Dear Dr. Herrera,

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PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: General Comments

The manuscript presents a comprehensive experimental study systematically evaluating the effects of bioactive substances, light wavelength, and CO₂ injection regimes—both individually and in combination—on the growth of four microalgae/cyanobacterium of biotechnological interest (Arthrospira platensis, Chlorella vulgaris, Ankistrodesmus falcatus, and Tetradesmus dimorphus).

The inclusion of low-cost alternatives (such as Aloe vera and coconut water) is relevant from an applied perspective, and all data are fully available in the supplementary files, in accordance with the journal’s data availability requirements.

Despite its experimental merit, the manuscript presents several important issues that need to be addressed before it can be considered for publication, including:

Textual clarity and English quality:

There are recurrent grammatical errors, inappropriate use of prepositions, agreement issues, and long or confusing sentence constructions. In several sections, the English does not fully compromise comprehension, but it does reduce scientific clarity, particularly in the Introduction and Discussion.

Conceptual coherence between objectives, methods, and conclusions:

The stated objective refers to growth “and/or nutritional quality,” but the nutritional analysis is limited and applied only to selected conditions. While the conclusions emphasize growth, the nutritional data show only modest changes.

Statistical interpretation and risk of inflated significance:

The extensive use of the LSD test in an experimental design involving many treatments increases the risk of Type I error. Although statistical assumptions were validated, the choice of the LSD test should be more clearly justified given the large number of comparisons.

The specific points that require clarification and revision are detailed below.

Abstract

1. The abstract presents a large number of percentage values, making it dense and difficult to read. Reducing the number of quantitative examples and retaining only the most representative ones is recommended.

2. The terms “CO₂ injection regimes,” “CO₂ injection times,” and “CO₂ injection” are used interchangeably. Terminology should be standardized throughout the manuscript.

3. The abstract suggests the investigation of synergistic effects; however, the results clearly indicate a lack of additive effects. This nuance should be stated more explicitly and with a less optimistic tone.

Introduction

1. There is repetition of ideas. The relevance of microalgae and growth optimization is reiterated across multiple paragraphs with limited conceptual progression. For example, lines 37–45 and 47–49 essentially address the same argument.

2. The final objective of the study (lines 65–70) refers to both growth and nutritional quality, which creates expectations of more in-depth nutritional analyses than those performed. The objective should be clarified, emphasizing that growth is the main focus and that nutritional analysis is exploratory or complementary.

Materials and Methods

1. This section contains excessive operational detail.

2. The description of culture isolation and cleaning procedures is too long for a research article.

3. It is recommended to condense operational details, retaining only what is essential for reproducibility.

4. It should be clearly stated that species identification was based on morphology only, without molecular confirmation.

5. The rationale for selecting the concentration ranges of bioactive substances is not clearly explained.

6. Some substances show positive effects only at very specific concentrations, but this is not discussed methodologically.

7. References should be included to justify the selected concentrations, or the authors should explicitly state that these were exploratory concentrations.

8. Terminology should be standardized, for example:

“Indol-3-butíric acid” vs. “Indole-3-butyric acid”;

“naphtalenacético” vs. “naphthaleneacetic”.

9. Light intensity is described as “estimated” (80–150 µmol m⁻² s⁻¹). Is there a limitation preventing direct measurement of this variable? If intensity was not controlled, conclusions based solely on spectral effects should be interpreted cautiously.

10. Why is biomass recovery from the culture medium described only in Section 2.5?

11. Was the same wavelength (630 nm) used for biomass determination for all species? This wavelength may not be optimal for all microalgae analyzed.

12. Is there a bibliographic reference supporting the method used to construct the calibration curves? Were the calibration curves established after 15 days of growth monitoring?

13. Why were only one initial point and one final point (12 days) used for the main tests?

14. Regarding statistical analysis, why was a more conservative post hoc test (e.g., Tukey) not applied?

Results

1. The resolution of Figures 2–9 should be improved.

2. Many figures contain excessive visual information (numerous LSD test letters), making interpretation tiring. Simplification of the graphical presentation is recommended, if possible.

3. Growth curves are not fully explored in the text, representing a missed opportunity to enrich the dynamic interpretation of growth.

4. There is excessive repetition of numerical values, p-values, and LSD letters in the text. Reducing numerical detail and emphasizing general trends is suggested.

5. The Results section contains limited interpretative synthesis, with extensive description and little integration across findings. Better integration of results is recommended.

6. Information presented in figures and tables is extensively repeated in the text. Reorganization is suggested to avoid redundancy.

7. It should be clearly stated that combined treatments are not necessarily advantageous and may induce physiological stress.

8. Why is no statistical analysis presented for the responses shown in Table 2?

9. The term “significant nutritional improvement” should be avoided unless supported by appropriate statistical analysis, which should be included if available.

Discussion

1. The Discussion includes repeated ideas, long paragraphs, and occasionally redundant language, which compromises fluency and objectivity. Revision is recommended to remove repetition and synthesize arguments.

2. In some passages, results are presented as robust evidence of physiological mechanisms, whereas the data are essentially observational. Such statements should be revised. For example, direct attribution of effects to cytokinins, vitamins, or antioxidants is made without direct biochemical measurements. More cautious language is recommended (e.g., “may be associated with,” “possibly related to”).

3. The relevance of Aloe vera and coconut water as sustainable alternatives is reiterated several times using similar arguments.

4. Light intensity was not rigorously controlled, only estimated, and CO₂ injection was based on time rather than dissolved concentration. These limitations should be more explicitly acknowledged to avoid overinterpretation of the results.

5. The discussion of combined factors (bioactives + light + CO₂) could be more concise and more directly connected to the data. The central argument (physiological interference and non-linear responses) becomes diluted in lengthy paragraphs.

Conclusions

1. The conclusions refer to “nutritional quality,” but the data show modest and inconsistent changes. Reducing the emphasis on this aspect or explicitly characterizing it as exploratory is recommended.

2. The conclusions could more strongly emphasize that the results are specific to the experimental conditions adopted in this study.

Reviewer #2: The manuscript entitled “Enhancing microalgal productivity through bioactive substances, light, and CO₂”. This manuscript need revision before publication

Comments

1. Line 86: and remove unwanted particles. What are the unwanted martials and how authors remove them in microalgae culture?

2. Line 86-90 are confusing need to improve it.

3. Lin 93 what was the concentration of microalgae (OD) in the start of the experiment.

4. Why authors use such low light intensity (100 μmol m⁻² s⁻¹,) for low 12 h:12 h light/dark photoperiod. The optimized is 18:6.

5. How authors determine the growth of microalgae.

6. Table 1..why authors choose such costly bioactive compound rather than any other economic material for imrove the growth of microalgae. Is this study is economic feasible.

7. Where is the growth curve. Provide it in supplementary material.

8. Authors may add the flowing publication in discussion to improve the quality of paper

10.1016/j.pbi.2025.102696, 10.1016/j.bcab.2024.103315, 10.3390/su16167075,

9. Figures are not in good resolution. Provide the figure with good resolution.

Reviewer #3: This article addresses an interesting and important topic: the search for conditions that enhance the productivity of microalgae, which in turn are a promising resource for the production of valuable products, biofuels, and other materials. The article presents a methodology and applies an interesting approach that allows to determine the influence of individual growth factors (stimuli) and to study the complex (simultaneous) impact of several factors.

The following comments need to be made:

1. The authors should note that the introduction, specifically the analysis of previously conducted studies, is extremely brief. It is important to present not only the general results of such studies but also a brief description of the methods used. It would be desirable to include in the introduction the results of the combined effects of various growth factors on microalgal biomass, if such studies have been conducted previously.

2. Given the low solubility of CO2, it is unclear what processes could have achieved any noticeable changes in microalgal biomass productivity with such short exposure times (30 to 120 s). Experiments assessing the effect of CO2 on microalgal growth are typically conducted with continuous bubbling of the culture fluid with a mixture of CO2 and air with an elevated carbon dioxide concentration. Can the authors explain why this particular mode of CO2 addition to the culture fluid was chosen for the experiments?

3. The authors should clarify the composition of the gases during aeration during the main part of the experiments – was it air with a standard CO2 concentration? In section 2.2, the authors write: “Aeration was continuous and maintained at 1.0 L/min.”

4. Figures labeled "A" are too information-heavy and difficult to understand and analyze. Perhaps a more detailed analysis should be provided in Table B in File S3?

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:Sofia KiselevaSofia KiselevaSofia KiselevaSofia Kiseleva

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Enhancing microalgal productivity through bioactive substances, light, and CO₂

PONE-D-25-63084R1

Dear Dr. Herrera,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Ashfaq Ahmad, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: In the revised submission the authors have satisfactorily addressed my comments and concerns raised on their original submission. Therefore, I recommend publication.

Reviewer #3: The authors provided adequate responses to all the reviewer's comments. I disagree with some of the author's judgments (comment No. 2), but I admit that this experimental design (short-term bubbling of a microalgae suspension with 100% carbon dioxide) could be considered in a scientific study.

The description of the experimental methodology is adequate and complete. The experiments were conducted in at least three replicates. Statistical processing of the results was performed. The results are presented in detail, analyzed, and discussed.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: No

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