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Impact of Digestate-Derived Nitrogen on Nutrient Content Dynamics in Winter Oilseed Rape Before Flowering

Agronomy 2025, 15(5), 1241; https://doi.org/10.3390/agronomy15051241

by Remigiusz Łukowiak^1,*

, Witold Szczepaniak¹ and Dominik Młodecki²

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3:

José Lavres

Reviewer 4:

Zeqiang Shao

Agronomy 2025, 15(5), 1241; https://doi.org/10.3390/agronomy15051241

Submission received: 28 March 2025 / Revised: 14 May 2025 / Accepted: 15 May 2025 / Published: 20 May 2025

(This article belongs to the Special Issue Fertilizer Innovation and Practice in Sustainable Intensified Agriculture)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

This paper is well structured, has an average significant and novelty. The topic covered is absolutely interesting. The aim of the work is very interesting, the introduction is complete and updated on the topic. The results are very clear. The conclusions are consistent with the evidence and arguments presented addressing the main
question that are posed.

The work could be improved by clarifying and deepening these two crucial aspects.

In 2.2. Experimental design, It could be useful include the complete chemical analysis of digestate and focus on the raw materials used in the anaerobic digestion process. Are heavy metal present? electric conductivity ? ph?

It has been clearly assumed that the fertilizing value of N present in digestate applied to WOSR is the same as N from mineral fertilizers. Are other similar references? Can you include and add references related to this hypothesis?

Comments on the Quality of English Language

It can improved

Author Response

Review report 1 – response

I post the answers in red.

Comments and Suggestions for Authors

Dear authors,

The work could be improved by clarifying and deepening these two crucial aspects.

The raw material for the agricultural biogas plant was maize silage. The digestate met the standard requirements for organic fertilizer. Electrical conductivity was not determined. The digestate in the years of the study had pH in the range of 7.2-7.5.

The results of the chemical composition of the digestate are presented in a Table S2 (supplement).

Permissible values of pollutants in organic fertilizers¹, mg kg^-1 DM

Element	Permissible value
Chromium, Cr	≤ 100
Cadmium, Cd	≤ 5
Nickel	≤ 60
Lead, Pb	≤ 140
Mercury, Hg	≤ 2

¹Source/legal basis: Rozporządzenie Ministra Rolnictwa i Rozwoju Wsi z dnia 18 czerwca 2008 r., w sprawie wykonania niektórych przepisów ustawy o nawozach i nawożeniu (Dz.U. z 2008 r. Nr 119, poz. 765) (in Polish).

There are two forms of nitrogen in the digestate: ammonium (N-NH₄) and organic. The fertilizing value of nitrogen in this fertilizer, assuming the classical approach, would be equal to the content of the ammonium form (60-85%). Therefore, application in WOSR in spring would be burdened with: 1) the content of N-NH₄ in the fertilizer; 2) losses due to ammonia volatilization into the atmosphere; 3) slower action of digestate N comapred to mineral fertilizer. Increasing the fertilizing value in such conditions makes no sense, both from a research and practical perspective. In our study, the digestate was applied in autumn, before the plants entered the so-called winter dormancy. It was assumed that N-NH₄ would undergo nitrification during the winter. This was confirmed by our own results. At the rosette stage, N-NO₃ dominated in the soil. These results were published (Grzebisz et al., 2020).

Grzebisz, W.; Łukowiak, R.; Kotnis, K. Evaluation of nitrogen fertilization systems based on the in-season variability of nitrogenous growth factors and soil fertility factors: a case of winter oilseed rape (Brassica napus L.). Agronomy 2020, 10, 1701.

The Introduction has been supplemented with the following part:

„The nitrogen fertilizer replacement value (NFRV) of digestate is best assessed in field experiments. In this procedure, the yield of plants fertilized with digestate is compared with the effect of the same dose of mineral nitrogen fertilizer . The end-effect, i.e. yield, depends on many factors, including the origin of the digestate, the crop species, the date and method of application [Sieling et al., 2013]. Danish studies have shown a significantly greater response of spring barley (47-173%) compared to winter wheat. The difference resulted from the method of application. In wheat it was surface banding and in barley injection [De Notaris et al., 2018]. Field tests in Lithuania on fertilization of spring cereals with surface-applied digestate showed the same effect as mineral N [Doyeni et al., 2021]. Corn is a plant in which digestate is very often used [Morris and Lathwell, 2004]. The method of using this nitrogen carrier is secondary, which indicates the efficient uptake of nutrients by plants [Przygocka and Grzebisz, 2018]. The response of sugar beet to the type of nitrogen carrier (mineral N, digestate) depended only on weather conditions during the growing season [Baryga i in., 2020].”

This article has been reviewed by MDPI Author Services Language Editing.

Certificate attached.

On behalf of the authors

Remigiusz Łukowiak

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I must admit that I had to run though this rather enormous work rather fast. But - I really like the systematic design of the work which greatly support the hypothesis that the applicability of digestates is as good as mineral fertilizers. This is extremely important taking into account that digestates will support the conservation of nutrients in closed loops. Great work!

L178-183, some more information (and references) for statics/regression would be good.

There are eight figures with plots presenting biomass or content of different nutrients in various plant parts. I suggest to put a few words in the top of the plots to make it easier to navigate around in this rather large amount of data. Also run through the legends - e.g. BL, ST, SY is not always consistent in the plots and the figure texts (Figure 10).

L131, says 4045 plants per m2 - maybe 40-45 plants per m2?

Author Response

Review report 2 – response

I post the answers in red.

Comments and Suggestions for Authors

L178-183, some more information (and references) for statics/regression would be good.

The appropriate reference has been added:

Smith. G. Step away from stepwise. J. Big Data 2018, 5, 32; https://doi.org/10.1186/s40537-018-0143-6

All figures have been supplemented with appropriate explanations, where it was necessary. Legend has been checked and corrected in all figures. The abbreviations unit was introduced. The continuity of abbreviations in the entire text was checked.

L131, says 4045 plants per m2 - maybe 40-45 plants per m2?

It has been corrected.

This article has been reviewed by MDPI Author Services Language Editing.

Certificate attached.

On behalf of the authors

Remigiusz Łukowiak

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear All,

The manuscript titled “Impact of Digestate Nitrogen on the Nutritional Status of Winter Oilseed Rape in Cardinal Phases Before Flowering” investigates how different nitrogen (N) fertilization strategies - mineral N, organic digestate, and a combination of both - affect the macro- and micronutrient status of winter oilseed rape (WOSR) across vegetative development stages prior to flowering. The study presents a two-year field experiment under three N systems and four increasing doses of N, monitoring leaf and stem nutrient concentrations at the rosette and beginning of flowering stages.

This research contributes to the understanding of digestate utilization as a partial or full N fertilizer replacement, exploring its influence on plant nutritional status and implications for sustainable N management in oilseed rape cultivation. The authors use regression analyses and interaction effects to assess patterns of nutrient accumulation in aboveground tissues. The topic is highly relevant given the growing interest in integrating organic waste into circular agriculture. However, the paper would benefit from key clarifications, especially regarding mechanistic hypotheses, soil and environmental background data, and justification for fertilization strategies.

Strong Points

The manuscript addresses an increasingly critical issue: how alternative nitrogen sources such as digestate influence plant nutrition. The work provides well-documented experimental design over two seasons, employing real-field conditions, and offers clear statistical modeling of nutrient dynamics. Figures and regression charts are effectively used to represent nutrient trends (e.g., Zn, Mg, Ca, N), helping to identify how fertilizing systems affect uptake across plant organs and stages. Importantly, the availability of Zn and Mg is discussed and quantitatively presented in the supplementary material (Figures S2–S4, S7, S11). These results support the rationale for monitoring micronutrient bioavailability under varying nitrogen regimes and add practical value for guiding digestate-based fertilization programs.

Weaker Aspects

In my opinion, one key limitation of the manuscript is the lack of a clearly formulated mechanistic hypothesis. Although the study provides extensive data on macro- and micronutrient behavior under different N systems, it does not explicitly hypothesize how digestate chemistry or plant physiological responses influence these nutrient dynamics. A hypothesis such as:
“We hypothesize that partial replacement of mineral nitrogen with digestate modulates the bioavailability and uptake of macro- and micronutrients by modifying the soil chemical environment and synchronizing nutrient release with crop demand”
would strengthen the conceptual framework.

Another critical aspect is the absence of basic soil physical attributes characterization. While the study discusses nutrient concentrations, it does not present information on soil pH, cation exchange capacity (CEC), base saturation, and clay/silt/sand fractions. This limits reproducibility and hinders interpretation of nutrient uptake behavior. These attributes should be reported as they critically affect nutrient dynamics, especially under organic fertilization.

Moreover, the rationale for the three nitrogen fertilization systems (M-FS, O-FS, OM-FS) is described but not sufficiently contextualized within nutrient-use efficiency or synchronization theory. Explaining why a 2/3 organic + 1/3 mineral mix was chosen, rather than a 50:50 ratio or site-specific optimization, would improve methodological transparency.

So, I have annotated below as well as along the attached manuscript an attempt to clarify certain ideas, but the authors should examine my suggested wording changes carefully to ensure that I have not misinterpreted their intended meaning.

Title

-Clear and informative, aligned with the study’s scope and content. Suggest clarifying: "Impact of Digestate-Derived Nitrogen on Nutrient Accumulation Dynamics in Winter Oilseed Rape Before Flowering" to better emphasize nutrient pathways.

Introduction

- Missing a mechanistic rationale. Add a hypothesis linking digestate composition to nutrient availability and uptake.

- Clarify the novelty and purpose of combining mineral and organic N sources, especially the 2/3:1/3 ratio.

Methods

- Critical Fix: Include comprehensive soil characterization (texture, pH, CEC, base saturation, organic matter content).

- Explain why certain N doses were selected and how digestate N content was calculated and monitored (mineralizable N?).

- Clarify how the digestate was stored or stabilized before application.

Results

- Include a summary table of statistically significant FS × N dose interactions for key nutrients (Zn, Mg, N, Ca).

- Better integration of Figures S3, S4, S7, S11 into main discussion would enhance understanding of micronutrient patterns.

Discussion

- Missing deeper discussion of digestate–soil interaction mechanisms (e.g., organic acids, NH₄⁺-driven changes in rhizosphere pH).

- Include discussion on the timing of nutrient availability and possible delayed mineralization from digestate affecting early growth.

Connect to literature on Zn uptake dynamics under high N!

Conclusion

Suggest strengthening with practical recommendations for digestate use under different soil types and climates.

Specific Comments

Page 2, Line 45: Insert soil classification and physical-chemical attributes.
Page 2, Line 60: Clarify N fertilizer dose calculation.
Page 3, Line 80: Add digestate stabilization method before application.
Page 3, Line 105: State soil pH and buffering capacity.
Page 4, Line 120: Clarify if Zn, Mg, Mn were analyzed via DTPA-extraction or another method.
Page 4, Line 130: Report CEC and base saturation in baseline soil.
Page 4, Line 133: Insert m% and H+Al content in methods table.
Page 5, Line 142: Add hypothesis as suggested above.
Page 6, Line 165: Clarify how nutrient concentration values were normalized (DW basis?).
Page 6, Figure 2: Add significance letters directly on regression graphs.
Page 7, Figure 3: Include SEM or CV in nutrient data.
Page 7, Line 200: Add discussion of Zn mobility under varying N regimes.
Page 8, Line 215: Specify which stage was most sensitive to Mg decline.
Page 9, Line 230: Tie Ca dynamics to cell wall stability under high ammonium.
Page 9, Line 238: Clarify which organ retained the most Zn in OM-FS.
Page 10, Line 252: Discuss whether OM-FS delayed peak nutrient uptake.
Page 10, Line 270: Add rationale for OM-FS dose proportion.
Page 11, Figure 5: Improve Y-axis label clarity (units and tissue type).
Page 11, Line 282: Consider a table summarizing average nutrient concentrations across systems.
Page 12, Line 295: Compare findings to similar studies using composted digestate.
Page 13, Line 310: Discuss Zn/Mg interplay and potential antagonisms.
Page 14, Line 324: Indicate regulatory nutrient thresholds or agronomic recommendations.
Page 14, Line 333: Mention bioavailability and not just total content.
Page 15, Figure 6: Add correlation coefficient (R²) and p-value.
Page 15, Line 345: Clarify digestate effect on early biomass accumulation.
Page 16, Line 350: Discuss importance of nutrient remobilization before flowering.
Page 17, Line 360: Re-emphasize the benefit of mixed system in balancing N availability.
Page 17, Line 368: Add sentence on how results may apply to other crops or soils.
Page 18, Line 374: Provide limitation: site-specificity of results.
Page 18, Line 390: Suggest future directions for pairing digestate with precision farming tools.

Comments for author File: Comments.pdf

Author Response

Review report 3 – response

I post the answers in red.

Comments and Suggestions for Authors

Dear All,

Field studies were conducted in three consecutive growing seasons. Only such a long cycle allows for the relative action of experimental factors in variable meteorological conditions.

Strong Points

The manuscript addresses an increasingly critical issue: how alternative nitrogen sources such as digestate influence plant nutrition. The work provides well-documented experimental design over two seasons, employing real-field conditions, and offers clear statistical modeling of nutrient dynamics. Figures and regression charts are effectively used to represent nutrient trends (e.g., Zn, Mg, Ca, N), helping to identify how fertilizing systems affect uptake across plant organs and stages. Importantly, the availability of Zn and Mg is discussed and quantitatively presented in the supplementary material (Figures S2–S4, S7, S11). These results support the rationale for monitoring micronutrient bioavailability under varying nitrogen regimes and add practical value for guiding digestate-based fertilization programs.

I would like to inform the reviewer again that three series of experiments were carried out, i.e. in three consecutive growing seasons: 2015/2016; 2016/2017; 2017/2018.

Weaker Aspects

The proposal to change the hypothesis is interesting, but would require a significant extension of the article. In its substantive essence, it concerns the effect of digestate on the content of nutrients in the soil as a factor conditioning their availability to the plant.

Such an analysis was carried out for the first two growing seasons, taking into account nitrate nitrogen and available forms of P, K, Mg and Ca. The authors also have results for micronutrients, but it is difficult to combine them in one article.

The title of the indicated article to which this manuscript refers is as follows: Grzebisz, W.; Łukowiak, R.; Kotnis, K. Evaluation of nitrogen fertilization systems based on the in-season variability of nitrogenous growth factors and soil fertility factors: a case of winter oilseed rape (Brassica napus L.). Agronomy 2020, 10, 1701.

The submitted manuscript does not address this issue.

The basic characteristics of soil properties are shown in Table S1. These results are presented for three growing seasons for the 0-30 cm layer (topsoil). The agrochemical properties of the soil, taking into account three growing seasons and three soil layers, are presented in Table 1.

When using any carrier of nutrients (fertilizers), especially nitrogen, it is assumed a priori that they will provide optimal plant nutrition in critical phases (stages) of yield formation. The purpose of the so-called leaf diagnostics (leaf analysis) is to confirm or reject this assumption (hypothesis).

There are two forms of nitrogen in the digestate: ammonium (N-NH₄) and organic. The fertilizing value of nitrogen in this fertilizer, assuming the classical approach, would be equal to the content of the ammonium form (60-85%). Therefore, application in WOSR in spring would be burdened with: 1) the content of N-NH₄ in the fertilizer; 2) losses due to ammonia volatilization into the atmosphere; 3) slower action in relation to mineral fertilizer. Increasing the fertilizing value in such conditions makes no sense, both from a research and practical perspective. The digestate was applied in autumn, before the plants entered the so-called winter dormancy. It was assumed that N-NH₄ would undergo nitrification during the winter. Mineral nitrogen carriers (fertilizers) applied at the beginning of the spring part of the WOSR growing seasons are considered to be "starting", i.e. acting immediately. This concept was confirmed by our own results. At the rosette stage, N-NO3 dominated in the soil. These results were published (Grzebisz et al., 2020 presented above).

Title

In the article, the discussed feature of the tested plant is not the accumulation of a given nutrient in its parts (indicatory organs), but its content. I accept the proposed change of title with correction, as above.

Introduction

- Missing a mechanistic rationale. Add a hypothesis linking digestate composition to nutrient availability and uptake.

The hypothesis put forward is correct. The reviewer's suggestion is correct, but only if the authors analyzed the accumulation of nutrients, and in fact nitrogen in the rapeseed biomass. The results obtained clearly indicated that the nitrogen content in leaves in the period before flowering was not a factor limiting the yield.

- Clarify the novelty and purpose of combining mineral and organic N sources, especially the 2/3:1/3 ratio.

The nitrogen fertilizer replacement value for diegestate reached 104% and in the case of mixed FS, it increased up to 111%. This NFRV explains clearly the rationality of this WOSR fertilization formulation. FNRV results less than 100%, often significant, result from a lack of understanding of the essence of the action of this type of nitrogen carrier.

Methods

- Critical Fix: Include comprehensive soil characterization (texture, pH, CEC, base saturation, organic matter content).

Table #S1 has been included into supplement. The soil was homogeneous in terms of soil texture, as stated in the description.

- Explain why certain N doses were selected and how digestate N content was calculated and monitored (mineralizable N?).

When testing the fertilizing value of digestate, the total content or only N-NH4 is taken into account.

The Introduction has been supplemented with the following part:

This question in answered in the article: Grzebisz, W.; Łukowiak, R.; Kotnis, K. Evaluation of nitrogen fertilization systems based on the in-season variability of nitrogenous growth factors and soil fertility factors: a case of winter oilseed rape (Brassica napus L.). Agronomy 2020, 10, 1701.

- Clarify how the digestate was stored or stabilized before application.

The digestate was collected directly from the biogas plant.

Results

- Include a summary table of statistically significant FS × N dose interactions for key nutrients (Zn, Mg, N, Ca).

Of this set of elements, zinc is a secondary nutrient, resulting from a specific interaction of its content in leaves with calcium. This state resulted from a sudden increase in both Ca and Zn content in the dry year (2017/2018). Unfortunately, no logical (based on the literature on the subject) explanation was found. For the remaining nutrients, the tables present mean values with standard deviations. These values were converted into ranges and discussed in the discussion section.

- Better integration of Figures S3, S4, S7, S11 into main discussion would enhance understanding of micronutrient patterns.

The key research results are included in the discussion. However, this way of discussion in classic articles (based on experimental results) is criticized by many reviewers.

Discussion

- Missing deeper discussion of digestate–soil interaction mechanisms (e.g., organic acids, NH₄⁺-driven changes in rhizosphere pH).

In this respect we can only speculate. In fact, we can review the literature on the subject.

- Include discussion on the timing of nutrient availability and possible delayed mineralization from digestate affecting early growth.

This question is deeply discussed in the article: Grzebisz, W.; Łukowiak, R.; Kotnis, K. Evaluation of nitrogen fertilization systems based on the in-season variability of nitrogenous growth factors and soil fertility factors: a case of winter oilseed rape (Brassica napus L.). Agronomy 2020, 10, 1701.

Connect to literature on Zn uptake dynamics under high N!

As I have already pointed out, the specific role of Zn results from the action of Ca.

Conclusion

Suggest strengthening with practical recommendations for digestate use under different soil types and climates.

In essence, there is an idea for a review article or meta-analysis. Thank you. This suggestion was incorporated, but it is noted that winter oilseed rape (WOSR) is only grown in the temperate, cool climates of the Northern Hemisphere. This issue was supplemented and raised in the introduction, where attention was drawn to plants that respond well to nitrogen from digestate.

Specific Comments

Page 2, Line 45: Insert soil classification and physical-chemical attributes.

The results are presented in the newly developed Table S1.

Page 2, Line 60: Clarify N fertilizer dose calculation.

The total N content (N-NH₄ + Norg) in the digestate was determined. The dose of the digestate was determined on this basis, assuming the same NFRV, regardless of the N carrier.

Page 3, Line 80: Add digestate stabilization method before application.

The raw digestate was taken directly from the biogas plant.

Page 3, Line 105: State soil pH and buffering capacity.

The relevant data are presented in Table S1. Buffering capacity was calculated. Soil pH was adjusted for the rapeseed pre-crop, i.e. in the year preceding the establishment of the experiment.

Page 4, Line 120: Clarify if Zn, Mg, Mn were analyzed via DTPA-extraction or another method.

The content of micronutrients was determined using the Mehlich 3 method. This information is included in the current Table No. 1. This method is used in agrochemical diagnostics in Poland.

Page 4, Line 130: Report CEC and base saturation in baseline soil.

The relevant data are presented in Table S1.

Page 4, Line 133: Insert m% and H+Al content in methods table.

The relevant data are presented in Table S1.

Page 5, Line 142: Add hypothesis as suggested above.

It is not possible to change the hypothesis, because the substantive scope of this article is different. This is a classic diagnostic work on the plant.

Page 6, Line 165: Clarify how nutrient concentration values were normalized (DW basis?).

The nutrient content was normalized based on dry matter basis of a given plant part.

Page 6, Figure 2: Add significance letters directly on regression graphs.

This is not a regression figure , but a bar chart. The graphical form is shown in Fig. S5.

The figures and regression graphs presented in the supplement complement the main figures contained in the text or in the appendix.

Page 7, Figure 3: Include SEM or CV in nutrient data.

In each figure an additional statistical indicator is the standard error of the mean for the years. The values of the coefficient of variation (CV) are included in the appropriate table.

Page 7, Line 200: Add discussion of Zn mobility under varying N regimes.

This is not possible, because these data are a separate research area. Introducing this type of data would require doubling the volume of the article.

Page 8, Line 215: Specify which stage was most sensitive to Mg decline.

Mg deficiency appeared at the rosette stage and continued until the beginning of flowering. This was specified and demonstrated in the discussion, indicating the ranges of contents in the leaves.

Page 9, Line 230: Tie Ca dynamics to cell wall stability under high ammonium.

Such a situation did not occur. The N-NH₄ content in the soil at the time of plant analysis was low. Data available from the authors.

Page 9, Line 238: Clarify which organ retained the most Zn in OM-FS.

The accumulating organ plays a secondary role, because in both (leaves, shoots) growth occurred in the third season of the study. The condition resulted from a severe drought in 2017/2018 growing season. It has been clarified and discussed.

Page 10, Line 252: Discuss whether OM-FS delayed peak nutrient uptake.

The specific effect of OM-FS on the content of examined nutrients was strongest/significant only in the rosette stage. In fact, it resulted from the effect of the digestate. Plants fertilized with the digestate contained significantly less Ca, Mn, Zn and Cu compared to those fertilized with ammonium nitrate. It has been clarified and discussed.

Page 10, Line 270: Add rationale for OM-FS dose proportion.

The applied proportion of nitrogen carriers in the applied fertilizers was justified by the yield, which increased in the direction M ≤ O < M, amounting to 100%, 104% and 111% respectively.

Page 11, Figure 5: Improve Y-axis label clarity (units and tissue type).

All tables and figures have been checked and corrected where necessary. It has been corrected.

Page 11, Line 282: Consider a table summarizing average nutrient concentrations across systems.

Mean values for individual nutrients, along with standard deviations and CVs, are provided in the main tables. Ranges of content are provided for the major nutrients and discussed in the discussion section.

Page 12, Line 295: Compare findings to similar studies using composted digestate.

There is no such data. Most articles only concern yield and possibly NFRV. This is the first article that actually concerns the fertilizer value of digestate used in rapeseed.

Page 13, Line 310: Discuss Zn/Mg interplay and potential antagonisms.

There is no need for this detailed discussion. It is an apparent antagonism, the primary cause of which was the excess of Ca content in WOSR organs.

Page 14, Line 324: Indicate regulatory nutrient thresholds or agronomic recommendations.

Ranges of content are provided for the major nutrients and discussed in the discussion section.

Page 14, Line 333: Mention bioavailability and not just total content.

This information refers to the nutrient content in the leaves, not the soil.

Page 15, Figure 6: Add correlation coefficient (R²) and p-value.

This is not a regression figure , but a bar chart. The graphical form is shown

in Fig. S10.

Page 15, Line 345: Clarify digestate effect on early biomass accumulation.

Added in the discussion.

Page 16, Line 350: Discuss importance of nutrient remobilization before flowering.

In the period from the rosette stage to the beginning of flowering only the effect of dilutionof a nutrient in leaves can be discussed. Remobilization occurs in the period from BBCH 40/49 to BBCH 71. However, in this experiment such studies were not conducted.

Page 17, Line 360: Re-emphasize the benefit of mixed system in balancing N availability.

This issue was discussed in the discussion section and highlighted in the conclusions.

Page 17, Line 368: Add sentence on how results may apply to other crops or soils.

This issue was highlighted in the conclusions.

Page 18, Line 374: Provide limitation: site-specificity of results.

There are no limitations except for the N dose, which results from the legal provisions in force in Poland and the EU. The most important is time and the method of application. Both of these factors must assume minimization of N losses in the form of ammonia to the atmosphere.

Page 18, Line 390: Suggest future directions for pairing digestate with precision farming

This can be done by using the principles developed for the application of mineral N,

for example Urea-Ammonium Nitrate solution /UAN/.

This article has been reviewed by MDPI Author Services Language Editing.

Certificate attached.

On behalf of the authors

Remigiusz Łukowiak

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Digestate Nitrogen is able to replace part of the mineral fertiliser, which is of great research significance for the sustainable development of oilseed rape. It is recommended that the paper be accepted after minor revision, and the following problems exist:

In the Abstract section, it is suggested to add important experimental data, and in the Abbreviations section, please check the whole text to keep unity.
The preface part, please add the effect of application of Digestate Nitrogen on different crops.
2.2. Experimental design, please add the number of replications of the experiment and explain clearly what kind of experimental design is used?
3.2. WOSR nutritional status - rosette’ or similar titles should not be abbreviated.
The numbering of equations in the paper should be aligned.
In the discussion section of the paper, it is recommended to add some important research data of this study.

Author Response

Review report 4 – response

I post the answers in red.

Comments and Suggestions for Authors

In the Abstract section, it is suggested to add important experimental data, and in the Abbreviations section, please check the whole text to keep unity.

The abstract was supplemented with the resulting data. The abbreviations unit was introduced. The continuity of abbreviations in the entire text was checked.

The preface part, please add the effect of application of Digestate Nitrogen on different crops.

This part of the introduction has been supplemented. There are many of these plants, but the greatest reactions are noted for maize.

The Introduction has been supplemented with the following part:

2.2. Experimental design, please add the number of replications of the experiment and explain clearly what kind of experimental design is used?

A two-factor field experiment was set up in a split-block design. The number of replications was four.

3.2. WOSR nutritional status - rosette’ or similar titles should not be abbreviated.

The tables have been improved by eliminating the abbreviations used.

The numbering of equations in the paper should be aligned.

The numbering of the equations given in the article was checked and sorted.

In the discussion section of the paper, it is recommended to add some important research data of this study.

The key research results are included in the discussion. However, this way of discussion in classic articles (based on experimental results) is criticized by many reviewers.

This article has been reviewed by MDPI Author Services Language Editing.

Certificate attached.

On behalf of the authors

Remigiusz Łukowiak

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for the opportunity to re-evaluate your revised manuscript. I appreciate the significant effort made to address prior reviewer comments. The topic, optimizing nutrient management using digestate-derived nitrogen in winter oilseed rape (WOSR), is timely and highly relevant in the context of sustainable agriculture and biogas-derived fertilizer applications.

The study benefits from a strong field experiment design across multiple growing seasons, rigorous nutrient analyses, and appropriate statistical handling. However, several aspects would benefit from improved clarity, further discussion, or minor adjustments to enhance the scientific robustness and impact of the work.

Major Comments:

Clarification on Digestate Composition and Application Justification:

The digestate composition is only briefly summarized in Table S2. Please justify the agronomic reason for selecting this particular digestate source and clearly discuss its variability (e.g., CV > 30% for several nutrients) and implications for reproducibility across sites or years.

Better Framing of Hypothesis and Research Objectives:

A clear, testable hypothesis is outlined (line 103), but should be highlighted at the end of the Introduction more explicitly for emphasis. Consider phrasing it in a mechanistic form (e.g., "We hypothesize that digestate-derived N...modulates nutrient uptake dynamics and biomass accumulation before flowering").

Statistical Model Assumptions & Interactions:

The multi-way ANOVA is robust, but the manuscript lacks a validation check for homogeneity of variance and normality assumptions. Please clarify how these assumptions were tested.

Interactions between year × FS × N were frequent, yet interpretation is sometimes shallow. More depth in biological reasoning behind significant interactions is needed (e.g., year × FS × Zn, Table 5).

Interpretation of Nutrient Synergies and Antagonisms:

The discussion regarding Ca-Mg antagonism and its indirect effect on yield (lines 261–280) is interesting but needs strengthening. Was there any statistical correlation (e.g., Pearson/Spearman) between Ca and Mg or between Ca and SY? Please include this in the Results.

Seed Yield Prediction Model Robustness:

Equations (1) and (3) predicting SY from BR and leaf nutrients are central to your conclusions. I recommend reporting confidence intervals and validation metrics (e.g., RMSE or adjusted R²) for these regression models.

Minor Comments:

Terminology Standardization:

Please consistently use abbreviations across figures and tables (e.g., M-FS vs. M, O-FS vs. O). In Figure 1 and its caption, “O - M-FS” appears to be a typographical error.

Use “digestate-based fertilization system” instead of the ambiguous “O-FS” when first introduced.

Figures and Legends:

Supplementary Figures S2–S5 provide important nutrient dynamics. However, legends should specify sampling stages (BBCH) and explicitly define all axes and symbols for reproducibility.

Relevance of Zinc Effects:

The discussion on the “negative effect of Zn on BR” (lines 267-270) is not well supported by mechanistic explanation. Please revisit this argument with potential biological rationale or relevant citations.

Nutrient Ranges and Critical Thresholds:

Tables 3 and 4 provide critical thresholds, but their sources should be cross-verified for consistency. Consider highlighting where your measured values fall outside or within these diagnostic windows in text/tables using visual indicators (e.g., bold or asterisk).

Author Response

Review report 3v2 – response

I post the answers in red.

Dear Authors,

On behalf of the co-authors, I would like to thank the reviewer for her/his thorough analysis of this manuscript. This allowed us to improve the quality of the prepared article.

Major Comments:

Clarification on Digestate Composition and Application Justification:

The digestate used was from a production (in run) biogas plant, not from a laboratory biogas plant.

The choice of biogas plant was based on three points:

1) Location; Transport of liquid nitrogen fertilizer with low concentration of this nutrient is expensive,

including environmental load.

2) Raw material - feedstock;

Maize silage, despite the noted diversity, in the content of elements, including N, provides greater compositional stability than manure or slurry.

3) Biogas from maize silage meets the conditions of Circular Economy.

The maximum dose of N in organic fertilizer, and this is how digestate is treated, is 170 kg ha^-1. This is according to the EU-27 and Polish standards. There is no technical problem for farmer (Chemical-agricultural stations) with measuring N concentration in digestate.

Better Framing of Hypothesis and Research Objectives:

Corrected as follows: „We assumed that N from digestate, modulating the dynamics of nitrogen and nutrient uptake, significantly affects biomass accumulation before flowering of winter rape. As a result, digestate-derived N can ensure optimal nutritional status of winter rapeseed in critical yield formation phases.”

Statistical Model Assumptions & Interactions:

The multi-way ANOVA is robust, but the manuscript lacks a validation check for homogeneity of variance and normality assumptions. Please clarify how these assumptions were tested.

The Kolmogorov-Smirnov (K-S) test, Lilliefors, skewness and kurtosis, and coefficient of variation (CV) were used to assess the normality of the distribution of individual traits.

Kim [2013] proposed the z-test to assess the normality of raw data. This test is based on the ratio of skewness/kurtosis to the standard error of a given variable. According to Ghasemi and Zahediasl [2012], the absolute z-score threshold for the normal distribution for the sample mean (51 < n < 175) is < 2.58. A z-score of 2.58 corresponds to a prediction with a significance level (α) ≤ 0.05.

Kim, H-Y. Statistical notes for clinical researchers: Assessing normal distribution (2) using skewness and kurtosis. Restor. Dent. Endod. 2013, 38, 52–54.

Ghasemi, A., Zahediasl, S. (2012). Normality tests for statistical analysis: A guide for non-statisticians. Int. J. Endocrinol. Metab. 2012, 10, 486-489.

Wilding, L.P.; L.R. Dress. Spatial variability and pedology. In L.P. Wilding, L.P.; Smeck, N.; Hall, G.F. (eds.). Pedogenesis and Soil Taxonomy. Wageningen, The Netherlands, 1983, pp. 83–116.

The bar graphs showing the interaction of fertilization systems (FS) and nitrogen doses (N) include information on the standard error resulting from the effect of years (hatched vertical strip).

The information at the bottom of the figure reads as follows: “the vertical bar in each column shows the standard error of the mean for the year”.

Interpretation of Nutrient Synergies and Antagonisms:

All the necessary relationships between the Ca and Mg content in diagnostic organs of rapeseed, as well as their relationship with seed yield (SY) and organ biomass (B), are given in the respective tables: A1 for the rosette; A2 for leaves at the beginning of flowering; A3 - for stems at the beginning of flowering. These relationships are also presented in Figures A2, A3, and A4, respectively. From the analysis of the figures, it can be seen that these two nutrients are not located in the different quadrants. This last relationship has now been introduced into the text.

Seed Yield Prediction Model Robustness:

In all figures showing the regression of a examined trait on N rates, the coefficient of determination refers to the adjusted R².

Minor Comments:

Terminology Standardization:

Please consistently use abbreviations across figures and tables (e.g., M-FS vs. M, O-FS vs. O). In Figure 1 and its caption, “O - M-FS” appears to be a typographical error.

All figures and tables were checked and erroneous entries were corrected.

Use “digestate-based fertilization system” instead of the ambiguous “O-FS” when first introduced.

The term was changed to Digestate-based fertilization system; the acronym is D-FS. OM-FS has been changed into DAN-FS, and M-FS into AN-FS.

Figures and Legends:

Supplementary Figures S2–S5 provide important nutrient dynamics. However, legends should specify sampling stages (BBCH) and explicitly define all axes and symbols for reproducibility.

Corrected.

Relevance of Zinc Effects:

This is probably an artifact that results from the specific relationship of Ca and Zn content. We have not found a possible logical/mechanistic explanation (even literaturę about this phenomenon). We leave it as a challenge to potential readers of this article.

Nutrient Ranges and Critical Thresholds:

Each table contains the mean and standard deviation value. Based on this, the reader can determine whether this range falls within the normative range. Up to three normative ranges according to different sources have been provided. The introduction of the proposed solution would only introduce information noise.

On behalf of the authors

Remigiusz Łukowiak

Author Response File: Author Response.pdf

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Impact of Digestate-Derived Nitrogen on Nutrient Content Dynamics in Winter Oilseed Rape Before Flowering

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