Review Reports - Anaerobic Bioconversion of Mixed Fruit Waste into Organic Acids and a Multifunctional Enzymatic Bioproduct in a Stirred-Tank Bioreactor Using <i>Wickerhamomyces</i> sp. UFFS-CE-3.1.2

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors' explanations for the comments are logical. However, the revised and deleted statements in the manuscript are the same. Please mark only the revised statements in red. This will make it easy to understand where the revisions were made.

Author Response

Dear Editor and Reviewers,

We would like to sincerely thank the Editor and the Reviewers for the careful evaluation of our manuscript and for the constructive and insightful comments provided throughout the review process. We greatly appreciate the time, effort, and expertise dedicated to improving the quality, clarity, and scientific rigor of our work.

We are especially grateful for the positive evaluation in this stage, recognizing that the manuscript has been substantially improved and is now considered suitable for publication.

In this revised version, we have carefully addressed the remaining comment regarding the presentation of revisions. As requested, all modified statements have now been clearly highlighted in red throughout the manuscript to facilitate identification of the changes introduced in response to the reviewers' suggestions.

We also note that, in addition to the highlighted revisions, minor editorial improvements were performed across the manuscript to enhance readability, consistency, and scientific clarity. However, only the statements directly modified in response to the reviewers' comments were marked in red, as requested.

Below, we provide our specific responses.

Reviewer 1

Comment:
"The authors' explanations for the comments are logical. However, the revised and deleted statements in the manuscript are the same. Please mark only the revised statements in red. This will make it easy to understand where the revisions were made."

Response:
We thank the reviewer for this important observation and for the careful evaluation of the revised manuscript.

In the current version, we have carefully revised the manuscript to ensure that only the modified statements are highlighted in red, as requested. All redundant or duplicated markings were removed, and unchanged text is no longer highlighted. This allows for clear, precise identification of the revisions introduced in response to the previous comments.

We believe that this adjustment improves the transparency and readability of the revision process.

Sincerely,
Helen

(on behalf of the coauthors)

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

I have already reviewed the previous version of this manuscript. In the current submission, I compared the revised version with both the earlier manuscript and my previous comments. The authors have adequately addressed the concerns raised during the previous review, and the manuscript has been substantially improved. In my opinion, the manuscript is now suitable for publication in its present form.

Author Response

Dear Editor and Reviewers,

We are especially grateful for the positive evaluation in this stage, recognizing that the manuscript has been substantially improved and is now considered suitable for publication.

Below, we provide our specific responses.

Reviewer 2

Comment:
"I have already reviewed the previous version of this manuscript. In the current submission, I compared the revised version with both the earlier manuscript and my previous comments. The authors have adequately addressed the concerns raised during the previous review, and the manuscript has been substantially improved. In my opinion, the manuscript is now suitable for publication in its present form."

Response:
We sincerely thank the reviewer for this positive evaluation and for acknowledging the improvements made in the manuscript. We greatly appreciate the constructive feedback provided throughout the review process, which was essential for strengthening the scientific quality and clarity of this work.

We once again thank the Editor and the Reviewer for their valuable comments and for their positive assessment of the revised manuscript. We believe that the current version meets the requirements for publication and represents a significantly improved, more robust scientific contribution.

We remain at your disposal for any further clarification.

Sincerely,
Helen

(on behalf of the coauthors)

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript entitled ‘Anaerobic bioconversion of mixed fruit waste into organic acids and a multifunctional enzymatic bioproduct in a stirred-tank bioreactor using Wickerhamomyces sp. UFFS-CE-3.1.2» addresses the valorization of agro-industrial residues (mixed fruit peel waste) to organic acids and a multifunctional enzymatic bioproduct by Wickerhammyces strain. The process was conducted without supplementation of synthetic medium and exhibited stable operation in a stirred-tank bio-reactor. This manuscript aligns well with the aims and scope of Microorganisms Journal. I am ready to recommend it for publication, subject to the following comments:

Unfortunately, the text submitted by the authors is presented in a formatting mode, which makes it extremely difficult to read.
In the Introduction, more attention should be paid to similar approaches to fruit processing, and the authors should indicate how their work differs from these approaches and what is novel about it.
The source of the strain must be provided, including where it was isolated from and from which culture collection it was obtained.
The section on analytical methods should be supplemented with information on the equipment used, the manufacturer, and the operating conditions of the equipment during the analyses.
Sections 3.1 and 3.2 contain no graphical material, making the data presented in the text very difficult to interpret.
The Results and Discussion section requires a more detailed analysis of the findings. Attention should be paid to the efficiency of processing different types of waste, with an explanation of which types of waste are processed more effectively.
Was an assessment of community contamination processes conducted? This is a very important issue in waste processing.
Attention should be paid to the reactor operating conditions. Was optimization of these conditions carried out? What parameters influence the improvement of product yield? Since this work has an applied character, such data could significantly improve the study.

Author Response

Dear Editor and Reviewers,

We would like to sincerely thank you for your continued evaluation of our manuscript and for the valuable comments provided throughout the review process. We greatly appreciate the opportunity to improve our work further, as well as the positive feedback recognizing the substantial improvements already achieved.

In response to the editorial comments, we have carefully revised the manuscript to address all the points raised.

First, the manuscript has been expanded to provide a more comprehensive and detailed description of the study. Additional information was included in the Introduction to strengthen the research background and better contextualize the study within the biorefinery and circular bioeconomy framework. The Materials and Methods section was also expanded to improve reproducibility by including further details on biomass selection, fermentation conditions, inoculum preparation, strain origin, and analytical procedures. These modifications increased the overall length of the manuscript and enhanced its scientific depth, in accordance with the journal’s recommendations.

Second, regarding the presentation of revisions, we have carefully revised the manuscript to ensure that all modified statements are clearly highlighted in red, as requested. Only the portions of the text that were effectively revised have been marked, while unchanged content remains unaltered. This approach allows for clear, precise identification of the changes introduced.

In addition to the highlighted revisions, minor editorial improvements were made throughout the manuscript to enhance clarity, readability, and consistency. These include refinements in wording, sentence structure, correction of formatting issues, and removal of typographical inconsistencies. However, only changes directly related to the revision process were highlighted, as requested by the reviewer.

Third, we have carefully reviewed the reference list and reduced the number of self-citations where appropriate. The remaining self-citations were maintained only when essential to support methodological or contextual aspects of the study.

We also thank the additional reviewer for their positive evaluation of the manuscript and their constructive suggestions. In response, the manuscript was further improved by:

revising formatting issues to enhance readability;
strengthening the Introduction to highlight novelty and differences from previous studies better;
including information on the origin and maintenance of the microbial strain;
expanding the description of analytical methods to include equipment, manufacturer, and operating conditions;
improving the clarity and depth of the Results and Discussion section;
clarifying the scope of the study regarding mixed waste processing and reactor operation;
emphasizing that aspects such as contamination assessment and process optimization were beyond the scope of the present study and are relevant directions for future research.

Below, we provide a point-by-point response to the reviewer’s comments.

Reviewer Comments and Responses

Comment 1:
“The text is presented in a formatting mode, which makes it difficult to read.”

Response:
We thank the reviewer for this observation. The manuscript formatting has been carefully revised to remove word breaks, formatting inconsistencies, and layout issues. The revised version now presents a clear and readable structure.

Comment 2:
“In the Introduction, more attention should be paid to similar approaches and novelty.”

Response:
We agree and have revised the Introduction to provide a more comprehensive background on fruit waste bioconversion and biorefinery approaches. Additional context has been included to highlight better how the present study differs from previous works, particularly in terms of (i) the use of mixed fruit residues, (ii) anaerobic fermentation, and (iii) operation in a stirred-tank bioreactor aimed at generating a multifunctional enzymatic bioproduct rather than a single metabolite.

Comment 3:
“The source of the strain must be provided.”

Response:
We thank the reviewer for this important comment. Information regarding the origin of Wickerhamomyces sp. UFFS-CE-3.1.2 has been added to the Materials and Methods section, including its isolation source and maintenance conditions.

Comment 4:
“Analytical methods should include equipment and operating conditions.”

Response:
We agree and have expanded the analytical methods section to include details on the equipment used (e.g., HPLC system and detectors), manufacturer information, and key operating conditions to improve reproducibility.

Comment 5:
“Lack of graphical material in Sections 3.1 and 3.2.”

Response:
We appreciate this suggestion. While graphical representation can be useful, the dataset comprises a limited number of discrete sampling points. It is presented entirely in tabular form, with statistical analysis. We have improved the textual description of temporal trends in the Results and Discussion to facilitate interpretation. We respectfully chose to maintain the tabular format to avoid redundancy.

Comment 6:
“Results and Discussion require more detailed analysis.”

Response:
We agree and have expanded the Results and Discussion section to provide a more detailed interpretation of the findings. Additional discussion has been included to contextualize the results within the literature better and to explain observed trends in metabolite formation and enzymatic activity.

Comment 7:
“Efficiency of processing different types of waste should be discussed.”

Response:
We thank the reviewer for this suggestion. In the present study, the objective was to evaluate the performance of a mixed fruit-residue system rather than individual waste fractions. We have clarified this in the manuscript and added a discussion emphasizing that the mixed-substrate approach reflects real agro-industrial waste streams, which are inherently heterogeneous.

Comment 8:
“Was contamination assessed?”

Response:
We acknowledge the importance of this aspect. The fermentation was conducted under controlled, sterile conditions in a closed bioreactor system, minimizing the risk of contamination. Although no specific microbial community analysis was performed, this limitation has been clarified in the manuscript and identified as a relevant aspect for future studies.

Comment 9:
“Reactor conditions and optimization.”

Response:
We thank the reviewer for this important comment. The aim of this study was not to optimize reactor conditions, but rather to evaluate metabolite formation and enzymatic activity under defined anaerobic conditions. We have clarified this in the manuscript. Additionally, we included a discussion indicating that parameters such as agitation, temperature, and substrate composition may influence product yields and should be investigated in future optimization studies.

We once again thank the Editor and the Reviewers for their valuable comments and for their positive evaluation of our work. We believe that the manuscript has been significantly improved and now meets the journal’s expectations.

We remain at your disposal for any further clarification.

Sincerely,
Helen

(on behalf of the coauthors)

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

To my opinion, the authors have significantly revised the manuscript and taken into account all my comments. I am ready to recommend the manuscript for publication in this form.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study states that the mixing ratio obtained from the optimization of the fruit mixture in a previous study (https://doi.org/10.1007/s12649-024-02718-7) was used. However, that study focused on bioethanol.

This study, on the other hand, focuses on enzymatic activity. The ratios determined in the previous study were for bioethanol. It is unclear whether they are suitable for determining the planned enzyme activities in the current study. In other words, this study lacks any experimental design. Enzyme content was determined in samples taken at different times in a specific culture medium. There is no information indicating that these analyses were repeated. Furthermore, there is no statistical evaluation in the study, and the results are not sufficiently discussed. In short, the content of the study is weak. Therefore, it is not acceptable in its current form.

Author Response

Dear Editor and Reviewers,

We would like to sincerely thank the editor and the Reviewers for their careful reading of our manuscript and for their constructive and insightful comments. We greatly appreciate the time and effort invested in evaluating our work. The suggestions were extremely valuable and allowed us to substantially improve the clarity, rigor, and overall quality of the manuscript.

In response to the comments, we have carefully revised the manuscript throughout. The main modifications include: (i) refinement of the research objective to better align with the scope of the experimental data; (ii) expansion of the Materials and Methods section to improve reproducibility; (iii) inclusion of statistical analysis and explicit reporting of triplicate experiments; (iv) revision of the Results and Discussion to avoid overinterpretation; (v) improvement of quantitative comparisons with the literature; and (vi) revision of the Conclusions so that all statements are fully supported by the data obtained.

Below, we provide a detailed point-by-point response to all comments.

Reviewer 1

We thank the reviewer for the thoughtful and critical evaluation of our manuscript.

Comment 1

"This study states that the mixing ratio obtained from the optimization of the fruit mixture in a previous study was used. However, that study focused on bioethanol."

Response

We thank the reviewer for this insightful comment. We agree that the fruit mixture ratio used in this study was originally optimized for bioethanol production in a previous work. However, in the present study, the objective was not to optimize enzyme production, but rather to evaluate the metabolic behavior and enzymatic profile of Wickerhamomyces sp. during anaerobic fermentation of a representative, previously characterized fruit-waste mixture in a stirred-tank bioreactor.

The selected mixture provides a reproducible, sugar-rich substrate that supports microbial growth and metabolite formation. Therefore, its use in this study enables consistent comparison with previous findings and the investigation of how fermentation conditions influence metabolite distribution and enzymatic activity.

To clarify this point, the manuscript has been revised to explicitly state that the substrate composition was not optimized for enzymatic production, but was used as a standardized model substrate to evaluate process performance under bioreactor conditions.

Changes made in the manuscript

A clarification was added to the Materials and Methods section indicating that the mixture composition was based on a previous bioethanol-oriented optimization study. However, it was used here as a standardized, reproducible substrate for evaluating metabolite formation and enzymatic activity.

Comment 2

"This study, on the other hand, focuses on enzymatic activity. The ratios determined in the previous study were for bioethanol. It is unclear whether they are suitable for determining the planned enzyme activities in the current study. In other words, this study lacks any experimental design. Enzyme content was determined in samples taken at different times in a specific culture medium. There is no information indicating that these analyses were repeated. Furthermore, there is no statistical evaluation in the study, and the results are not sufficiently discussed. In short, the content of the study is weak. Therefore, it is not acceptable in its current form."

Response

We thank the reviewer for this detailed and critical evaluation of our manuscript.

We want to clarify that the main objective of this study was not to design or optimize enzymatic production, but rather to evaluate the metabolic behavior and enzymatic profile of Wickerhamomyces sp. during anaerobic fermentation of a representative fruit-waste substrate in a stirred-tank bioreactor. The use of a previously established substrate composition aimed to ensure reproducibility and comparability with earlier studies, while focusing on process performance under bioreactor conditions.

Regarding experimental replication and statistical analysis, we agree that this information was insufficiently described in the original version. In the revised manuscript, we explicitly state that all fermentation experiments were performed in triplicate (three independent biological replicates) and that all analytical determinations were carried out in triplicate. In addition, results are now presented as mean ± standard deviation, and statistical differences between fermentation times were evaluated using one-way ANOVA followed by Tukey's post hoc test (p < 0.05).

Furthermore, the Results and Discussion section has been expanded to provide a more robust and critical interpretation of the observed trends, and the manuscript has been carefully revised to improve clarity and avoid unsupported extrapolations.

We believe these revisions significantly strengthen the manuscript's scientific rigor.

Changes made in the manuscript

The Materials and Methods, Results and Discussion, and Statistical Analysis sections were revised to include biological and analytical replicates, mean ± standard deviation, ANOVA/Tukey analysis, and a more detailed interpretation of the results.

Reviewer 2 Report

Comments and Suggestions for Authors

This article addresses the current and interesting topic of fruit waste valorization using unconventional yeasts and bioreactor fermentation. In its current form, the manuscript requires major revision before possible publication.

General comments:

The research objective is clearly stated, but somewhat overly ambitious given the scope of the data presented – the authors declare an analysis of carbon flux modulation, while the focus is primarily on the final metabolite profile and enzymatic activities.
The Materials and Methods section requires significant refinement – many parameters necessary to replicate the experiment are missing, including the precise fermentation conditions, the method for maintaining anaerobiosis, inoculum standardization, the number of biological replicates, and more detailed information about enzymatic assays.
Appropriate statistical analysis is lacking – reporting deviations below 15% does not replace providing means, standard deviations, the number of replicates, and significance tests. 4. The Results and Discussion section contains overinterpretations – this particularly concerns the conclusions regarding oxidative stress, bioremediation, contaminant degradation, and bioherbicidal potential.
The presentation of the results is too sparse – for the bioprocess work, there are no graphs showing the dynamics of changes in metabolites, enzymatic activities, and basic process parameters over time.
The conclusions are too broad in relation to the obtained data – the statements regarding process scalability and industrial potential are particularly problematic.

Specific comments:

Lines 25–26: "with potential for contaminant degradation and treatment of lipid-rich effluents" – the conclusion is too broad. Please change it to: "with potential for future evaluation in contaminant degradation and treatment of lipid-rich effluents."

Lines 74–83: the section regarding the research gap is too general. The authors should further demonstrate what was missing in previous research and how the present work addresses it. Lines 84–88: "Evaluate how anaerobic fermentation in a stirred-tank bioreactor modulates carbon flux and enzymatic functionality..." – this formulation suggests a mechanism analysis that is not actually presented in the paper. Please simplify, e.g., to: "To evaluate metabolite formation and enzymatic activities during anaerobic fermentation..."

Lines 92–97: The substrate description is too general. It would be helpful to supplement it with basic chemical characteristics, such as dry matter content, sugar content, and initial pH.

Lines 107–111: The inoculum preparation description is too brief. Information on cell count, OD, or CFU/mL, as well as the method of inoculum standardization, is missing.

Lines 114–119: The description of fermentation in the bioreactor is insufficient. Please provide the stirring speed, method of achieving anaerobic conditions, pH control, stirrer type, and the number of independent fermentations. Lines 121–123: “Determined according to the methodologies described by Longo et al.” – Key methodological details should not be referred to another publication. Please provide a full description or at least the most important test parameters.

Lines 126–130: The description of the amylase and cellulase assay requires further clarification.

Lines 199–201: “This behavior is consistent with previous studies…” – Please provide a more specific quantitative comparison with previous work.

Lines 218–223: The interpretation regarding pH reduction is weak if pH was not monitored during the process.

Lines 224–230: “Oxidative bioremediation” and “bioherbicidal activity” – These applications are hypothetical at this stage and should not be presented as directly resulting from the conducted experiments.

Lines 241–243: The statement “all experimental deviations were lower than 15%” is insufficient. Please provide means ± SD, n, and statistical analysis.

Lines 245–250: If the authors use the term "significantly higher," they must demonstrate statistical significance. Otherwise, please change it to "higher than."

Lines 252–256: The relationship between catalase/SOD activity and pollutant degradation potential is too simplistic.

Lines 277–285: This entire section needs to be softened. Statements about bioremediation of contaminated environments, treatment of fatty wastewater, and bioherbicide formulations are currently too far-reaching.

Lines 27–299: The conclusions partially repeat overinterpretations from the discussion. Please limit them to what has actually been experimentally demonstrated.

Author Response

Dear Editor and Reviewers,

Below, we provide a detailed point-by-point response to all comments.

Reviewer 2

We thank the reviewer for the constructive evaluation and for recognizing the topic's relevance and timeliness.

General Comment 1

"The research objective is clearly stated, but somewhat overly ambitious given the scope of the data presented – the authors declare an analysis of carbon flux modulation, while the focus is primarily on the final metabolite profile and enzymatic activities."

Response

We thank the reviewer for this important comment. We agree that the original objective was formulated too broadly, given the scope of the data presented. Since the study did not include a mechanistic analysis of carbon flux, the objective has been revised better to reflect the experimental approach and the obtained results.

The expression "carbon flux modulation" was replaced with a more precise formulation focused on metabolite formation and enzymatic activities during anaerobic fermentation in a stirred-tank bioreactor.

Changes made in the manuscript

The final paragraph of the Introduction was revised. The objective now reads:

"In this context, the present study aimed to evaluate metabolite formation and enzymatic activities during anaerobic fermentation of mixed fruit peel waste in a stirred-tank bioreactor using Wickerhamomyces sp. UFFS-CE-3.1.2. Rather than targeting the production of a single metabolite, the study focuses on generating a multifunctional enzymatic bioproduct under bioreactor conditions."

General Comment 2

"The Materials and Methods section requires significant refinement – many parameters necessary to replicate the experiment are missing, including the precise fermentation conditions, the method for maintaining anaerobiosis, inoculum standardization, the number of biological replicates, and more detailed information about enzymatic assays."

Response

We thank the reviewer for this important comment regarding the completeness and reproducibility of the experimental procedures.

The Materials and Methods section has been substantially revised to provide a more detailed description of the experimental conditions. Specifically, we have included: (i) fermentation parameters, including temperature (30 °C) and agitation speed (150 rpm); (ii) the method used to establish anaerobic conditions, achieved by maintaining the reactor closed without aeration and confirmed through dissolved oxygen monitoring, which indicated anaerobiosis approximately 30 min after the start of fermentation; (iii) inoculum standardization at approximately 1 × 10⁶ CFU mL⁻¹; (iv) the number of biological replicates (n = 3); and (v) expanded methodological details for reproducibility.

Changes made in the manuscript

Sections 2.2, 2.3, and 2.4 were revised to include the initial soluble sugar concentration and initial pH of the medium, inoculum standardization, fermentation conditions, anaerobic operation, dissolved oxygen monitoring, and the number of independent biological replicates.

General Comment 3

"Appropriate statistical analysis is lacking – reporting deviations below 15% does not replace providing means, standard deviations, the number of replicates, and significance tests."

Response

We thank the reviewer for this important comment. We agree that the original manuscript lacked sufficient statistical detail. This issue has now been corrected. All results are presented as mean ± standard deviation from triplicate experiments, and statistical differences in fermentation times were evaluated using one-way ANOVA followed by Tukey's post hoc test, with significance set at p < 0.05.

The previous statement referring to deviations below 15% was removed.

Changes made in the manuscript

A new statistical analysis subsection was added, and Table 1 was revised to include mean ± standard deviation and statistical groupings.

General Comment 4

"The Results and Discussion section contains overinterpretations – this particularly concerns the conclusions regarding oxidative stress, bioremediation, contaminant degradation, and bioherbicidal potential."

Response

We appreciate the reviewer's observation and agree that the original wording in some sections was too speculative. The Results and Discussion sections have been carefully revised to avoid unsupported claims. Statements related to oxidative stress, bioremediation, contaminant degradation, and bioherbicidal applications have been reformulated to indicate that these are potential future applications rather than direct outcomes of the present study.

The revised discussion now focuses strictly on what was experimentally demonstrated, while framing possible applications more cautiously as perspectives for future work.

Changes made in the manuscript

Sections 3.2, 3.3, 3.4, the Abstract, and the Conclusions were revised to soften the language and eliminate unsupported extrapolations.

General Comment 5

"The presentation of the results is too sparse – for the bioprocess work, there are no graphs showing the dynamics of changes in metabolites, enzymatic activities, and basic process parameters over time."

Response

We thank the reviewer for this valuable suggestion.

We agree that graphical representation can be useful in many cases. However, in the present study, the dataset consists of a limited number of discrete sampling points (0, 18, 24, 48, and 72 h) and a relatively small number of variables measured over time. These data are already fully and clearly presented in tabular form as mean ± standard deviation (n = 3).

We carefully considered including graphs; however, given the limited number of time points and the straightforward trends, we concluded that graphical figures would not provide additional analytical value beyond what is already clearly conveyed in the table. Instead, they would largely duplicate the same information, increasing the manuscript length. To address the reviewer's concern, we strengthened the textual description of the temporal behavior of both metabolites and enzymatic activities in the Results and Discussion section.

Changes made in the manuscript

The Results and Discussion section was expanded to clarify temporal trends and better guide the reader through the data presented in Table 1.

General Comment 6

"The conclusions are too broad in relation to the obtained data – the statements regarding process scalability and industrial potential are particularly problematic."

Response

We thank the reviewer for this important comment.

We agree that the original Conclusions contained statements that were too broad in relation to the experimental evidence. The Conclusions section has been revised so that the data directly support all statements. Claims regarding scalability and industrial applicability were moderated, and the revised text now states that, although stable operation in a stirred-tank bioreactor was demonstrated, further studies are required to assess scalability and industrial feasibility.

Changes made in the manuscript

The Conclusions section was rewritten to remove overstatements and align the final claims with the actual experimental results.

Specific Comments from Reviewer 2

Comment

"Lines 25–26: 'with potential for contaminant degradation and treatment of lipid-rich effluents' – the conclusion is too broad. Please change it to: 'with potential for future evaluation in contaminant degradation and treatment of lipid-rich effluents.'"

Response

We agree and have revised the sentence accordingly. The text now indicates that these applications represent potential future evaluations rather than direct outcomes of the present study.

Comment

"Lines 74–83: the section regarding the research gap is too general. The authors should further demonstrate what was missing in previous research and how the present work addresses it."

Response

We thank the reviewer for this comment. The Introduction has been revised to define the research gap better, emphasizing the limited studies on multifunctional enzymatic extracts from mixed fruit residues under anaerobic conditions and in stirred-tank bioreactors, and clarifying how the present study addresses this gap.

Comment

"Lines 84–88: 'Evaluate how anaerobic fermentation in a stirred-tank bioreactor modulates carbon flux and enzymatic functionality...' – this formulation suggests a mechanism analysis that is not actually presented in the paper. Please simplify."

Response

We agree and have simplified the objective accordingly. The statement regarding carbon flux modulation was replaced with a more precise formulation focused on metabolite formation and enzymatic activities.

Comment

"Lines 92–97: The substrate description is too general. It would be helpful to supplement it with basic chemical characteristics, such as dry matter content, sugar content, and initial pH."

Response

We appreciate this suggestion. Additional information regarding the substrate was included in the Materials and Methods section, including the initial soluble sugar concentration of the liquid fraction and the initial pH.

Comment

"Lines 107–111: The inoculum preparation description is too brief. Information on cell count, OD, or CFU/mL, as well as the method of inoculum standardization, is missing."

Response

We agree and have expanded the description of inoculum preparation to include the inoculum concentration used in the experiments, which was standardized to approximately 1 × 10⁶ CFU mL⁻¹.

Comment

"Lines 114–119: The description of fermentation in the bioreactor is insufficient. Please provide the stirring speed, method of achieving anaerobic conditions, pH control, stirrer type, and the number of independent fermentations."

Response

We thank the reviewer for this comment. The fermentation description has been expanded to include agitation speed (150 rpm), anaerobic conditions (closed reactor without aeration), dissolved oxygen monitoring to confirm anaerobiosis, pH control information (pH not controlled), and the number of independent biological replicates (n = 3).

Comment

"Lines 121–123: 'Determined according to the methodologies described by Longo et al.' – Key methodological details should not be referred to another publication."

Response

We agree. The manuscript was revised to include the most important methodological details directly in the text rather than relying only on previous references.

Comment

"Lines 126–130: The description of the amylase and cellulase assay requires further clarification."

Response

The descriptions of the amylase and cellulase assays were clarified by explicitly specifying the substrate, incubation conditions, assay principle, and the basis for activity calculation.

Comment

"Lines 199–201: 'This behavior is consistent with previous studies...' – Please provide a more specific quantitative comparison with previous work."

Response

We thank the reviewer for this suggestion. This section was revised to include a more explicit quantitative comparison with previous literature, indicating that citric acid production in the present study falls within the range reported for similar anaerobic fermentations of agro-residues under stress conditions.

Comment

"Lines 218–223: The interpretation regarding pH reduction is weak if pH was not monitored during the process."

Response

We acknowledge this limitation. The discussion was revised to adopt a more cautious wording, indicating that the accumulation of organic acids likely influenced the medium conditions, without directly asserting measured pH reduction during the process.

Comment

"Lines 224–230: 'Oxidative bioremediation' and 'bioherbicidal activity' – These applications are hypothetical at this stage and should not be presented as directly resulting from the conducted experiments."

Response

We agree and have revised this section to remove direct claims regarding oxidative bioremediation and bioherbicidal activity. These aspects are now presented only as possible future research perspectives.

Comment

"Lines 241–243: The statement 'all experimental deviations were lower than 15%' is insufficient. Please provide means ± SD, n, and statistical analysis."

Response

This statement has been removed and replaced by appropriate statistical reporting, including mean ± standard deviation, number of replicates (n = 3), and ANOVA followed by Tukey's test.

Comment

"Lines 245–250: If the authors use the term 'significantly higher,' they must demonstrate statistical significance. Otherwise, please change it to 'higher than.'"

Response

We agree and revised the text so that claims of statistical significance are used only where supported by the applied statistical analysis. In other cases, wording was changed to "higher than."

Comment

"Lines 252–256: The relationship between catalase/SOD activity and pollutant degradation potential is too simplistic."

Response

We agree and revised this section to provide a more balanced and cautious interpretation of the observed enzymatic activities, avoiding direct extrapolation to pollutant degradation.

Comment

"Lines 277–285: This entire section needs to be softened. Statements about bioremediation of contaminated environments, treatment of fatty wastewater, and bioherbicide formulations are currently too far-reaching."

Response

This section was extensively revised to soften the interpretation and to clearly distinguish between experimentally supported findings and potential applications that require future investigation.

Comment

"Lines 27–299: The conclusions partially repeat overinterpretations from the discussion. Please limit them to what has actually been experimentally demonstrated."

Response

We agree. The Conclusions section has been revised to eliminate overinterpretation and to ensure that the experimental results directly support all statements.