodology has been added to the abstract in L. 26-27 of the revised version.

1.3. The field and lab experiments were not clear.

Response: We have added statements to indicate more clearly that our study is of a field experiment in nature not a lab experiment.  This is indicated by the origin of data used which were from the field. Please refer to the statements in the Introduction in L. 137-138 And in the Materials and methods L. 147-149.

1.4. Soil samples were collected from the year of 2007-2008, how the soil samples were

preserved?

Response: When the soil samples were collected in the year 2007-2008, they were brought back to the laboratory and immediately extracted for DOM. The extract solution samples were immediately frozen at –20 ◦C until analysis. Freezing of the extract solution is a common technique employed by many researchers for preserving DOM (Kawahigashi et al., 2006; Mavi et al., 2012; Scott and Rothstein, 2014; Soong et al., 2015; Bowering et al., 2023). The process of freeze/thaw of DOM samples similar to that used in this study was not expected to have substantial effect on both bulk quantities and molecular structure (quality) of DOC as revealed by Cook et al. (2016). Furthermore, this approach has no effect on DOC loss via flocculation as observed by Bowering et al. (2023). For dissolved organic N, it was found that the concentrations did not significantly decrease after freezing (Fellman et al. 2008). We have added a statement to this effect in L. 220-224 of the current revised manuscript.

Additional references:

Bowering, K. L., Edwards, K. A., Wiersma, Y. F., Billings, S. A., Warren, J., Skinner, A., & Ziegler, S. E. (2023). Dissolved organic carbon mobilization across a climate transect of mesic boreal forests is explained by air temperature and snowpack duration. Ecosystems, 26(1), 55-71.

Cook, S., Peacock, M., Evans, C. D., Page, S. E., Whelan, M., Gauci, V., & Khoon, K. L. (2016). Cold storage as a method for the long-term preservation of tropical dissolved organic carbon (DOC). Mires and Peat, 18(25), 1-8.

Fellman, J. B., D'Amore, D. V., & Hood, E. (2008). An evaluation of freezing as a preservation technique for analyzing dissolved organic C, N and P in surface water samples. Science of the total environment, 392(2-3), 305-312.

Kawahigashi, M., Kaiser, K., Rodionov, A., & Guggenberger, G., 2006. Sorption of dissolved organic matter by mineral soils of the Siberian forest tundra. Global Change Biology, 12(10), 1868–1877. https://doi.org/10.1111/j.1365-2486.2006.01203.x.

Mavi, M. S., Marschner, P., Chittleborough, D. J., Cox, J. W., & Sanderman, J. (2012). Salinity and sodicity affect soil respiration and dissolved organic matter dynamics differentially in soils varying in texture. Soil Biology and Biochemistry, 45, 8-13.

Scott, E. E., & Rothstein, D. E. (2014). The dynamic exchange of dissolved organic matter percolating through six diverse soils. Soil Biology and Biochemistry, 69, 83-92.

Soong, J. L., Parton, W. J., Calderon, F., Campbell, E. E., & Cotrufo, M. F. (2015). A new conceptual model on the fate and controls of fresh and pyrolized plant litter decomposition. Biogeochemistry, 124, 27-44.

 

1.5. Figure 1 should be put ahead of the table 2. You should describe the data before the data analysis. In fig. 1 (b), what was the organic residue used ? where is the other 4 kinds ?

Response: The current Table 3 (previously Table 2) showing statistical analysis which provide the information on the significance or non-significance of main and interactive effects which was used as guidelines for the presentation of the results to follow.  Fig. 1 showed only the main effects of residue treatments (Fig. 1a) and decomposition time (Fig. 1b) as they were significant.  On the other hand, the interaction was not significant as shown in Table 3 so the results of the interaction on DOC content were not shown. Thank you for your useful comments which prompt us to add a statement in L. 269-271 to inform the readers why Fig. 1 is shown.

            Fig. 1b shows the effect of decomposition time on DOC contents. The values of DOC content at each time is the average of all residue treatments (5 treaments).

1.6. Fig. 2 is not clear. You can change the scatter to column

Response: Based on your suggestion, we have changed Fig. 2 from the line graph to the column graph to make the result presentation clearer.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors studied the effect of the entry of monoculture residues of common agricultural plants into sandy soil on the composition of soluble organic matter. The study of the stoichiometric ratio of dissolved nitrogen and carbon at different stages of decomposition of plant residues demonstrated the role of plant species composition (quality of plant material) and microbiological activity in the formation of the material composition of soluble soil organic matter.

At the same time, in our opinion, to confirm the adequacy of the chosen method of isolating organic matter, it is necessary to add clarification of the correctness of the use of passing oxygen through an eluate containing water-soluble organic matter.

Line 155: purging with pure oxigen gas to remote present inorganic C. Confirmation is required (perhaps in the form of a reference to a previous study) that this process did not affect the composition of the substance being studied

 

Author Response

Rvw 2 comments with responses from the authors:

Suggestions for Authors

The authors studied the effect of the entry of monoculture residues of common agricultural plants into sandy soil on the composition of soluble organic matter. The study of the stoichiometric ratio of dissolved nitrogen and carbon at different stages of decomposition of plant residues demonstrated the role of plant species composition (quality of plant material) and microbiological activity in the formation of the material composition of soluble soil organic matter.

2.1 At the same time, in our opinion, to confirm the adequacy of the chosen method of isolating organic matter, it is necessary to add clarification of the correctness of the use of passing oxygen through an eluate containing water-soluble organic matter.

Response: We have confirmed the adequacy of the chosen method of isolating dissolved organic matter by clarifying the correctness of the use of passing oxygen through an eluate containing water-soluble organic matter as seen in detail in the response to comment # 2.2 below.

2.2 Line 155: purging with pure oxigen gas to remote present inorganic C. Confirmation is required (perhaps in the form of a reference to a previous study) that this process did not affect the composition of the substance being studied

Response: We have found out that the use of pure oxygen gas to purge the CO₂ gas from the solution samples of water extracted DOM does not significantly change the composition of the substances in our study, namely DOC and DN which are termed non-purgeable organic C (NPOC) and non-purgeable DN (NPDN), respectively. Regarding NPOC, it is accepted as the equivalent of total organic C (TOC). It has been shown that NPOC values in surface water is close to TOC as found through the technique of separately determining total C (TC) and inorganic C (IC) which are subsequently subtracted from each other (TOC = TC – IC) or the technique TOC by difference (Wallace et al., 2002). In addition, NPOC is regarded as equivalent to TOC because of the low purgeable organic C (POC) content, which is usually less than 1% of the TOC value in drinking/surface water (Wallace et al., 2002).

Oxygen is not the only gas used for purging, other forms of gas are used in various published work (Table A). The other gasses have oxygen as one of their composition, however all purging gases must be carbon free, such as purified air, ultrahigh purity oxygen and nitrogen (Mopper and Qian, 2006),

As for DN, the simultaneous determination of DOC and DN by the non-purgeable C and N technique has been proved to produce TNb values identical to those obtained by wet chemical digestion (K₂S₂O₈-digested N) (Ammann et al., 2000). Using the non-purgeable C and N technique, the samples in the current study were strictly acidified to pH < 2, to drive inorganic C as CO₂ gas and to prevent loss of ammonia while purging with oxygen.

In fact, DOC measurement by using a technique of non-purgeable organic carbon (NPOC) is a common technique that is used in DOC measurement derived from various sources including plant residue (Kothawala et al., 2008; Hömberg et al., 2020), soil (Sowerby et al., 2010; Mavi et al. 2012; Wagai, et al., 2020; Sae-Tun et al., 2023), and water (Wallace et al., 2002; Cook et al., 2016; 2017; Varonka et al., 2020). The simultaneous determination of DOC and DN by the non-purgeable technique was also conducted in soil solution samples (Mavi et al., 2012; Fellman et al., 2008), decomposing litter extract (Brandstatter et al., 2013), and surface and wastewater (Ammann et al., 2000).

We have added a statement to confirm no substantial change in DOM due to oxidation by the use of oxygen gas in the purging of inorganic C from DOM samples at the end of section 2.4 (Materials and methods) as follows: “The purging of inorganic C from DOM samples by oxygen gas during the NPOC technique did not produce substantial change in DOM as proved by the published results of a previous study [32] (Table S1)” in L. 234-236 of the current revised manuscript.

 

 

 

Table A Different types of gases and their composition used for purging inorganic carbon (IC) as measured DOC by non-purgeable organic carbon (NPOC) technique

No.	Sources of DOC	Types of gases used for purging	Gas composition	References
1	Water	Purified air	N2, O2, and Ar1	Cook et al. (2017). Quantifying tropical peatland dissolved organic carbon (DOC) using UV-visible spectroscopy. Water Research, 115, 229-235.
2	Water	C-free air	Gases without carbonaceous gases	Aitkenhead-Peterson et al. (2009). Dissolved organic carbon and nitrogen in urban and rural watersheds of south-central Texas: land use and land management influences. Biogeochemistry, 96, 119-129.
3	Soil	C-free air	Gases without carbonaceous gases	Wagai, et al. (2020). Iron and aluminum association with microbially processed organic matter via meso-density aggregate formation across soils: organo-metallic glue hypothesis. Soil, 6(2), 597-627.
4	Groundwater and sediment	Oxygen (O2)	O2	Steube et al. (2009). First attempts towards an integrative concept for the ecological assessment of groundwater ecosystems. Hydrogeology Journal, 17(1), 23.
5	Soil water (collected by using lysimeters)	Oxygen (O2)	O2	Sowerby, et al. (2010). The response of dissolved organic carbon (DOC) and the ecosystem carbon balance to experimental drought in a temperate shrubland. European Journal of Soil Science, 61(5), 697-709.
6	Water	Carrier gas	Not specified	Wallace et al. (2002). Total organic carbon analysis as a precursor to disinfection byproducts in potable water: Oxidation technique considerations. Journal of Environmental Monitoring, 4(1), 35-42.

¹(Nara et al., 2012).

Ammann, A. A., Rüttimann, T. B., & Bürgi, F. (2000). Simultaneous determination of TOC and TNb in surface and wastewater by optimised high temperature catalytic combustion. Water Research, 34(14), 3573-3579.

Brandstätter, C., Keiblinger, K., Wanek, W., & Zechmeister-Boltenstern, S. (2013). A closeup study of early beech litter decomposition: potential drivers and microbial interactions on a changing substrate. Plant and soil, 371, 139-154.

Cook, S., Peacock, M., Evans, C. D., Page, S. E., Whelan, M., Gauci, V., & Khoon, K. L. (2016). Cold storage as a method for the long-term preservation of tropical dissolved organic carbon (DOC). Mires and Peat, 18(25), 1-8.

Fellman, J. B., D’Amore, D. V., Hood, E., & Boone, R. D. (2008). Fluorescence characteristics and biodegradability of dissolved organic matter in forest and wetland soils from coastal temperate watersheds in southeast Alaska. Biogeochemistry, 88, 169-184.

Hömberg, A., Obst, M., Knorr, K. H., Kalbitz, K., & Schaller, J. (2020). Increased silicon concentration in fen peat leads to a release of iron and phosphate and changes in the composition of dissolved organic matter. Geoderma, 374, 114422.

Kothawala, D. N., Moore, T. R., & Hendershot, W. H. (2008). Adsorption of dissolved organic carbon to mineral soils: A comparison of four isotherm approaches. Geoderma, 148(1), 43-50.

Mavi, M. S., Marschner, P., Chittleborough, D. J., Cox, J. W., & Sanderman, J. (2012). Salinity and sodicity affect soil respiration and dissolved organic matter dynamics differentially in soils varying in texture. Soil Biology and Biochemistry, 45, 8-13.

Mopper, K., & Qian, J. (2006). Water analysis: Organic carbon determinations. Encyclopedia of analytical chemistry: Applications, Theory and Instrumentation.

Sae-Tun, O., Keiblinger, K. M., Rosinger, C., Mentler, A., Mayer, H., & Bodner, G. (2023). Characterization of aggregate-stabilized dissolved organic matter release-A novel approach to determine soil health advances of conservation farming systems. Plant and Soil, 488(1), 101-119.

Sowerby, A., Emmett, B. A., Williams, D., Beier, C., & Evans, C. D. (2010). The response of dissolved organic carbon (DOC) and the ecosystem carbon balance to experimental drought in a temperate shrubland. European Journal of Soil Science, 61(5), 697-709.

Varonka, M. S., Gallegos, T. J., Bates, A. L., Doolan, C., & Orem, W. H. (2020). Organic compounds in produced waters from the bakken formation and three forks formation in the Williston Basin, North Dakota. Heliyon, 6(3).

Wagai, R., Kajiura, M., & Asano, M. (2020). Iron and aluminum association with microbially processed organic matter via meso-density aggregate formation across soils: organo-metallic glue hypothesis. Soil, 6(2), 597-627.

Reviewer 3 Report

Comments and Suggestions for Authors

The language and explanations/justifications throughout the manuscript (literature review and the present study findings) need clarification. The wording is often unclear or vague. The main ideas of the discussion need to be clarified and emphasized, also through revising language. The very poor use of the English language made it difficult to understand the flow of thoughts, so I may have misunderstood some of them.

Line 27, what is the balance DOC/DN ratio?

Line 26-29, is really opposite as described in literature. However, justification is required for this result.

Line 29-31 seems hypothesis but there is no exact outcome of the study. What is your main finding could be your conclusive statement.

Line 50-51, poor writing.

Line 50-59, the meaning of this paragraph is unclear. What authors want to deliver is not clear. Stoichiometry balance or imbalance mean? What is the ratio?

Line 103-104, how did you measure microbial growth? Is that mentioned in methodology?

Line 103-104, alternate use of microbial CUE with qCO2 is underestimation, for better result it is required to measure microbial CUE on experimental basis or you can determine the stoichiometric microbial nutrient use efficiencies.

Line 108-109 not clear, rewrite it.

What about NPK fertilizers? If only crop residue has been applied then microbes may face nitrogen limitation (higher stoichiometry) that can cause decreased microbial CUE?

Conclusion part is too large

Discussion section need a thorough story, no need of sub-headings.

 

 

Comments on the Quality of English Language

The language and explanations/justifications throughout the manuscript (literature review and the present study findings) need clarification. The wording is often unclear or vague. The main ideas of the discussion need to be clarified and emphasized, also through revising language. The very poor use of the English language made it difficult to understand the flow of thoughts, so I may have misunderstood some of them.

Line 27, what is the balance DOC/DN ratio?

Line 26-29, is really opposite as described in literature. However, justification is required for this result.

Line 29-31 seems hypothesis but there is no exact outcome of the study. What is your main finding could be your conclusive statement.

Line 50-51, poor writing.

Line 50-59, the meaning of this paragraph is unclear. What authors want to deliver is not clear. Stoichiometry balance or imbalance mean? What is the ratio?

Line 103-104, how did you measure microbial growth? Is that mentioned in methodology?

Line 103-104, alternate use of microbial CUE with qCO2 is underestimation, for better result it is required to measure microbial CUE on experimental basis or you can determine the stoichiometric microbial nutrient use efficiencies.

Line 108-109 not clear, rewrite it.

What about NPK fertilizers? If only crop residue has been applied then microbes may face nitrogen limitation (higher stoichiometry) that can cause decreased microbial CUE?

Conclusion part is too large

Discussion section need a thorough story, no need of sub-headings.

 

 

Author Response

Rvw 3 comments:

Suggestions for Authors

3.1 The language and explanations/justifications throughout the manuscript (literature review and the present study findings) need clarification. The wording is often unclear or vague. The main ideas of the discussion need to be clarified and emphasized, also through revising language. The very poor use of the English language made it difficult to understand the flow of thoughts, so I may have misunderstood some of them.

Response: Following your comments, the manuscript was extensively revised and then edited thoroughly by a knowledgeable native English-speaking scientist. We hope that the unclear or vague wording and grammar have been improved so that the flow of thoughts can be perceived accurately.

 

3.2 Line 27, what is the balance DOC/DN ratio?

Response: The balance DOC/DN ratio is a shorthand writing of the balance DOC-and-DN ratio. It means that the DOC-and-DN ratio of dissolved organic matter is close to the microbial biomass C-to-N ratio. We have removed this shorthand writing and used the full ‘DOC-and-DN ratio’ throughout the revised manuscript.

 

3.3 Line 26-29, is really opposite as described in literature. However, justification is required for this result.

Response: Do you mean the contradictory results between the high-N residues in the literature leading to high CUE and that in this study, i.e., groundnut stover, which produced low CUE? If so, the justification can be provided as follows: The groundnut stover in the current study led to imbalanced DOC-to-DN stoichiometry leading to its lower CUE than its lower-N counterparts, however those high-N residues in the literature produced balanced DOC-to-DN stoichiometry leading to their higher CUE than their lower-N counterparts.

3.4 Line 29-31 seems hypothesis but there is no exact outcome of the study. What is your main finding could be your conclusive statement.

Response:   The exact outcome of the study which is its main finding is that

“Our study has produced a novel finding that the balanced/imbalanced DOC-and-DN stoichiometry, in addition to the initial chemical quality of the organic residues applied to soil, control microbial processing. Similar quality organic residues can result in different microbial processing, e.g., CUE, depending on the resulting balanced/imbalanced DOC-and-DN stoichiometry.” We have added this statement to the conclusions in L. 598-602 of the revised manuscript.

3.5 Line 50-51, poor writing.

Response: It has been rewritten. Please refer to the response to comment 3.6 immediately below.

 

3.6 Line 50-59, the meaning of this paragraph is unclear. What authors want to deliver is not clear. Stoichiometry balance or imbalance mean? What is the ratio?

Response: The whole paragraph has been rewritten to make it clearer as follows: “Because microbes need to maintain their own internal stoichiometric balance or homeostasis of the C and N nutrients, the contents of DOC and DN in soils and their interaction influence their microbial uptake. The DOC-and-DN stoichiometry exerts control over the microbial uptake.  Microbial stoichiometric homeostasis means that microbes maintain their chemical composition close to constant in the face of variations in the chemical composition of their resources (substrates) [11].  Thus, the C and N nutrients in microbes are maintained constant as indicated by their C-to-N ratio despite the changes in C-to-N ratio of the substrates. Considering DOC and DN as the microbial substrates in soils, their C-to-N ratio relative to that of microbes indicates whether they have balanced or imbalanced stoichiometry. The balanced DOC-and-DN stoichiometry of substrates implies that the DOC-to-DN ratios of substrates are close to the C-to-N ratios of microbes [9,12]. In contrast, the imbalanced DOC and DN stoichiometry implies that the DOC-to-DN ratios of substrates are either substantially lower or higher than the C-to-N ratios of microbial biomass [9,12]. The balanced DOC and DN stoichiometry enhances microbial C use efficiency (CUE) in contrast to the imbalanced DOC and DN stoichiometry [7].” Please refer to L 59-73 in the revised version.

 

 

3.7 Line 103-104, how did you measure microbial growth? Is that mentioned in methodology?

Response: The word microbial growth has been changed to ‘microbial biomass’ in L. 132 of the current version.  The method employed to measure microbial biomass is the fumigation-extraction technique (Amato and Ladd, 1988).  The method is mentioned in the revised version of the manuscript in the Materials and methods (section 2.1) L. 163-164.

 

3.8 Line 103-104, alternate use of microbial CUE with qCO2 is underestimation, for better result it is required to measure microbial CUE on experimental basis or you can determine the stoichiometric microbial nutrient use efficiencies.

Response:

You are correct that direct conversion of qCO₂ to CUE is an underestimation of CUE as

qCO₂ = CO₂-C/ MBC while CUE is calculated as MBC/ (CO₂-C + MBC).  According to Xu et al. (2017) who studied global pattern and factors controlling qCO₂, the association of qCO₂ and CUE is confirmed to have negative relationships.  The negative relationship between qCO₂ and CUE was verified by Sinsabaugh et al. (2017) and later by Fang et al. (2020) and Li et al. (2021).  We have added a statement to explain this in the manuscript as follows “The qCO₂, defined as microbial respiration per unit of microbial biomass, is used as a proxy for CUE [21] as they have been found to be negatively correlated to each other [7,22].” in L. 102-104 of the current version of the manuscript.

Our current study did not have the objective to quantitatively determine CUE on an experimental basis but to use qCO₂ determined quantitatively as a proxy for CUE to compare their differences among residue treatments. As the negative correlation between the two parameters has been scientifically established, the use of qCO₂ as a proxy for CUE is deemed acceptable. The suggested use of the stoichiometric microbial nutrient use efficiencies, e.g., the ratio of CUE to NUE is relevant, however in our current study we have used the stoichiometry of substrates (DOM), i.e., indicated by the ratio of DOC-to-DN, and that of microbial stoichiometry, i.e., the ratio of MBC to MBN to discuss the balanced or imbalanced stoichiometry of nutrient C and N in the DOM substrate.  This suffices to test our hypothesis. The framework of the current paper did not involve NUE which needs data on mineral N.

References

Fang, Y., Singh, B. P., Collins, D., Armstrong, R., Van Zwieten, L., & Tavakkoli, E. (2020). Nutrient stoichiometry and labile carbon content of organic amendments control microbial biomass and carbon-use efficiency in a poorly structured sodic-subsoil. Biology and Fertility of Soils, 56, 219-233.

Li, J., Sang, C., Yang, J., Qu, L., Xia, Z., Sun, H., ... & Wang, C. (2021). Stoichiometric imbalance and microbial community regulate microbial elements use efficiencies under nitrogen addition. Soil Biology and Biochemistry, 156, 108207.

Sinsabaugh, R. L., Moorhead, D. L., Xu, X., & Litvak, M. E. (2017). Plant, microbial and ecosystem carbon use efficiencies interact to stabilize microbial growth as a fraction of gross primary production. New Phytologist, 214(4), 1518-1526.

Xu, X., Schimel, J.P., Janssens, I.A., Song, X., Song, C., Yu, G., Sinsabaugh, R.L., Tang, D., Zhang, X. and Thornton, P.E., 2017. Global pattern and controls of soil microbial metabolic quotient. Ecological Monographs, 87(3), pp.429-441.

3.9 Line 108-109 not clear, rewrite it.

Response: It has been rewritten to “In this research, DOM samples studied were immediately extracted from fresh soils sampled which had been collected in year 13 (2007-2008) of a long-term field experiment initiated in 1995.” in L. 147-149 of the current version.

3.10 What about NPK fertilizers? If only crop residue has been applied then microbes may face nitrogen limitation (higher stoichiometry) that can cause decreased microbial CUE?

Response: A statement on the use of inorganic fertilizers to supplement low quality residues to reduce N limitation through adjusting the C-and-N stoichiometry of the microbial substrate was added into the Discussion section in L. 566-568 of the current version.

 

3.11 Conclusion part is too large

Response: We have rewritten the conclusions to show more clearly the results which have proved the hypotheses, the novel findings and implications, although the length of the section is not reduced.

 

3.12 Discussion section need a thorough story, no need of subheadings.

Response: Discussion sections (4.1 and 4.2 in the previous version) have been merged together as suggested. However, we have put headings at some intervals to make it easier to the readers to follow the story.

 

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

In my opinion, this manuscript does not have the characteristics of a scientific article as it has the appearance of a re-elaboration of data deriving from articles already published by some of the authors. It is not clear the novelity of this paper perhaps represented by the determination of DOC and DN and the calculation of their ratio?

 

Author Response

Rvw 4 comments with responses from the authors:

Suggestions for Authors

4.1 In my opinion, this manuscript does not have the characteristics of a scientific article as it has the appearance of a re-elaboration of data deriving from articles already published by some of the authors. It is not clear the novelity of this paper perhaps represented by the determination of DOC and DN and the calculation of their ratio?

Response: Thank you for your comments. You are correct about the sources of the data used in this article. One source was published data in the literature, but another source was newly generated by determining the DOC and DN in stored samples and the calculation of their ratio. However, we respectfully disagree with you on the lack of novelty of this article. In our view, the novelty of a scientific article is based on the analysis and interpretation of its data not the source of the data.  By analyzing the relationships of these two sets of data, this paper has come up with a novel finding which is stated in the conclusions part of the paper in L. 598-602 (current version) as follows “Our study has produced a novel finding that the balanced/imbalanced DOC-and-DN stoichiometry, in addition to the initial chemical quality of the organic residues applied to soil, control microbial processing. Similar quality organic residues can result in different microbial processing, e.g., CUE, depending on the resulting balanced/imbalanced DOC-and-DN stoichiometry.” 

Reviewer 5 Report

Comments and Suggestions for Authors

I'm enclosing it in separate file

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Some phrases or sentences could be improved, but I do not observe numerous errors in English 

Author Response

Rvw 5 comments with responses from the authors:

 

Suggestions for Authors

Review of manuscript titled:

5.1 Contrasting quality decomposing plant residues influence dissolved organic matter stoichiometry resulting in different soil microbial processing (ID: soilsystems-2772517)

In the manuscript entitled: Contrasting quality decomposing plant residues influence dissolved organic matter stoichiometry resulting in different soil microbial processing, the authors take up the interesting topic of dissolved organic matter (actually dissolved organic carbon and nitrogen and the ratio of these components) against the background of microbial transformations depending on the different quality of organic residues introduced into soils. The research is based on a long-term field experiment described in the methodology section. The manuscript is prepared in accordance with the requirements of the journal Soil systems. Literature is cited appropriately but not excessively. The research topic undertaken by the authors fits very well into the scope of the journal.

Response: We have chosen the journal Soil Systems after we have read its scope and determine that our manuscript fits the journal. Thank you for pointing this out.

 

5.2 However, the manuscript needs deep consideration and improvement.

Response: We have thoroughly revised the manuscript along the line suggested by all the reviewers and editor.  We are confident that we have made significant improvements.

 

5.3 My most important objections concern the quality of the results presented, which mostly do not support the relationships indicated by the authors. The relationships are weak or not statistically significant at all. The results should be interpreted here with great caution. One can probably point to the possible presence of some trends that still need to be confirmed. Discussion and inference built on such results may constitute overinterpretation.

Response: We agree with your comments. We have deleted many results which are considered weak due to weak relationships and/or non-significant and now only moderate to strong and statistically significant relationships are presented. More detailed response is in the response to comment # 5.9.

 

5.4 Without an improvement in the quality of the results presented and a profound rewriting of the results and the discussion, I do not see how the paper can be published in this form.

Response: We are confident that after the improvement of the results as explained above in responses to comment # 5.3 and 5.9 and the rewriting of the results and discussion, the paper has been much improved.

 

5.5 I believe that the title of the paper also needs to be modified so that it corresponds to the substantive content of the manuscript and, above all, to the conclusions formulated.

Response: We have modified the title to “Different-quality classes of decomposing plant residues influence dissolved organic matter stoichiometry resulting in different soil microbial processing”. We believe that this title reflects the revised content of the manuscript and the conclusions.

5.6 I am opposed to the term "contrasting quality" often used by authors and suggest that it be removed from the entire paper. I refer to this further below.

Response: We thank you for your constructive suggestions. Please refer to the response to comment #5.26 below where we explain the term ‘chemical quality.’ We would like to inform  the reviewer that an assessment of the chemical quality of the different residues used in the study  has been made in the revised Table 1 in the form of ‘chemical quality class’.

 

5.7 I leave the final decision on the fate of the manuscript to the Editor, I believe that after thorough improvement of the manuscript, it is possible to significantly increase its quality and publish it in an revised, better form.

Response: We thank the reviewer for pointing out that the quality of the paper can be significantly increased to the level publishable. We have thoroughly revised the manuscript along the line suggested by all the reviewers and the academic editor.  We are confident that we have made significant improvements, and we hope that the paper is now in an acceptable form for publication in Soil Systems.

 

Comments and general suggestions for authors:

5.8 Abstract: The abstract should be revised extensively. It is supposed to illustrate the entire paper, so only the most important information should be selected and, in addition, presented in a way that is understandable to a reader who is not familiar with the subject. This is not helped by the large number of abbreviations introduced into the abstract by the authors, perhaps this should be modified as it makes it very difficult to read. In my opinion, the aim of the study should be clearly stated, the most important results and possible statistically significant correlations should be briefly and simply presented and special attention should be paid to the conclusions, which must be corrected in the abstract.

Response: The abstract has been thoroughly revised along the lines of your and the other reviewers’ suggestion.  The abbreviations have been replaced by full words. The purpose of the study is clearly stated, a brief description of the methodology added, the most important results presented, and a statement of our main conclusionsadded.

 

5.9 I have great doubts about the quality of the results. Please verify the validity of relationships that are weak or statistically insignificant in the graphs. I believe this is one of the weaker points of this manuscript. Leading a discussion on the basis of poor quality results is unjustified and/or must be very cautious, this applies especially to the discussion on microbial activity. I would very much ask you to reconsider especially the presentation of results relating to soil microbiological properties (also included in the supplementary materials), especially as it is based on results from another publication.

Response: We have removed those individual graphs showing the relationships between dissolved nitrogen (DN) and some microbiological parameters which are weak or non-significant statistically from Figure 3. The microbiological parameters for these relationships retained are CO₂-C, qCO₂ and microbial biomass N pertaining to the early stage of decomposition. The relationships during the later stages of decomposition are mostly weak or non-significant and consequently have been removed from Figure 3. In addition, the supplementary material Figure S1 pertaining to the relationships of DOC-to-DN ratios with various microbiological parameters during the three stages of decomposition have been removed as it contained mostly weak and non-significant relationships.  Now Figure 4 illustrating the relationships of DOC-to-DN ratios with qCO₂ during the early stage of decomposition which is the only significant relationship is retained. Please also refer to the response on the same subject in the response to comment # 5.34.

            The discussion of this part has been thoroughly revised to highlight the early stage of decomposition which showed the relatively strong and significant relationships or influences compared to the later stages of decomposition in L 471-480 of the current revised version.

5.10 Please remove the repeated of the same statements from the text.

Response: The statement “The long-term field experiment was carried out in 1995.” from L. 124 (previous version) has been removed from the section 2.2 in the current version.

 

Specific comments:

5.11 Line 89 - please start with a new paragraph. Research hypotheses should be presented after first formulating the aim of the research. It is necessary to reconsider and clearly formulate the research objective(s), so the last section of the introduction should be carefully thought through and strongly reworked and the research objectives formulated accordingly (the objective cannot be to conduct determinations). The research objectives are carried out with the assumptions we are to verify – this role is played by the research hypotheses, so please place them below the formulated objectives.

Response: We have put the objectives followed by the hypotheses in a new paragraph as suggested. Please find it in L. 128-144 of the revised version.

5.12 Line 108: the sentence needs linguistic improvement.

Response: It has been rewritten to “DOM samples studied were immediately extracted from fresh soils samples which had been collected in year 13 (2007-2008) of a long-term field experiment initiated in 1995.” in L. 147-149 of the revised version.

5.13 Line 124: please avoid repetition.

Response:  We have deleted this sentence all together. Please refer to section 2.2 of the revised version.

5.14 Line 122: it seems to me that it makes much more sense to give the average temperature and precipitation for each season, rather than year-round averages. This is just a suggestion.

Response: We agree with your suggestion and have inserted the new information in L. 182-183 of the revised version,

5.15 Line 125: the introduction of basic soil information needs to be supplemented with important properties that will determine the direction of the chemical and microbiological transformation processes in soils, e.g. pH, macronutrient content such as P, CaCO3 (inorganic C) content.

Response: We agree with your suggestion and have incorporated the new information on soil properties in L. 185-186 of the revised version. The statement now reads “Initial soil properties were pH of 5.5, SOM 0.36%, total N 0.02%, Bray II P 47.2 mg kg-1, exchangeable K 0.077 cmol kg-1, and CEC 3.53 cmol kg^-1”

5.16 Line 144: instead of "different decomposition stages" I propose to introduce rather: "subsequent decomposition stages".

Response: We have followed your suggestion by changing "different decomposition stages" to "subsequent decomposition stages" in L. 211 of the current version.

5.17 Line 144: Are you sure "contrasting" organic residues is the right term here? Why are these organic residues contrasting? How do we know if they are contrasting? Or are they rather just different, distinct etc.?

Response: These organic residues are contrasting because they have different chemical composition notably nitrogen, lignin, cellulose and polyphenols.  They are categorized into different chemical quality classes as shown in Table 1 of the current revised version. The word “different” may be used, however, the use of “contrasting” is a stronger word than “different”. Due to this reason we have elected to retain the word “contrasting” which was used interchangeably with “different”. However, in many places in the revised manuscript we have modified the “contrasting organic residues” to “contrasting organic residue classes” or “high/low quality classes of residues”., for examples, in L. 18, 128-129, 238, 482, 484, 499, and 583 of the current version.

5.18 Line 151: Please add “samples” after soil.

Response: We have done this as in L. 218 of the current version.

5.19 Line 155: Please explain the procedure for removing inorganic C with pure oxygen.

Response: The procedure used in the current study consists of the acidification of the sample and sparging (gas flushing) with C-free air (pure oxygen), in which the inorganic carbon (IC) is converted to carbonic acid (H₂CO₃), also known as dissolved carbon dioxide (CO₂) (Wallace et al., 2002). The dissolved CO₂ was driven off in this process. The sparging time used was 3 minutes. After removal of inorganic carbon by sparging, DOC as non-purgeable organic C (NPOC) and dissolved N (DN) as and non-purgeable DN (NPDN) were immediately analyzed with oxidative-high temperature combustion (800 °C) using a TOC/TN_b analyzer (Multi N/C 2100s, Analytik Jena, Jena, Germany) equipped with a non-dispersive infrared (NDIR) detector for CO₂ measurement for DOC and chemical luminescence detector (CLD) for DN.

We have modified the passage starting with the sentence “After filtration….” to the end of the paragraph to explain the procedure in more detail as follows.  “After filtration, the extractants were acidified to a pH < 2 by adding 2 M HCl and purging with pure oxygen gas for 3 minutes per sample to remove present inorganic C. The purging with oxygen gas converted inorganic C to dissolved CO₂ which was driven off. After removal of inorganic carbon by sparging (gas flushing), DOC as non-purgeable organic C (NPOC) and DN as NPDN were simultaneously analyzed immediately with oxidative-high temperature combustion (800 °C) using a TOC/TN_b analyzer (Multi N/C 2100s, Analytik Jena, Jena, Germany) equipped with a non-dispersive infrared (NDIR) detector for C and a chemical luminescence detector (CLD) for N.” Please find this passage in L. 226-233 of the current version.

 

5.20 Line 159: Table 1: I suggest dropping the designation a and b and introducing another table number, i.e. designate Tables 1 and 2 and convert them into completely separate tables.

Response: Done. We have converted Table 1a and b into completely separate tables, i.e., Table 1 and 2.  Please find them  in L. 173 and L. 188 of the current version, respectively.

 

5.21 Table 1a - whether the results presented are averages or individual measurements; no information on measurement error or standard deviation. Table 1b has these data but is not explained in the description. Please add appropriate labels and explanations.

Response: Regarding Table 1a (currently Table 1), the results in Table 1a are averages, however the error measurement (standard error of the means) are only available for the parameters C, N and C-to-N ratio. Consequently, we have elected not to show the errors for all parameters.  We had to do the same in our other previously published papers (Puttaso et al., 2011; 2013; Kunlanit et al., 2014; 2020; Poosathit et al., 2023) using this set (year 13) of residue chemical quality data without the errors indicated.  However, the most recently published paper from our research group (Laub et al., 2024) has shown the results of chemical composition of the studied residues from year 13 accompanied by standard errors (SE) computed from residue chemical composition applied in four different years of the long-term experiment to show the errors as shown in the Table A below. 

 

Table A Chemical characteristics of applied organic litter (Puttaso et al., 2013). Values within the same column that share the same capital letter are not significantly different (p < 0.05).

Treatments	Quality1	Carbon (g kg–1)		Nitrogen (g kg–1)	Lignin (g kg–1)	Cellulose (CL)	C-to-N ratio	Polyphenols
Rice straw	Low	367A	4.7A		28.7A	507A	78A	6.5A
Groundnut stover	High	388A		22.8B	67.6A	178AB	17B	12.9A
Dipterocarp	Low	453B	5.7A		175.5B	306AB	80A	64.9B
Tamarind	Medium	427B	13.6C		87.7C	143B	32C	31.5C
SE		7	0.8		19	46	3.4	5.6

¹ Palm et al. (2001); SE, standard error computed based on published measured values from different years (year 1995, 2004, 2007, 2012) (Vityakon et al., 2000; Samahadthai et al., 2010; Puttaso et al., 2013; Sanghaw et al., 2017).

            Regarding Table 1b (currently Table 2), the explanation of these data has been put in the text of the revised version in L. 166-172.

            The labels and descriptions were already complete.

Additional references:

Laub, M., S. Blagodatsky, M. Van de Broek, S. Schlichenmeier, B. Kunlanit, J. Six, P. Vityakon,

and G. Cadisch. 2024. SAMM version 1.0: A numerical model for microbial-mediated

aggregate formation. Geosci. Model Dev., 17, 931–956.

doi.org/10.5194/gmd-17-931-2024

Palm, C.A. et al. 2001. Organic inputs for soil fertility management in tropical agroecosystems: Application of organic resource database. Agriculture, Ecosystems and Environment 83: 27-42.

 

5.22 Line 170 - what does it mean: RCBD needs clarification.

Response: It means randomized complete block design.  We have changed it to RCB in this sentence (L. 240).  We write it in the abbreviated form as it is the second use of this term. The first use of this term in full name was in L. 202 (section 2.3). 

 

5.23 Line 175: Please enter this information in all tables.

Response: The information on no. of observations equals to 3 (n = 3) has been entered in Tables 2 in the current revised manuscript. The other tables (Table 1 and 3) do not require such information.

5.24 N=3?

Response: Yes, n = 3, i.e., in Table 2 (formerly Table 1b) and Fig. 2. In this Table and Fig., we have stated that mean values are derived from 3 observations (n = 3)

5.25 Previously written about 2 replicates???

Response: Do you mean the statement in L. 142-143 (previous version) with the statement “Soil samples were collected at 0-15 cm depth in two replications per plot using a bucket auger..”? If so, we offer the explanation as follows: The field experiment has three replications or blocks as indicated in section 2.3. However, soil collection was conducted in each plot with two systematic replications per plot as indicated in section 2.4. We have added the phrase ‘or blocks’ behind ‘replications’ in L. 202 to distinguish it from the ‘replications per plot’ in section 2.4 in L. 210. Regarding the calculation of the means of DOC or DN contents, it was done as follows: the means of the two replicates/plot were calculated to represent the means of each plot followed by the calculation of the final mean of each treatment involving the 3 blocks as presented in the results in the manuscript.

 

5.26 Lines 168; 193: it is unclear how the organic matter chemical quality (Q) was evaluated this absolutely needs to be clarified as otherwise it is not clear what the relationships studied are based on; in Table 1a the properties presented do not classify the study variants into specific Q quality groups, so what specifically is Q?

Response: We thank you for the constructive comments on the use of chemical quality to mean each of the residue treatments. In our original manuscript we had overlooked the need to clarify this which may confuse some readers who are unfamiliar with literature on decomposition process in soils. The ‘chemical quality (Q)’ is the standard term originally used in the literature on decomposition of organic materials in soils (Swift et al., 1979; Palm et al., 2001). This term permeates throughout the literature in the field of decomposition in soils.  Briefly, quality or chemical composition of organic materials, more prominently N, lignin, cellulose, and polyphenol, is one of the three factors, i.e., quality, decomposer organisms, and physico-chemical environment, regulating decomposition. The chemical quality is distinguished from ‘physical quality’, e.g., leaf litter thickness, hardness, litter size, which also controls the rate of decomposition, but it is outside the scope of this manuscript. Organic materials used for the purpose of soil fertility improvement, emphasizing on N release, have been classified into four categories based on three chemical composition parameters, N, lignin and polyphenols (Palm et al., 2001). Later studies emphasizing soil organic matter build-up have added cellulose as another quality parameter to effectively classify organic residues (Puttaso et al., 2013; Kunlanit et al., 2014). Organic residues are classified into a spectrum of low to high quality classes mainly based on their low to high N contents. The spectrum of residue quality classes has its variants based on the composition of carbonaceous compounds, i.e., lignin and cellulose which are resistant and labile C, respectively.  For example, a low N but high lignin residue and its low N but high cellulose counterpart are variants of low-quality residues.

We have added this explanation of chemical quality in the Introduction (L. 83-92) and have changed the first sentence of section 2.5 (Statistical analysis) (L. 238 current version) to ‘Testing of the effects of organic residue chemical quality classes (Q),….’ as well as the first sentence of section 3.1 to ‘The chemical quality classes of organic residues (Q)…’ in L. 265 of the current version.

References (additional):

Swift, M.J. et al.,1979. Decomposition in terrestrial ecosystems. Blackwell Scientific Publications, Oxford, 372 p.

Palm, C.A. et al. 2001. Organic inputs for soil fertility management in tropical agroecosystems: Application of organic resource database. Agriculture, Ecosystems and Environment 83: 27-42.

 

5.27 If, however, these are simply different types of organic matter introduced into the soil (RS, GN, DP, TM), differing in their chemical properties, this should be made clear

Response: It has been made clear in the revised Table 1 as to their ‘quality classes’ reflecting their contrasting chemical composition.

5.28 In my opinion, the use of the term "chemical quality" should be abandoned, because no qualitative assessment of the properties of the samples from the different variants has been made here, only the properties of these groups have been investigated and are presented in Table 1, so we can only speak here of different groups/treatments or of differences between these groups, not of quality, much less of "contrasting quality" as already stated in the title of the paper.

Response: Based on your constructive comments, we have added the qualitative assessment of the properties of the studied residues to Table 1 (revised version).  We think this addition should allow us to discuss these residues  in terms of chemical quality and compare their differences.

5.29 I propose to modify these terms throughout the manuscript, or provide a way to evaluate the quality of organic residues.

Response: Thank you for your helpful suggestion. We have opted to provide a way to evaluate the quality of the residues. This is done by adding the qualitative assessment of the properties of the studied residues in Table 1. Having done so, we have elected to retain the term ‘chemical quality’. In addition, this is a conventional term used in the literature on decomposition in soils as explained in detail in the response to comment #5.26 above.

 

5.30 Line 200: please explain all symbols in Table 2.

Response: Done

 

5.31 Line 251: Fig 2. Please explain the abbreviation WAI in the tables; why in the column for 52 WAI in tables a and b are the test results only for sampling time (capital letters), is this a mistake?

Response: We have elected to delete the abbreviation WAI from the revised Fig. 2 and to use the full term instead. In addition, following your suggestion, Fig. 2 has been completely revised to column (bar) graphs and the letters, both uppercase and lowercase, used for mean separation are placed to directly accompany the columns. 

5.32 Figure 2 seems unreadable to me when the x data is only: 0, 2, 8 and 52 it seems that the Fig type is not properly chosen, I suggest using another graphical rearrangement of the results, e.g. like Fig. 1 or other column charts.

Response: Based on your suggestion, we have changed Fig. 2 from line graphs to column graphs which illustrate the results more clearly.

 

5.33 Line 278 - mental abbreviation; add 'of decomposition'.

Response: Done in L. 348.

5.34 Line 276 - section on soil microbiological parameters: the results discussed in this section are taken from another paper, most of them do not show strong or statistically significant correlations, and the graphs showing these results alone take up as much as 1 page. I consider these data to be of poor quality and the relationships unconfirmed, so I would remove this entire section from the manuscript.

Response: We have drastically revised this paragraph following your suggestion. The revision was based on the deletion of many relationships involving soil microbiological parameters that were weak and non-significant.  As a result, Fig. 3 has only 3 individual graphs retained out of the original 12 graphs.  All the retained individual graphs are in the early stage of decomposition. They all show significant moderate to strong relationships. We have elected to retain Fig. 4, which show significant although not strong relationship. Nevertheless, Fig. 4 supports the results of Fig. 3 in highlighting the early stage as the significant period of decomposition where the role of DN and the ratio of DOC-to-DN in microbial processing were the most evident.

5.35 Section 3.3. should be rethought.

Response: We have drastically revised this section following your suggestion. The revision was based on the maintenance of only the relationships between DN and the ratio of DOC-to-DN with the microbiological parameters, CO₂-C and qCO₂ in Fig. 5. These two microbiological parameters indicate the loss from and retention of C in the soil systems. This change follows that of Fig. 3 in which these two microbiological parameters are retained.

 

5.36 section 4.1. title: instead of "contrasting quality", I propose "different types of...".

Response: Section 4.1 and 4.2 have been combined following the suggestion of reviewer 3, as a result the section title was omitted.  The assessment of quality of residues using ‘chemical quality class’ in Table 1 has solved this problem.

5.37 Line 322: instead of “prominent”, I propose to replace with main, prevailing… or similarly.

Response: We have elected to replace it with ‘main’ as suggested (L. 389).

5.38 Line 374: 'residue N'?

Response: It has been replaced with ‘N contents in the residues’ (L. 452  of the current version).

5.39 Line 402: instead of I suggest: various types of residues. 

Response: We have changed it to ‘different quality class residues’ in L. 499 of the current version.  This is because we have introduced the chemical quality classes of residues in Table 1 which categorizes these residues.

5.40 Please use abbreviations consistently in the manuscript, e.g.: lines 440,442 - GN; lines 452; 459 TM.

Response: Done as in L. 525, 527, 537, 544 of the current version.

5.41 Line 505 - please replace "higher quality" with "higher content of..."

Response: This statement has been modified and the ‘higher quality’ has been replaced by ‘higher quality class’ in L. 583 of the current version.  In general, the high or low quality residue have been replaced by high or low quality class residues to reflect the residue types in the revised version.

5.42 Line 507 - Lower quality - as above

Response: Similar to the response to the comments in 5.41, this statement has been modified and the ‘lower quality’ has been replaced by ‘lower quality class’ in L. 585 of the current version.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have successfully revised the manuscript as per suggestions but conlusion is still too large. It need to be shorten with main and clear findings . conlsuion should not be another abstract but it should depict clear findings and outcomes and future perspecticves etc. It can be accpepted after revising conclusion section.

Comments on the Quality of English Language

Englis is clear and readability is also good.

Author Response

Response to Rvw 3 (R2)

Suggestions for Authors

The authors have successfully revised the manuscript as per suggestions but conlusion is still too large. It need to be shorten with main and clear findings . conlsuion should not be another abstract but it should depict clear findings and outcomes and future perspecticves etc. It can be accpepted after revising conclusion section.

Responses: We are pleased that the revision was mostly acceptable to the reviewer.  As for the conclusions, we have shortened it by deleting some detailed descriptions of results to show only important findings that support or contradict the hypotheses. We also reduced the elaboration on the novel findings. Now the conclusions section has been reduced to 172 from 348 words (reduced by 49%).

 

Comments on the Quality of English Language

Englis is clear and readability is also good.

Responses: We are pleased that the revised version is clearer and more readable. 

Once again we would like to thank the reviewer for the attention, constructive comments and time spent on our manuscript which has greatly improved its quality.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have deeply revised the manuscript following the suggestions of the reviewers therefore the manuscript can be accepted in this latest version

Author Response

Response to Rvw 4 (R2)

Suggestions for Authors

The authors have deeply revised the manuscript following the

suggestions of the reviewers therefore the manuscript can be

accepted in this latest version

Responses: We are pleased that our revision has been received positively. We would like to thank the reviewer for the attention and time spent on our manuscript.

Reviewer 5 Report

Comments and Suggestions for Authors

Please find the file attached.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please find the file attached.

Author Response

Responses to reviewer 5 (R2)

ID: soilsystems-2772517

Responses to the authors (round 2):

I would like to thank the authors for all their insightful and thorough responses to my comments. I accept them all positively. I believe that the quality of the manuscript after the revision is much

higher.

Responses: We are pleased that the revision is taken positively by the reviewer.  We would like to thank you again for your attention, constructive comments and time spent on our manuscript which has greatly improved it.

 

There are a few typos, but these do not detract from the substantive evaluation of the

manuscript.

Responses: The manuscript has been reviewed again and the typo errors have been corrected.

However, below are my comments on the hypotheses, it seems to me that there is still a problem with them and that it would be beneficial for the authors to reformulate them.

Responses: We have reformulated the hypotheses along the lines suggested by the reviewer. Please see below.

Comments on the section 128-144:

The rewritten section unfortunately still needs, in my opinion, to be tidied up or to formulate the

thoughts in a more appropriate way. Below are some of my suggestions:

Line 130:

“In addition, the influence of the resulting DOC and DN as well as their DOC-to-DN ratio on

microbial processes including microbial respiration, microbial biomass, and microbial CUE was

studied.”

I would change it to:

We also aimed to investigate the influence of the resulting DOC and DN as well as their DOC-toDN ratio on microbial processes including microbial respiration, microbial biomass, and microbial CUE.

Responses: We have changed to “We also aimed to investigate the influence of the resulting DOC and DN along with their DOC-to-DN ratio on microbial processes …” as suggested in L. 130-133 of the current version.

 

Lines 133-134:

„The study significantly advances understanding of the role of DOC, DN and their interaction in

microbial processing during decomposition of different quality classes of organic residues.”

The sentence quoted below does not introduce relevant information regarding the purpose of the

research or the hypotheses; in its current form it is like a review, an evaluative description, praising the research carried out. The sentence needs to be removed from this place of introduction. If it is necessary, this sentence can only be an introductory sentence to the presentation of the purpose of the research, which is the most important in this section. It could also have an introductory function in the abstract if needed.

Responses: We have deleted this sentence as suggested. We agree that it does not introduce relevant information regarding the purpose of the research or the hypotheses. A similar statement has been put in the Materials and methods section 2.1 (L. 155-157 of the current version)

 

Lines 135-138: „While the data on DOC and DN were newly generated in the current study, the

data on microbial parameters were taken from our previously published results. Both sets of data

were derived from the same set of samples from the year 13 of the long-term field experiment

described above.” This piece needs to be moved to the methods section.

 

Responses: We have deleted the sentence as it is a repetition of that in the Materials and methods (section 2.1) L. 153-155 of the current version

 

142 – I suggest the early stages... than „the earlier stages”

Responses: It has been changed to early stages as suggested (L. 134 of the current version).

 

lines 138-144: „These findings were used to test the hypothesis that the DN generated by mediumto high-quality classes of residues (medium to high N), i.e., TM and GN, respectively, would be higher and the DOC-to-DN ratio would be lower than in the lower quality classes counterparts, such as N-poor RS and DP, especially during the earlier stages of decomposition. In addition, the high quantity of DN and a low DOC-to-DN ratio under N-rich GN would enhance microbial processes, but reduce microbial CUE, as compared to medium-N TM and low-N RS and DP.”

I think the hypotheses need to be reformulated, re-ordered and well thought out again. They are in the current version not very understandable and tangled. Please compare version one and what came out in version two. Hypotheses must provide answers to the objectives set and should be formulated as such. They must also be formulated in a simple and understandable way. It should also be taken into account that they have to be confirmed by research results or rejected in the following.

For example, the formulations can be used (but of course there are also many other ordering and

improving options for the reader):

We have assumed that....

or

In order to achieve the stated objectives, the following hypotheses were verified/ tested:

1...

Responses: We have modified the hypotheses as suggested. They strictly follow the objectives that came before them.  In comparing with version one of the manuscript, we are of the opinion that the hypotheses clearly provide answers to the objectives set out. Please find the modified version in L. 133-139 of the current version.