A Novel Biostimulant–Biochar Strategy for Improving Soil Quality and Salinity Tolerance in Medicinal Mint (Mentha longifolia L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors:

 

The study is commendable for its integrative approach, combining a carbon-rich soil amendment (biochar) with a biological stimulant (Spirulina) to address the challenges of saline irrigation. The inclusion of both soil and plant physiological parameters strengthens the impact of the work.

However, the following areas need attention:

1. Abstract

The abstract is a bit long; consider condensing it to focus only on the most critical findings. It could benefit from clearer structuring—perhaps splitting into background, methods, results, and conclusions.

Suggested edit (first sentence):

"This study evaluated the combined application of biochar (BC) and Spirulina platensis (SP) as a sustainable strategy to improve soil quality and salinity tolerance in mint (Mentha longifolia L.) cultivated in sandy soils."

2. Introduction

The introduction includes repetitive elements; the discussion of salinity effects and biochar’s benefits could be condensed. The transition between paragraphs could be smoother; some sentences seem abruptly placed.

Scientific concern:

It would be helpful to briefly mention why Mentha longifolia was chosen (i.e., salinity tolerance threshold, economic value).

3. Materials and Methods

The biochar characterization lacks some important details—e.g., surface area (BET), porosity, or functional groups—which are critical for understanding interaction with soil/salts.

Please clarify how Spirulina was applied (e.g., was it dissolved in water? mixed dry into soil?).

It's unclear if environmental conditions during the pot experiment (temperature, humidity, photoperiod) were controlled or monitored.

The statistical section should include the assumptions checked (e.g., normality, homogeneity of variances) and what post hoc tests were used if ANOVA was significant.

Use consistent units formatting, e.g., "mg kg⁻¹" vs. "mg/kg" should be unified throughout.

 

4. Results

While the results are rich in data, interpretation sometimes creeps into this section (e.g., “highlighting the potential of…”).

The use of letter groupings (a, b, c…) for significance in graphs should be explained in figure legends.

Consider breaking results into more sub-sections for clarity (e.g., “Soil Physicochemical Properties”, “Ionic Balance”, “Plant Nutrient Uptake”, “Growth Parameters”).

Data on microbial biomass carbon (MBC) is intriguing; a follow-up on specific microbial shifts (e.g., functional groups, activity) would enhance scientific value.

5. Discussion

The discussion is quite long and would benefit from clearer organization and focus.

Some points (e.g., pH-buffering effect, Na⁺ adsorption by biochar) are repeated multiple times; streamline for clarity.

More critical analysis of limitations is needed—e.g., small-scale pot study, lack of microbial community profiling, unknown long-term effects.

Scientific suggestion:

Consider proposing mechanisms (biochemical or physiological) for the reduced Na⁺ uptake with BC+SP (e.g., ion exclusion, root membrane transporters).

The role of Spirulina as a biostimulant could be expanded with references to hormonal effects or gene expression.

Author Response

Reviowere1#

1. The study is commendable for its integrative approach, combining a carbon-rich soil amendment (biochar) with a biological stimulant (Spirulina) to address the challenges of saline irrigation. The inclusion of both soil and plant physiological parameters strengthens the impact of the work.

Authors' Response: We thank the reviewer for the encouraging feedback and appreciation of our integrative approach using biochar and Spirulina to enhance salinity tolerance.

2. Abstract

The abstract is a bit long; consider condensing it to focus only on the most critical findings. It could benefit from clearer structuring—perhaps splitting into background, methods, results, and conclusions.

Suggested edit (first sentence):

"This study evaluated the combined application of biochar (BC) and Spirulina platensis (SP) as a sustainable strategy to improve soil quality and salinity tolerance in mint (Mentha longifolia L.) cultivated in sandy soils."

Authors' Response: We thank the reviewer for this helpful suggestion. We have revised the abstract to be more concise and clearly structured into background, methods, results, and conclusions. The opening sentence was updated following the reviewer’s recommendation to better summarize the study’s objective. Additionally, we shortened the abstract to focus on the most critical findings.

3. The introduction includes repetitive elements; the discussion of salinity effects and biochar’s benefits could be condensed. The transition between paragraphs could be smoother; some sentences seem abruptly placed.

Authors' Response: We appreciate this constructive feedback. We carefully revised the introduction to remove repetitive elements and condensed the discussion on salinity effects and biochar benefits. Additionally, we improved the flow between paragraphs for smoother transitions and better readability.

4. Scientific concern: It would be helpful to briefly mention why Mentha longifolia was chosen (i.e., salinity tolerance threshold, economic value).

Authors' Response: This species was selected for this study due to its moderate salt tolerance (up to 7.5 dS m⁻¹) and significant economic importance in salt-affected regions, despite its susceptibility to growth reduction and decreased essential oil yield under salinity stress [2–4].

5. Materials and Methods
   • The biochar characterization lacks some important details—e.g., surface area (BET), porosity, or functional groups—which are critical for understanding interaction with soil/salts.

Authors' Response: We added Figure 1 contains “Figure 1. Biochar Physicochemical Characterization. (A) Scanning Electron Microscopy (SEM) image showing the porous surface morphology of corn cob-derived, (B) BET analysis by nitrogen adsorption-desorption isotherms, and (C) Fourier Transform Infrared (FTIR) spectrum revealing functional groups present on the biochar surface”.

• Please clarify how Spirulina was applied (e.g., was it dissolved in water? mixed dry into soil?).

Authors' Response: Spirulina was applied in powdered form and thoroughly mixed into the soil prior to planting.

• It's unclear if environmental conditions during the pot experiment (temperature, humidity, photoperiod) were controlled or monitored.

Authors' Response: The pot experiment was conducted under open-field conditions from January to April 2024. According to local meteorological data, average maximum and minimum temperatures during the experimental period ranged from 18 to 30 °C and 10 to 15 °C, respectively. Relative humidity averaged between 40–60%, and the daily sunshine duration was approximately 9-10 hours.

• The statistical section should include the assumptions checked (e.g., normality, homogeneity of variances) and what post hoc tests were used if ANOVA was significant.

Authors' Response: The experimental design of the pot experiment was a factorial randomized complete block design (RCBD) with three replicates per treatment. Data were subjected to analysis of variance (ANOVA) using the Statistix software (version 8.1). Prior to ANOVA, assumptions of normality and homogeneity of variances were verified using the Shapiro–Wilk and Levene’s tests, respectively. When significant differences were detected (p < 0.05), treatment means were compared using the Least Significant Difference (LSD) test at a 5% probability level.

• Use consistent units formatting, e.g., "mg kg⁻¹" vs. "mg/kg" should be unified throughout.

Authors' Response: All units have been standardized throughout the manuscript to ensure consistency. Concentration units are uniformly expressed as mg kg⁻¹

6. Results

6.1. While the results are rich in data, interpretation sometimes creeps into this section (e.g., “highlighting the potential of…”).

Authors' Response: We will revise the Results section to focus strictly on reporting data, removing interpretative statements such as “highlighting the potential of…”. Interpretations will be reserved for the Discussion section to maintain clarity and scientific rigor.

6.2. The use of letter groupings (a, b, c…) for significance in graphs should be explained in figure legends.

Authors' Response: We did.

6.3. Consider breaking results into more sub-sections for clarity (e.g., “Soil Physicochemical Properties”, “Ionic Balance”, “Plant Nutrient Uptake”, “Growth Parameters”).

Authors' Response: We did

6.4. Data on microbial biomass carbon (MBC) is intriguing; a follow-up on specific microbial shifts (e.g., functional groups, activity) would enhance scientific value.

Authors' Response: We appreciate the reviewer’s insightful comment regarding microbial biomass carbon (MBC). While this study focused primarily on MBC as a broad indicator of microbial activity and soil health, we recognize that analyzing specific microbial functional groups and their activity would provide deeper mechanistic insights. We recommend that future research include metagenomic or enzymatic assays to elucidate microbial community shifts in response to biochar and Spirulina amendments.

7. Discussion

7.1. The discussion is quite long and would benefit from clearer organization and focus.

Authors' Response: We modified the discussion and improved it as possible.

7.2. Some points (e.g., pH-buffering effect, Na⁺ adsorption by biochar) are repeated multiple times; streamline for clarity.

Authors' Response: We corrected that.

7.3. More critical analysis of limitations is needed—e.g., small-scale pot study, lack of microbial community profiling, unknown long-term effects.

Authors' Response: We added that in the conclusion.

7.4.Scientific suggestion:

Consider proposing mechanisms (biochemical or physiological) for the reduced Na⁺ uptake with BC+SP (e.g., ion exclusion, root membrane transporters). The role of Spirulina as a biostimulant could be expanded with references to hormonal effects or gene expression.

Authors' Response: We added this part to the discussion.

The combined application of biochar (BC) and Spirulina (SP) significantly reduced Na⁺ uptake in plants, indicating improved ionic homeostasis under saline soil conditions. This effect may be attributed to several biochemical and physiological mechanisms. One possible explanation is the ion exclusion mechanism at the root level, where biochar's porous structure and surface functional groups (e.g., carboxyl and hydroxyl) adsorb excess Na⁺ ions in the rhizosphere, reducing their availability for plant uptake [14,15]. In parallel, Spirulina may enhance root membrane integrity and selectivity, possibly through modulation of ion transporters, such as high-affinity potassium transporters and sodium/proton antiporters, which help regulate Na⁺ influx and compartmentalization [14,15]. Moreover, Spirulina’s role as a biostimulant could be linked to its content of plant growth regulators such as auxins, cytokinins, and gibberellins, which have been shown to influence root architecture and ion transporter activity [8,17]. Recent studies also suggest that algal biostimulants may upregulate stress-responsive genes involved in ionic regulation and antioxidant defense systems, contributing to improved salt tolerance [8,17]. Collectively, these mechanisms indicate that BC+SP treatment supports plants not only by modifying the soil environment but also by enhancing physiological resilience at the cellular level. Further research into gene expression profiling and transporter activity assays could elucidate these interactions more clearly.

Reviewer 2 Report

Comments and Suggestions for Authors

Major Revision

Provide a clear rationale for the selection of Spirulina as a treatment amendment, addressing why it was chosen over other potential soil amendments. Include comparative references to support this decision.

Clarify how the concentration of Spirulina was finalized, including the specific criteria used for determining optimal dosage and potential agricultural applications.

Discuss potential environmental implications of Spirulina application, particularly in relation to marine resource sustainability and potential conflicts with marine demands.

Consider future directions for utilizing Spirulina waste in soil amendments to minimize waste and enhance sustainability.

Include a rationale for the selection of specific seaweed species, focusing on their known bioactive compounds and how they relate to the observed outcomes in the study.

Modify Figure 2 to use a more professional and less flashy color scheme, maintaining clarity without distracting visual elements.

Address potential limitations of the study, including variability in seaweed composition, environmental factors, and scalability of the proposed amendments.

The conclusion effectively summarizes key findings but should also suggest specific future research directions. Highlight areas that require further investigation, such as molecular mechanisms underlying observed physiological changes.

Consider a comprehensive proofreading and language editing to improve readability .

Comments on the Quality of English Language

Consider a comprehensive proofreading and language editing to improve readability.

Author Response

Reviowere2#

1. Provide a clear rationale for the selection of Spirulina as a treatment amendment, addressing why it was chosen over other potential soil amendments. Include comparative references to support this decision.

Authors' Response: We added this part to the introduction:

Spirulina platensis was selected as a treatment amendment due to its well-documented effectiveness in alleviating the negative impacts of abiotic stresses, particularly salinity [8,9]. As natural and organic resource rich in bioactive molecules, Spirulina enhances plant stress tolerance by improving both physiological and biochemical functions [8,9]. Prior research has demonstrated that Spirulina application increases photosynthetic pigment levels, stabilizes cellular membranes, reduces ion leakage, and promotes water retention in plants under stress conditions [8,16–18]. These traits make Spirulina a valuable alternative to synthetic soil amendments, particularly in systems irrigated with saline or low-quality water. Despite the individual advantages of biochar and Spirulina, their combined influence on salinity tolerance in mint remains largely unexplored. The integration of these amendments may result in synergistic effects, improving soil physicochemical properties and enhancing plant performance. Therefore, the selection of Spirulina in this study is based on its potential as a sustainable, eco-friendly biostimulant that complements biochar in promoting salinity resilience in mint cultivation.

2. Clarify how the concentration of Spirulina was finalized, including the specific criteria used for determining optimal dosage and potential agricultural applications.

Authors' Response: We added this part to the materials and methods

Spirulina was applied at 1% w/w, a rate commonly used in pot experiments, which approximately corresponds to 15-20 t/ha when scaled to field conditions based on typical topsoil bulk density and depth. This dosage aligns with standard soil amendment practices and ensures effective delivery of bioactive compounds to enhance plant stress tolerance [8,9,16–18].

3. Discuss potential environmental implications of Spirulina application, particularly in relation to marine resource sustainability and potential conflicts with marine demands.

Authors' Response: We added this part to the discussion

Spirulina is widely recognized for its sustainability and agricultural benefits, but large-scale production may raise environmental concerns due to high water and nutrient demands, potentially affecting marine resource sustainability and ecosystem balance [16].

4. Consider future directions for utilizing Spirulina waste in soil amendments to minimize waste and enhance sustainability.

Authors' Response: We added this part to the conclusion

5. Include a rationale for the selection of specific seaweed species, focusing on their known bioactive compounds and how they relate to the observed outcomes in the study.

Authors' Response: We added this part to the discussion

The selected seaweed species were chosen based on their documented content of bioactive compounds such as alginates, laminarins, phenolics, and plant growth regulators (e.g., auxins and cytokinins), which are known to enhance nutrient uptake, improve soil microbial health, and stimulate plant physiological responses—factors closely linked to the outcomes observed in this study [16-18].

6. Modify Figure 2 to use a more professional and less flashy color scheme, maintaining clarity without distracting visual elements.

Authors' Response: We did.

7. Address potential limitations of the study, including variability in seaweed composition, environmental factors, and scalability of the proposed amendments.

Authors' Response: We included the limitation of the study in the end of conclusion.

8. The conclusion effectively summarizes key findings but should also suggest specific future research directions. Highlight areas that require further investigation, such as molecular mechanisms underlying observed physiological changes.

Authors' Response: we did.

The increasing reliance on low-quality saline water for irrigation demands innovative strategies to sustain crop productivity, especially in arid and semi-arid regions with limited freshwater resources. Medicinal plants such as Mentha longifolia L. (mint) demonstrate strong potential for cultivation under these challenging conditions. This study showed that saline water with an electrical conductivity up to 5 dS m⁻¹ can be effectively used to irrigate mint when supported by soil amendments. The combined application of biochar and the biostimulant Spirulina platensis significantly alleviated salinity stress, resulting in an 80% increase in mint biomass compared to untreated saline controls. This synergistic effect enhanced nutrient uptake, reduced sodium accumulation, and improved key soil quality parameters, including pH buffering capacity, microbial activity, organic matter content, and cation exchange capacity. These benefits were especially pronounced in sandy soils, highlighting the suitability of this integrated approach for salt-affected environments. Overall, the findings emphasize the potential of combining biochar with algal biostimulants as a sustainable and practical strategy to enhance plant performance and soil resilience under saline irrigation. Future research should focus on optimizing soil amendment protocols, evaluating salt-tolerant mint cultivars, and integrating beneficial microbial inoculants to further improve productivity in saline conditions. However, several limitations must be considered. Variability in seaweed composition due to species differences, harvest timing, and environmental factors may affect consistency. Environmental conditions such as soil type, moisture, and climate could also influence amendment effectiveness in field settings. Moreover, assessing the economic and logistical feasibility of large-scale application of seaweed-based amendments remains essential. Further studies should investigate the use of Spirulina processing residues as soil amendments to promote circular sustainability by reducing waste and enhancing long-term soil health. Additionally, exploring gene expression profiles and transporter activities could provide deeper insights into plant responses under salinity stress and amendment treatments.

9. Consider a comprehensive proofreading and language editing to improve readability .

Authors' Response: we did.

Reviewer 3 Report

Comments and Suggestions for Authors

Interesting scientific work must be improved from a graphic point of view but substantially it must be more engaging in its explanation to allow a wide and widespread diffusion of the information.

Author Response

Reviowere3#

Interesting scientific work must be improved from a graphic point of view but substantially it must be more engaging in its explanation to allow a wide and widespread diffusion of the information.

Authors' Response: We thank the reviewer for the encouraging comment and helpful suggestion. In response, we have revised several graphical elements in the manuscript to improve clarity and visual appeal. This includes enhancing figure resolution, simplifying labels, and improving consistency in color schemes. We have also revised sections of the text, particularly in the Introduction, Results, and Discussion, to adopt a more engaging and accessible tone, while maintaining scientific rigor. These changes aim to broaden the reach and impact of the study by making the content clearer and more compelling for a wider scientific and professional audience.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made extensive and commendable efforts in addressing the major revision comments, resulting in a much clearer and scientifically sound manuscript. Only a few minor corrections remain

Revise the title of Table 1 for clarity.

Ensure abbreviations such as biochar (BC) and Spirulina (SP) are defined only at their first mention; repeated expansions should be removed throughout the manuscript.

Double-check the manuscript for repeated full forms of abbreviations. Use the abbreviation only after the first mention to maintain consistency and scientific style.

Remove repeated Fourier Transform Infrared (FTIR), Scanning Electron Microscopy (SEM), etc.,

Add BET abbreviation

Author Response

We are grateful to Reviewer 2 for careful reading and insightful recommendations, which helped us improve the overall quality of the manuscript. 

In response to Reviewer 2’s comments, we have:

1. Revised the title of Table 1 to improve clarity.
2. Defined all abbreviations (e.g., biochar [BC], Spirulina [SP], FTIR, SEM, BET) only at their first mention and removed repeated full forms to ensure consistency and proper scientific style.
3. Properly introduced the BET abbreviation in the methods section.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is accepted

Author Response

We a sincerely thank Reviewer 3 for positive evaluation and support of the manuscript.