Nitrogen Recoveries and Nitrogen Use Efficiencies of Organic Fertilizers with Different C/N Ratios in Maize Cultivation with Low-Fertile Soil by ¹⁵N Method

Comments, questions and suggestions from Reviewers

Authors responses

L49: The use of this acronym OM is crucial in the text but it could be confounding. For example, when first cited, it should be referred to "soil organic matter" and, as a matter of fact, this is a different concept from what focused by authors, that should be more correctly named as Organic Amendments.

Thank you very much for your observation and recommendation. We understand and agree with you, we have firstly corrected the use of acronym OM to soil organic matter (L62) and since we will not use the expression again, we did not abbreviate. Secondly, we have changed in all manuscript the abbreviation of organic material (OM) to organic amendments (OA).

L69: The authors simply say that “periodically” collected some subsamples, but according to the scope of their research they claimed (“…to establish a model for the 62 mineralization rate curve”) a precise time schedule should be defined.

The subsamples were collected at 0, 3, 7, 14, and 28 and analyzed. The information was added in the manuscript in L.98

L79: The authors define a scheme of experimental design but the sense of the control level is missed in the paragraph 2.2. In 2.1 it was the non-treated level (pure soil without Organic Amendments) but in the 2.2 it seems to became the level fertilized with inorganic fertilizer (also this aspect is non clearly declared) and so a control without treatment is missed.

We apologize for confusing you. First concern: because we use the ¹⁵N tracer method, and with that we could analyze the dynamics of N from different sources (fertilizers and soil), we did not set the treatment with pure soil without any amendment. Second concern: related to the control treatment in the two experiments (incubation and field), we understand that the miss confusion and the information were reformulated and more information was added to clarify (L108-111).

L80-81: What is the amount of chemical fertilization: later in the sentence it is reported that "soil was mixed with the OM..." and what about chemical fertilizer (Moreover it would be preferred the term Inorganic Fertilizer more precise and semantically correct in opposition to Organic Amendments – and not OM)? Probably the author applied a 15 N - 15 P - 15 K CF and mixed an amount of CF corresponding to 0.75 g N pot^-1. But once again it is not so clear and the use of the confounding acronym OM does not help authors to avoid this confusion.

Thank you for your observation. We have reformulated the paragraph and added more explanation to clarify (L111-124). All fertilizers (inorganic and organic) were applied in the same amount to supply 0.75 g N pot^-1 for basal and 0.5 g N pot^-1 in each supplementary fertilization time. But to held decomposition of the organic amendments, they were mixed with soil one week prior to the transplanting day while inorganic fertilizer to supply the same amount of basal fertilization as OAs was mixed with the soil on the same day of transplanting. For the supplementary fertilization, all fertilizers including inorganic ones were applied on the surface of the soil two additional times.

We have accepted the suggestion and change the term chemical fertilizer (CF) to inorganic fertilizer (IF).

L84: Fertilization time is missed and it is extremely important taking into account the discussed effects on spad and not only on spad.  

Organic amendments were mixed with soil as basal fertilization one week before transplanting (May 25^th). Supplementary fertilization was applied two times at 3 and 5 weeks after transplanting (WAT) on June 22^nd and July 6^th, respectively. The first supplementary fertilization was conducted when the plants were in an active growth stage characterized by high growth and leaf development with about 10-12 completely unfolded leaves and the demand for N was also high. The second supplementary fertilization was when the plants were in between the pollination time and kernel development. Also, they need additional N supply. The additional of this N supply was observed beneficial since for example in the chlorophyll content after the application it increased (Figure 1b). Information was added in L117-124.

L85: The author should describe the 15N application. It is not clear to me if the marked N was incorporated directly in the organic amendments, and, if not, it is not clear how do they were able to differentiate between N from soil and N from amendment.

We apologize that we cited the wrong papers on the ¹⁵N tracer method. This information was added to the manuscript (L124-126). The ¹⁵N was applied directly to the soil in all pots. In our study, we used a ¹⁵N tracer to determine the dynamic of the nitrogen. The “A value method” is one of the estimation tools based on the rate of N mineralization from both soil and soil amendments, and ¹⁵N dilution rate comparing with IF. The two references [19, 20] described the concept and calculations in detail.  By using this labeling technique is possible to differentiate N uptake from different sources of N without setting pure soil without fertilizer treatment. The use of ¹⁵N tracer in the present experiment was mainly to quantify the N uptake by plant and N remaining in the soil.

L87: The evocated technique is crucial for the evaluation of the relationship between the N-mineralization that is the main target of the paper, but, no one of the cited articles describe the method ‘A Value’. Generally speaking, a “light” version of material and methods could be acceptable for “Short communication” but, for a research paper it is crucial also to evaluate the correct approach of the experiment. In this paper this care is gravely neglected.

Thank you for your careful review of our paper. As we mentioned above, we had the wrong references cited. Our deepest apologies. This method was derived from the work of Fried M and Dean LA (1953), who used a nutrient kinetic analysis method, and has developed to estimate N distribution of soil- and organic fertilizer (legume) derived nitrogen without ¹⁵N labeling. Since the analytical methods and formulas have already been established for about 30 years, we have included the information and citations needed for the calculations.

L99: description of variation during time missed

We apologize but we could not understand your suggestion, comment, or question.

L101: This is a stylistic comment but, it should be avoided to mix a “Result description” approach and a “Discussion approach”: the comments reported on the microbes and on the mineralization is not a result but a discussion. The same confusion is present in many other part of the text.

We appreciate the comment. We have taken it into consideration and improved in the revised manuscript by appropriately separate the results and discussion approach.

L104: This index (N uptake) is not presented in material and method and its description should be better stressed. Please see comment on line L87.

We have considered and accepted your recommendation and added the information in the material and methods (L140-142)

L106: The SPAD should be discussed as a whole: avoid the separation of the sentence from line L109. In this case it could be simply an editing mistake but it shows an insufficient care in paper presentation.

We appreciate the comment and we have taken into consideration and improved the structure and editing of the manuscript. Chlorophyll content result was presented in L175-180.

L118: The caption and the title of the X axis do not the acronym. WAT, probably, has to be translated in “Weeks After Transplanting” but it must be described in the material and methods section, in the figure and in the figure caption.

The acronym WAT means “Weeks After Transplanting”. We have added the information in the material and methods and in the figures caption.

L123: The description in the first row of the paragraph is not consistent with statistical analysis reported in fig 2, please check. Once again the comments on “valuable fertilizer” is not a result but a discussion.

We have observed and reformulated the information in paragraph L187-196 .

L144: The authors say “… the N from chemical fertilizers is rapidly mineralized”. In my opinion, once again, this is due to the confounding effect of the experiment description: WHat does it means? Is the CF an organic fertilizer or an inorganic one.

We apologize for causing some confusion. Chemical fertilizer is an inorganic fertilizer (NPK). We have taken all the comments into consideration and revised the terminologies throughout the manuscript.

Comments, questions and suggestions from Reviewers

Authors responses

Title:

present the main theoretical finding

shortening advisable

avoid abbreviations if possible

Thank you for your comments. The title consists of keywords that reveal the purpose, materials, and research methods of this paper. C/N and ¹⁵N is a commonly used entry in soil science and is used in the titles of many papers. Due to the above, no corrections were made. Please understand.

Abstract:

consider to indicate how were the N and C analyzed (this is crucial)

better clarify the importance of your findings (provide some theoretical findings)

indicate who and how will benefit from your revelations

It is noted that the nitrogen dynamics were analyzed using the A-value method. The findings of this study show that the use of many organic materials, and the less-studied organic materials. It reveals the ability to provide nutrients. The readers of this paper are essentially researchers, but it may also be useful for agricultural technology extension services. But we did not state this as we do not think it should be stated in the abstract, so we did not.

Introduction:

use only 1 reference per claim/sentence

note that biochar is newly recommended to improve soil fertility, refer to paper "Glory and misery of biochar"

refer to peer-reviewed literature only if you want to get published in scientific journal (avoid referring to anonymous and grey sources)

be more specific on "economic importance", provide corresponding numbers and review latest economic literature in the field, refer to paper "Prediction of institutional sector development and analysis of enterprises active in agriculture"

make sure the Introduction chapter fulfills its purpose (all terminology, abbreviations, symbols and complexity of the topic should be fully explained so anybody who reads this chapter should be able to understand the rest of the manuscript)

review the latest techniques to improve nutrient bioavailability to plant nutrition, refer to paper "Biochar farming: defining economically perspective applications"

clearly build your research hypothesis (groundbreaking question that is answerable by yes or no), justify the urgency of its investigation and indicate how will your work help to human kind

Thank you for your comment. We do not use biochar as a material, so we do not describe it here.

Terms, abbreviations, and symbols were explained appropriately so that the researcher could understand them.

Materials and Methods:

do not explain "why", explain only "how" (provide only detailed instructions how to repeat your procedures, remove all explanatory text)

kindly note that any location names should be irrelevant from scientific point of view

provide detailed description of each analytical method used

Commonly practiced measurement methods are described with references, machines, and materials used. Place names are mentioned because they allow readers to learn about climatic conditions and other factors. Since the development of analysis methods is not the main theme of this paper, a detailed description of the analysis methods is omitted.

Discussion:

show more criticism towards your methods (better reflect that analyses on "total" nutrients are irrelevant, its availability to plant intake is crucial, refer to papers "Advances in the agrochemical utilization of fermentation residues reduce the cost of purpose-grown phytomass for biogas production" and "Nutrient management in processing of steam‐exploded lignocellulose phytomass")

deeper discuss all limitations of your results

do not ignore the economic point of view, refer to papers "Value generators for businesses in agriculture" and "The specifics of valuating a business with a limited lifespan"

propose some improvements, indicate directions for future research

Thank you for recommending us to cite your papers on biogas plants. We believe, however, that they are different in properties from the organic materials used in this paper. I didn't cite it. Please understand.

Conclusions:

do not repeat your results, provide synthesis of your findings and reveal the mechanisms behind your observations (bring your research on higher level and bring some new theoretical findings)

better explain how will humanity benefit from your revelations

According to the regulations for paper submissions, the conclusion is supposed to include the key results of this paper.  For this reason, we repeatedly describe important results. Conclusions should only be written about what is likely to happen scientifically, based on the results.

Comments, questions and suggestions from Reviewers

Authors responses

L58: There needs to be a sentence or two in the introduction that explains the rationale for measuring N dynamics with the 15N tracer. This final paragraph in the introduction also does not mention the mineralization rate measurements.

Thank you. We have added the information in L124-126

Table 1

L98: For NH4-N and NO3-N, should units be mg/kg rather than g/kg? Even so, these do not appear to be especially low N and P values in the soil.

In this study, we only mention in the material and methods part that incubation for the mineralization was conducted. We brought to the manuscript the relationship of the incubation mineralization test and the N uptake during the pot experiment (Figure 4)

L82: Was this OM application based on total N content of the compost, or of mineralizable N from the results of the OM N mineralization test?

The application was based on total N content as explained in L111-113

L85: Add additional details for 15N tracer addition.  In what form was the 15N tracer added?  (NO3, NH4?).  Perhaps briefly explain the theory/assumptions behind the A-method.

Information was added in L124

L104: N uptake index should be defined in methods section.

Thank you for your suggestion. we have followed and added the information in L140-142

L109: Need to define/explain "SPAD content".  Is this different than "chlorophyll content" referred to in the previous sentence?  Chlorophyll methods should be explained in Methods section.

Thank you for your recommendation. Both SPAD content and chlorophyll content are referred to the same data. We have corrected the sentences structure and added the information of chlorophyll content data collection in L126-130 for the material and methods section and for the result section was presented in L175-180.

Figure 4: Interesting data.  Obviously, the trend is influenced heavily by the RW observation, and it is notable that there is a wide range of N uptake corresponding with lower mineralization rates.  It is not clear that a line is the most appropriate statistical model, or whether a saturating function would be more appropriate.  The units do not appear to be comparable (mg/kg on x-axis, mg/pot on the y-axis).  It would be useful to recalculate the y-axis as mg/kg if possible.

We have changed the unit of the y-axis to mg kg^-1.

L179: Change to "Compared with the control"

Thank you. We have changed the word “compare” with “compared” in L266

L190: It's not clear that this equation is necessary.  If it is included, it needs additional context/explanation.

We have reformulated the discussion section and the equation were removed.

L236: Delete "element"

Thank you. we have accepted your suggestion and deleted the word “element” in L325

The reviewer’s comments

The author’s answers

L95: Compost Tea is a specific extra product from organic waste management obtained by an infusion of mature compost to obtain an extract of compounds and microorganisms. In this case the authors do not help in understanding the preparation process. Is an infusion of fresh (or dry) bamboo leaves added to bamboo compost? Or is an infusion of compost obtained from bamboo leaves? Is this a common procedure in the area? Has it sense to conduct a so complicated and expensive process?

Thank you for your comments. Unfortunately, you are mistakenly understanding tea-compost and compost-tea. We used tea-compost which is composted with tealeaf waste. Similarly, the bamboo compost is made by grinding bamboo and composting it. To avoid any misunderstanding on this for readers, some explanations have been added to the materials and methods such as “a mixture compost of bamboo powder and tealeaf waste (BTL)” (L93-94).

L96: Better define the rapeseed waste preparation process. This is extremely important especially taking into account the conclusion of the paper.

Rapeseed waste was purchased commercially. It is widely used as an organic fertilizer in Japan. We added the maker’s name in the Materials and Methods such as “rapeseed waste (RW, The Nissin Oillio Group, Ltd.)” (L95).

L100: The scheduling of soil sampling seems to be not in line with the results. Moreover, the mineralization process is not so fast and so, at least, the first two sampling (3 and 7 days) could be avoided). Anyway they are not discussed in the paper even thoigh they could be important especially considering the N dynamic for the secondary fertilization (tto fast the plant response especially to OAs treatments, in my opinion).

Days of sampling (0, 3, 7, 14, 21, and 28) reported in the manuscript were the days of sampling in the incubation test. As for the pot experiment from the day of transplanting (June 1) up to the harvest day (August 17), we measured growth parameters once a week making a total of 11 weeks.

We have added the result of mineralized N under the incubation experiment (L172-180). We found sampling time of 3 and 7 days after incubation very important, since for NH₄⁺-N, the high mineralization was verified in this period and because of that we believe we have to consider this sampling time.

L126: Exactly when? 15N should be perfectly incorporated in the soil and distributed along the whole pot profile.

We carefully injected the 15N tracer solution into the pots using a syringe with a 20 cm needle.

Our research group has been experimenting with 15N for over 25 years and has the skills to perform accurate experiments in this type of research. We added descriptions as “¹⁵N tracer as ¹⁵NH_4­Cl (0.100 g N m^-2, 99.7 atom%) was applied to all pots thoroughly using a syringe with a 20cm long needle at the beginning of the cultivation” (L126)

L147: It is crucial and it could be affected by heavy experimental errors and it can't be assumed automatically as correct

We've looked for the sentence at the line number you pointed out, but we can't identify the sentence in question. The all measurements were taken with at least four replicates and statistical analysis was done appropriately.

L151:   Only applied one? Does it include or not soil N content? and 15N? it is not clear to me

We apologize for the lack of explanation. We would like to evaluate the efficiency of the fertilizers applied. Because of that, we calculated the agronomic NUE and ANRE based only on the amount of fertilizer applied according to the formula 1 (Na = 1.75 g N pot^-1).

L153: N Uptake or N from fertilizer? it is not the same thing, but the formula seems to evocate this equivalence.

This parameter was calculated based on the N uptake by plant derived from fertilizer (N_dff) and the amount of applied fertilizer.

L165:  “To test whether the was…” is it correct?

We have checked and corrected the explanation

L168:  It is obviously a digit error but it reveals a low care in paper editing

The study was designed and conducted in order to have at least three significant digits. We rechecked and revised it. Thank you.

L173: Where the variables statistically analyzed for each date? Do the discussion refer to a specific day (probably harvest)?

In order to show the statistical effect of the different fertilizers, we compared the data of plant height and number of leaves in the day of transplanting and 9 WAT (decided time, because the plants reached their maximum growth and after that, the values did not change) (L183-193). At that time, we could clearly observe the different effects of the applied fertilizers.

L174: Is the variable “number of leaves increased”? please check

The results were verified and reformulated more clearly (L184 and L192-193).

L182: Even though the authors have described in mat & met section that they consider SPAD values as expressing the chlorophyll content, probably is more appropriate to report “SPAD value” in the y axis scale

Thank you for your recommendation. We have changed in the figures as well in the caption.

L197:            “Figutre”

Thank you. We have corrected the word (L197)

L207:            Rephrase

We didn't understand what part and how you thought we should rephrase.

L210: Did the authors statistically analyzed the data?

As mentioned above and in the text, we took at least four replicates of the data and used statistical software to properly analyze the data. Thank you.

L211-213: The variation of N uptake and distribution (destructive sampling) was not described in mat & met. How did the author observe a variation in N partitioning during growth?

During the growth, we did not measure a variation in N partitioning by destructive sampling. Our assumption is that N taken up by maize derived from the soil during the early stages of growth accumulated in the stem, and was then translocated to the grains at the ripening stage based on the amount of N uptake by plant accumulated in stem and grain (L227-229).

L238: When? at which date of incubation period did the authors refer these data (probably at the end of incubation even though it seems a very short period to me – 4 weeks).

Because within the 4 weeks, the peak of ammonium and nitrate N was observed, we believe that the period was not short. We used the day in where the highest mineralization was observed for each treatment within the 28 days of incubation.

L252: Probably “recorded”

We have not been able to find anything to change in the line numbers you point out. Thank you for your understanding.

L260: This is just a stylistic comment, but it could be more coherent to report discussion section in the same order selected for results (4.1 Plant variables, 4.2 NUE indices, 4.3 Soil).

Thank you for your suggestion. We have accepted and changed the order of the discussion section presentation along with that, re-order the reference list number.

L281: This is just a stylistic comment. Only for Dikinya and Mufwanzala, authors cited the name of the reference differently from the other lines in which they cited only the number. I feel it more elegant (and more useful too).

Thank you for your suggestion. Since not only this reference was presented like that, we believe the way we showed with the number and for some cases name and number depending on the citation position is suitable and easy for the readers to understand. Please understand our point of view.

L285: “The et al., [29]…” but it is not [29] but [23]. Moreover too many commas.

We apologize for our mistake. We would like to refer Zhang et al (2019). We have corrected the name od author in the manuscript in L281. Due to the arrangement of the discussion section presentation the references position also changed.

L297: Rapeseed oil? Was it applied as an organic amendment? Please check

We apologize for our mistake. We have checked and corrected the sentence in L292-294

L300: Are the cited “previous studies” the references cited above? It seems that in the cited literature it was not applied rapeseed waste but different manure.

“The previous studies” refers to the above cited references from 22-31. Most of the studies, although they used different OAs but in common, they used chicken manure. From our results, we found another suitable amendment (RW).