Biofertilizers for Enhanced Nitrogen Use Efficiency: Mechanisms, Innovations, and Challenges

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

 

Title: Biofertilizers for Enhanced Nitrogen Use Efficiency: Mechanisms,
Innovations, and Challenges

Dear Authors and Editors

The topic of the manuscript is original and relevant to the field of agricultural sciences.

The content of the manuscript falls within the publishing profile of Nitrogen journal.

The manuscript was well planned and written. The topic of this review manuscript addresses current research trends worldwide. Extensive references were skillfully and appropriately used.

The primary goal of this manuscript submitted for review was to synthesize current knowledge on the mechanisms underlying increased NUE. The authors considered microbial inoculants and biofertilizer-based strategies.

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

 

Remarks:

1. Introduction: This section is written in sufficient detail to reflect the topic of the manuscript.
2. After the Introduction section, I propose an additional section "Agrotechnical methods for increasing the efficiency of fertilizer nitrogen use".
3. Section 2. Mechanism for Improving Nitrogen Use Efficiency. Lines 141-143 When discussing nitrate reductase V and III, it is important to mention a good supply of molybdenum and iron to plants. These elements are components of nitrate and nitrite reductase.
4. Section 3. Biological nitrogen fixation (BNF). Subsection 3.1. (Subsubsections 3.1.2., 3.1.3., 3.1.4 - Please add the numerical ranges of quantitative possibilities of biological reduction of molecular nitrogen N₂ by particular types of symbiotic bacteria.You can use information from the articles: https://doi.org/10.1080/03650340.2020.1817398  https://doi.org/10.5601/jelem.2018.23.3.1684
5. In the subheadings of subsections 3.1.2.; 3,1,3.; 3.1.4 - groups of plants that live in symbiosis with bacteria of the genera Bradyrhizobium and Sinorhizobium and with Cyanobacteria should be added.
6. Subsection 3.2. - How much N₂ can association bacteria (Azospirillum, Herbaspirillum, Methylobacterium, Bacillus) bind?
7. Conclusions section - should be: Summary and Conclusions.

Specific remarks:

1. Line 443- Alves et al.(2021) [?], line 463- Madhaiyan et al. (2015) [?], line 740- Giller et al. (2025) [?] Please provide the appropriate References numbers instead of the manuscript publication year.

Best regards

 

 

Comments and Suggestions for Authors

 

Title: Biofertilizers for Enhanced Nitrogen Use Efficiency: Mechanisms,
Innovations, and Challenges

Dear Authors and Editors

The topic of the manuscript is original and relevant to the field of agricultural sciences.

The content of the manuscript falls within the publishing profile of Nitrogen journal.

The manuscript was well planned and written. The topic of this review manuscript addresses current research trends worldwide. Extensive references were skillfully and appropriately used.

The primary goal of this manuscript submitted for review was to synthesize current knowledge on the mechanisms underlying increased NUE. The authors considered microbial inoculants and biofertilizer-based strategies.

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

 

Remarks:

1. Introduction: This section is written in sufficient detail to reflect the topic of the manuscript.
2. After the Introduction section, I propose an additional section "Agrotechnical methods for increasing the efficiency of fertilizer nitrogen use".
3. Section 2. Mechanism for Improving Nitrogen Use Efficiency. Lines 141-143 When discussing nitrate reductase V and III, it is important to mention a good supply of molybdenum and iron to plants. These elements are components of nitrate and nitrite reductase.
4. Section 3. Biological nitrogen fixation (BNF). Subsection 3.1. (Subsubsections 3.1.2., 3.1.3., 3.1.4 - Please add the numerical ranges of quantitative possibilities of biological reduction of molecular nitrogen N₂ by particular types of symbiotic bacteria.You can use information from the articles: https://doi.org/10.1080/03650340.2020.1817398  https://doi.org/10.5601/jelem.2018.23.3.1684
5. In the subheadings of subsections 3.1.2.; 3,1,3.; 3.1.4 - groups of plants that live in symbiosis with bacteria of the genera Bradyrhizobium and Sinorhizobium and with Cyanobacteria should be added.
6. Subsection 3.2. - How much N₂ can association bacteria (Azospirillum, Herbaspirillum, Methylobacterium, Bacillus) bind?
7. Conclusions section - should be: Summary and Conclusions.

Specific remarks:

1. Line 443- Alves et al.(2021) [?], line 463- Madhaiyan et al. (2015) [?], line 740- Giller et al. (2025) [?] Please provide the appropriate References numbers instead of the manuscript publication year.

Best regards

 

 

Author Response

Dear Reviewer,

We sincerely thank the reviewer for their thoughtful comments and constructive suggestions. Their observations have been extremely valuable in improving the clarity, scientific depth, and overall quality of the manuscript. We have carefully considered all of the points raised and revised the text accordingly, which we believe has significantly strengthened the work. We are grateful for the time and effort the reviewer dedicated to evaluating our study.

 

REVIEWER COMMENTS 

REVIEWER #1

[Comment 1] The topic of the manuscript is original and relevant to the field of agricultural sciences.

The content of the manuscript falls within the publishing profile of Nitrogen journal.

The manuscript was well planned and written. The topic of this review manuscript addresses current research trends worldwide. Extensive references were skillfully and appropriately used.

The primary goal of this manuscript submitted for review was to synthesize current knowledge on the mechanisms underlying increased NUE. The authors considered microbial inoculants and biofertilizer-based strategies.

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

 [Response 1] We sincerely thank the Reviewer for the positive and encouraging evaluation of our manuscript. We greatly appreciate the acknowledgment of the originality, relevance, and overall quality of the work, as well as the recognition of our efforts in compiling extensive and appropriate references. All suggested improvements have been carefully considered, and the corresponding revisions have been incorporated into the updated manuscript to further enhance its scientific value and clarity.

[Comment 2] Introduction: This section is written in sufficient detail to reflect the topic of the manuscript.

 [Response 2] We sincerely thank the Reviewer for the positive assessment of the Introduction section and for the encouraging feedback.

[Comment 3] After the Introduction section, I propose an additional section "Agrotechnical methods for increasing the efficiency of fertilizer nitrogen use".

 [Response 3] We thank the reviewer for the suggestion. Following their advice, we have added an additional section describing the agrotechnical methods for improving NUE.

[Comment 4] Section 2. Mechanism for Improving Nitrogen Use Efficiency. Lines 141-143 When discussing nitrate reductase V and III, it is important to mention a good supply of molybdenum and iron to plants. These elements are components of nitrate and nitrite reductase.

 [Response 4] We appreciate the reviewer’s constructive comment. The role of molybdenum and iron in nitrate reductase activity has now been explicitly cited in the text as follows:

“Iron and molybdenum are also critical micronutrients for biological nitrogen reduction, as iron is a structural component of nitrate reductase and nitrogenase, while molybdenum forms part of the nitrogenase active site [22,23].”

References added:

• Rutkowska, B.; Szulc, W.; Spychaj-Fabisiak, E.; Pior, N. Prediction of molybdenum availability to plants in differentiated soil conditions. Plant Soil Environ. 2017, 63, 491–497. https://doi.org/10.17221/616/2017-PSE
• Symanowicz, B.; Kalembasa, S. Effect of iron, molybdenum and cobalt on the amount of nitrogen biologically reduced by Rhizobium galegae. Ecol. Chem. Eng. A 2012, 19(11).

[Comment 5] Section 3. Biological nitrogen fixation (BNF). Subsection 3.1. (Subsubsections 3.1.2., 3.1.3., 3.1.4 - Please add the numerical ranges of quantitative possibilities of biological reduction of molecular nitrogen N₂ by particular types of symbiotic bacteria.

You can use information from the articles: https://doi.org/10.1080/03650340.2020.1817398  https://doi.org/10.5601/jelem.2018.23.3.1684

 [Response 5] We appreciate the reviewer’s valuable comment and the additional references provided. Following their suggestion, numerical ranges have now been incorporated across the relevant BNF sections, and the recommended scientific studies have been added to the manuscript.

3.1.1 Rhizobium-legume system

“In terms of agronomic contribution, faba bean cultivation can introduce approximately 80–200 kg N ha⁻¹ to the soil within a single growing season [71–73]. However, the magnitude of fixed nitrogen is strongly influenced by environmental conditions and crop management practices.”

References added:

• Kalembasa, D.; Szukała, J.; Symanowicz, B.; Kalembasa, S.; Faligowska, A.; Becher, M. Amount of Biologically Nitrogen Fixed by Faba Bean and Its Uptake by Winter Wheat Determined by 15N ID Method. Arch Agron Soil Sci 2020, 67, 1–14. doi:10.1080/03650340.2020.1817398.
• Mabrouk, Y.; Hemissi, I.; Ben Salem, I.; Mejri, S.; Saidi, M.; Belhadj, O. Potential of Rhizobia in Improving Nitrogen Fixation and Yields of Legumes. Symbiosis 2018. doi:10.5772/intechopen.73495.
• Serafin-Andrzejewska, M.; Falkiewicz, A.; Wojciechowski, W.; Kozak, M. Yield and Seed Quality of Faba Bean (Vicia faba L. var. minor) as a Result of Symbiosis with Nitrogen-Fixing Bacteria. Agriculture 2025, 15, 960. doi:10.3390/agriculture15090960.

3.1.2 Bradyrhizobium

“The soybean–Bradyrhizobium symbiosis can fix approximately 300 kg N ha⁻¹ under favourable field conditions [86]. The amount of nitrogen fixed is influenced by several factors, including soil mineral N levels, the genetic compatibility of the symbiotic partners, and the absence of other yield-limiting stresses.”

Reference added:

• Keyser, H.H.; Li, F. Potential for Increasing Biological Nitrogen Fixation in Soybean. In: Biological Nitrogen Fixation for Sustainable Agriculture. 1992, pp. 119–135. doi:10.1007/978-94-017-0910-1_7.

3.1.3 Sinorhizobium

“Sinorhizobium meliloti associated with alfalfa roots can fix between 150 and 350 kg N ha⁻¹ under typical field conditions [98]. In trials combining constant mineral fertilization with NPK and diversified supplementation with Fe and Mo micronutrients, nitrogen fixation levels were reported to reach up to 618 kg N ha⁻¹ [99]. “

Reference added:

• Symanowicz, B.; Skorupka, W. Effect of Mineral Fertilization on Nitrogenase Activity, Yield, Nitrogen Content and Uptake with Alfalfa (Medicago sativa L.) Yield. Elem. 2019, 24, 181–191. doi:10.5601/jele m.2018.23.3.1684.

3.1.3 Cyanobacteria

“In paddy-field systems, cyanobacteria are reported to fix about 20–30 kg N ha⁻¹ yr⁻¹ as part of the biological nitrogen fixation pool [107,108]. “

References added:

• Issa, A.A.; Abd-Alla, M.H.; Ohyama, T. Nitrogen-Fixing Cyanobacteria: Future Prospect. In: Advances in Biology and Ecology of Nitrogen Fixation. doi:10.5772/56995.
• Subramanian, G.; Shanmugasundaram, S. Uninduced Ammonia Release by the Nitrogen-Fixing Cyanobacterium Anabaena. FEMS Microbiol. Lett. 1986, 37, 151–154. doi:10.1111/j.1574-6968.1986.tb01784.x.

[Comment 6] In the subheadings of subsections 3.1.2.; 3,1,3.; 3.1.4 - groups of plants that live in symbiosis with bacteria of the genera Bradyrhizobium and Sinorhizobium and with Cyanobacteria should be added.

 [Response 6] We thank the reviewer for the suggestion. Following their advice, the subheadings now read as follows:

Bradyrhizobium–soybean system

Sinorhizobium–alfalfa system

Cyanobacterial nitrogen fixation in rice systems

[Comment 7] Subsection 3.2. - How much N₂ can association bacteria (Azospirillum, Herbaspirillum, Methylobacterium, Bacillus) bind?

 [Response 7] We appreciate the reviewer’s valuable comment. Following their recommendation, the following text has been added to the revised version of the manuscript:

“Although their contributions to plant nitrogen supply are generally smaller and less direct than those of rhizobial symbioses, associative diazotrophs can nevertheless provide meaningful nitrogen inputs under low-fertility conditions, offering a distinct ecological advantage [120]. Among these, Azospirillum spp. are the most extensively studied, typically contributing 20–40 kg N ha⁻¹ in under favourable conditions [121] , with values decreasing to 2–3 kg N ha⁻¹ in suboptimal environments [122]. Comparable contributions (≈ 40 kg N ha⁻¹) have been reported for Herbaspirillum in sugarcane [123]. Other diazotrophic genera, such as Methylobacterium and Bacillus, generally contribute more modest nitrogen inputs, although it remains difficult to accurately estimate their field-level fixation rates due to the limited number of agronomic studies and the predominance of laboratory-based evaluations.”

[Comment 8] Conclusions section - should be: Summary and Conclusions.

 [Response 8] We appreciate the reviewer’s suggestion. The section has now been retitled “Summary and Conclusions.”

[Comment 9] Specific remarks:

1. Line 443- Alves et al.(2021) [?], line 463- Madhaiyan et al. (2015) [?], line 740- Giller et al. (2025) [?] Please provide the appropriate References numbers instead of the manuscript publication year.

 [Response 9] We thank the reviewer for this helpful remark. The cited references have been updated throughout the manuscript to use the appropriate reference numbers instead of publication years, as requested.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The paper entitled "Biofertilizers for enhanced nitrogen use efficiency: mechanisms, innovations, and Challenges” authored by Joaquin Herrero et al., a collaborative group from Spain and China in the field of agrobiotechnology, provides useful information on current knowledge on the biological foundation of nitrogen use efficiency with main focus on the role of microbial biofertilizers and biostimulants.

After a consistent introduction on the state-of-the-arts in this field, the original contributions are referred to the mechanism for nitrogen use efficient improvement, explaining nutrient mobilization by means of enhancement solubilization of bound nutrients which improves nitrogen uptake synergy, regulation of nutrient cycling by suppressing nitrification or denitrification which reduce nitrogen loses, root growth stimulation since larger and healthier roots improve nutrient absorption, stress mitigation as biofertilizers help plants to cope with abiotic stress (salinity, draught) which maintains nitrogen metabolism efficiency.

Other aspects are related to biological nitrogen fixation are referred rhizobium – legume interactions, Brady-rhizobium, sinorhizobium and cyanobacteria – based biofertilization.

Moreover, unlike symbiotic nitrogen fixers or strictly free-living soil diazotrophs, other associative nitrogen-fixing bacteria established non-modulating relations with plants. Among these one can mention the microorganisms like azospirillum, herbaspirillum, methylobacterium and bacillus.

The next section of this paper presents recent advances in biofertilizer development referred to multifunctional strains and microbial consortia, biofertilizer formulation and alternative carriers that prolong microbial survival, interaction with soil and rhizosphere microbiota, biotechnology and gene editing. The integration of multi-omics data, genome editing and synthetic circuits provides a powerful toolkit for developing next generation biofertilizers with enhanced agronomic performance.

The last parts of the study are referred to bottlenecks and challenges reflected by host specificity and symbiotic compatibility, colonization efficiency and competition, biosafety and regulatory approval, quality control, farmer awareness and adoption.

The paper is finalized by market landscape and future perspectives, and these data are quantified in an overview of commercial biofertilizer products to enhance nitrogen use efficiency.

One may conclude that microbial biofertilizers and biostimulants provide adequate, nature-based solutions for improving nitrogen use efficiency in order to achieve a sustainable balance between productivity, profitability and environmental care.

The paper is generally well written, with a paper structure for a review of scientific work, and with explanation of the technical specialty concepts used. It might be useful for engineers interested in biological innovations, agrochemical optimization and management of nitrogen biofertilizers.

For these reasons the paper is recommended for publication with excellent appreciation.

Author Response

Dear Reviewer,

The authors sincerely thank the reviewer for their thorough evaluation, constructive comments, and valuable suggestions. We also express our gratitude for the positive assessment and encouraging feedback, as well as for the recommendation for publication.

 

REVIEWER #2

[Comment 1] The paper entitled "Biofertilizers for enhanced nitrogen use efficiency: mechanisms, innovations, and Challenges” authored by Joaquin Herrero et al., a collaborative group from Spain and China in the field of agrobiotechnology, provides useful information on current knowledge on the biological foundation of nitrogen use efficiency with main focus on the role of microbial biofertilizers and biostimulants.

After a consistent introduction on the state-of-the-arts in this field, the original contributions are referred to the mechanism for nitrogen use efficient improvement, explaining nutrient mobilization by means of enhancement solubilization of bound nutrients which improves nitrogen uptake synergy, regulation of nutrient cycling by suppressing nitrification or denitrification which reduce nitrogen loses, root growth stimulation since larger and healthier roots improve nutrient absorption, stress mitigation as biofertilizers help plants to cope with abiotic stress (salinity, draught) which maintains nitrogen metabolism efficiency.

Other aspects are related to biological nitrogen fixation are referred rhizobium – legume interactions, Brady-rhizobium, sinorhizobium and cyanobacteria – based biofertilization.

Moreover, unlike symbiotic nitrogen fixers or strictly free-living soil diazotrophs, other associative nitrogen-fixing bacteria established non-modulating relations with plants. Among these one can mention the microorganisms like azospirillum, herbaspirillum, methylobacterium and bacillus.

The next section of this paper presents recent advances in biofertilizer development referred to multifunctional strains and microbial consortia, biofertilizer formulation and alternative carriers that prolong microbial survival, interaction with soil and rhizosphere microbiota, biotechnology and gene editing. The integration of multi-omics data, genome editing and synthetic circuits provides a powerful toolkit for developing next generation biofertilizers with enhanced agronomic performance.

The last parts of the study are referred to bottlenecks and challenges reflected by host specificity and symbiotic compatibility, colonization efficiency and competition, biosafety and regulatory approval, quality control, farmer awareness and adoption.

The paper is finalized by market landscape and future perspectives, and these data are quantified in an overview of commercial biofertilizer products to enhance nitrogen use efficiency.

One may conclude that microbial biofertilizers and biostimulants provide adequate, nature-based solutions for improving nitrogen use efficiency in order to achieve a sustainable balance between productivity, profitability and environmental care.

The paper is generally well written, with a paper structure for a review of scientific work, and with explanation of the technical specialty concepts used. It might be useful for engineers interested in biological innovations, agrochemical optimization and management of nitrogen biofertilizers.

For these reasons the paper is recommended for publication with excellent appreciation.

[Response 1] We sincerely thank Reviewer for their detailed and thoughtful summary of our manuscript and for recognizing the relevance of our work to the aims and scope of Nitrogen. We truly appreciate the reviewer’s positive assessment of the scientific content, structure, and clarity of the review, as well as their acknowledgement of the usefulness of our discussion for researchers and professionals in agrobiotechnology and nutrient management. The encouraging feedback and recommendation for publication are greatly valued by all authors.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Reviewer comments nitrogen-3987156

The review manuscript entitled „biofertilizers for enhanced nitrogen use efficiency: Mechanisms, innovations, and challenges“ represents a comprehensive review on nitrogen as a key macronutrient for plant nutrition. Key topics including nitrogen use efficiency (NUE), microbial biofertilizers including bacterial and cyanobacterial nitrogen fixators, and biostimulants are discussed. The manuscript thus represents a complex review on the topic of nitrogen as a key crop macronutrient and nitrogen fertilization.

I can therefore recommend the manuscript for publication in Nitrogen.

I have only a few formal comments on the present manuscript related to English language and style which are provided below:

Line 443: Use the reference number instead of the publication year in the reference „Alves et al. [123]….“

Line 526: Add a comma following the words „for example,…“

Line 716: Add a comma following the words: „In practical,…“

Line 719: Correct the typing error in the word „response“ (not „respons“).

Line 740: Use the reference number instead of the publication year in the reference „Giller et al. [115]….“

Line 806: Modify the statement as follows: „The positive impact of diazotrophic microorganisms on agriculture has driven rapid growth that has opened the biofertilizer market.“

Lines 821-822. Add commas in the statement: „In few years, the biofertilizer market has grown, and, at present, many nitrogen-fixing microorganisms are marketed as biofertilizers.“

 

Final recommendation: Accept after a minor revision.

 

 

Author Response

Dear Reviewer,

We sincerely thank you for your thoughtful comments and constructive suggestions. Your observations have been extremely valuable in improving the clarity and overall quality of the manuscript. All points raised have been carefully considered, and the corresponding revisions have been incorporated throughout the text. We believe that these changes have significantly strengthened the work. We are truly grateful for the time and effort you dedicated to evaluating our study.

 

REVIEWER #3

[Comment 1] The review manuscript entitled „biofertilizers for enhanced nitrogen use efficiency: Mechanisms, innovations, and challenges“ represents a comprehensive review on nitrogen as a key macronutrient for plant nutrition. Key topics including nitrogen use efficiency (NUE), microbial biofertilizers including bacterial and cyanobacterial nitrogen fixators, and biostimulants are discussed. The manuscript thus represents a complex review on the topic of nitrogen as a key crop macronutrient and nitrogen fertilization.

I can therefore recommend the manuscript for publication in Nitrogen.

[Response 1] We sincerely thank the Reviewer for their positive evaluation and encouraging feedback. We are grateful for the recognition of the relevance of the topics covered in the manuscript, and we appreciate the recommendation for publication in Nitrogen. We have carefully addressed the points suggested for refinement, and the corresponding changes have been incorporated into the revised version to further improve the quality and clarity of the manuscript.

[Comment 2]  Line 443: Use the reference number instead of the publication year in the reference „Alves et al. [123]….“

[Response 2] In accordance with the reviewer’s recommendation, the citation format has been revised to use reference numbers rather than author names and publication years.

[Comment 3] Line 526: Add a comma following the words „for example,…“

[Response 3] We sincerely thank the Reviewer for their appreciation. Following this suggestion, the comma has been added to the mentioned sentence.

[Comment 4] Line 716: Add a comma following the words: „In practical,…“

[Response 4] We thank the reviewer for this observation. The suggested comma has been added.

[Comment 5] Line 719: Correct the typing error in the word „response“ (not „respons“).

[Response 5] We apologize for the typographical error. It has now been corrected in the revised manuscript.

[Comment 6] Line 740: Use the reference number instead of the publication year in the reference „Giller et al. [115]….“

[Response 6] We thank the reviewer for this suggestion. The reference has been updated accordingly. The sentence now reads:

“Regulatory frameworks should mandate rigorous evidence of nitrogen-fixing efficacy prior to commercialization to ensure product reliability and avoid unfounded claims.”

 

[Comment 7] Line 806: Modify the statement as follows: „The positive impact of diazotrophic microorganisms on agriculture has driven rapid growth that has opened the biofertilizer market.“

[Response 7] We appreciate the reviewer’s suggestion, and the proposed modification has been incorporated into the revised manuscript.

[Comment 8] Lines 821-822. Add commas in the statement: „In few years, the biofertilizer market has grown, and, at present, many nitrogen-fixing microorganisms are marketed as biofertilizers.“

[Response 8] We thank the reviewer for the grammatical suggestion, which has improved the clarity of the section. The commas have been added as recommended.

 

 

 

Author Response File: Author Response.docx