Sustainable Bioconversion of Cashew Apple Bagasse Hemicellulosic Hydrolysate into Xylose Reductase and Xylitol by Candida tropicalis ATCC 750: Impact of Aeration and Fluid Dynamics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study evaluated xylose reductase production by Candida tropicalis ATCC 750 using cashew apple bagasse hemicellulosic hydrolysate (CABHM) as an alternative carbon source, along with the effects of temperature, aeration, and fluid dynamics on the bioprocess. Below are comments/concerns:

1. Including microscopic images or flask photographs would enhance the manuscript by visually illustrating the phenotype of the strain.
2. Although some parameters (temperature, aeration, fluid dynamics) were varied, a full factorial design or response surface methodology was not applied, potentially missing synergistic effects or optimal conditions.
3. The XR enzyme is described in terms of molecular mass and pH/temperature activity, but detailed kinetic parameters (Km, Vmax), stability, or substrate specificity data are lacking, which are important for industrial enzyme applications.
4. The manuscript would benefit from calculating and reporting the conversion efficiency of substrate to product.
   

Author Response

Comment 1. Including microscopic images or flask photographs would enhance the manuscript by visually illustrating the phenotype of the strain.

Response: We appreciate the reviewer’s suggestion and acknowledge that visual documentation of the microorganism’s phenotype would strengthen the manuscript. However, during the experimental phase, we did not capture microscopic or macroscopic images of the cultures. While we recognize the value of such visual representations, unfortunately, these records are not available for inclusion at this stage.

 

Comment 2. Although some parameters (temperature, aeration, fluid dynamics) were varied, a full factorial design or response surface methodology was not applied, potentially missing synergistic effects or optimal conditions.

Response: We thank the reviewer for this insightful observation. At the time of experimental planning, a factorial design or response surface methodology (RSM) was not considered, primarily due to the sequential and exploratory nature of our study. Given the interdependence of variables such as temperature, aeration, and agitation, it is currently not feasible to retrospectively apply a comprehensive statistical design without compromising experimental consistency. Nonetheless, in the revised manuscript, we have made an effort to explain and correlate the effects of these variables in the discussion section to provide a better understanding of their individual and combined influences on the bioprocess.

 

Comment 3. The XR enzyme is described in terms of molecular mass and pH/temperature activity, but detailed kinetic parameters (Km, Vmax), stability, or substrate specificity data are lacking, which are important for industrial enzyme applications.

Response: We appreciate the reviewer’s comment regarding the importance of detailed biochemical characterization for potential industrial applications. However, the main objective of this study was to investigate the production of xylose reductase using cashew apple bagasse hemicellulosic hydrolysate as a carbon source, along with the evaluation of certain cultivation parameters. Only preliminary characterizations were performed. We agree that an in-depth kinetic and stability analysis, including parameters such as Km, Vmax, and substrate specificity, would indeed add value and may be addressed in a future study. Due to time constraints related to the manuscript revision process, it was not possible to conduct these additional experiments within the current scope.

 

Comment 4. The manuscript would benefit from calculating and reporting the conversion efficiency of substrate to product.

Response:

We agree with the reviewer’s recommendation. The conversion efficiency of substrate to product (yield) was calculated and incorporated into the revised manuscript. Some yield values were already reported in the original version (e.g., Y_P1/S1 in Table 2, referring to xylitol yield from xylose using FM medium). Additionally, we have now calculated the ethanol yield based on glucose and xylose consumption for the experiments using the CABHM medium. The following sentence has been added to the manuscript:

New sentence in the manuscript: "The ethanol yield at each temperature was calculated based on both the consumption of glucose alone and the combined consumption of glucose and xylose. When considering only glucose, the ethanol yields were 0.30, 0.35, 0.16, and 0.21 g_ethanol·g_glucose⁻¹ at 25, 30, 35, and 40 °C, respectively. However, when calculated based on the total consumption of glucose and xylose, the yields decreased to 0.15, 0.19, 0.08, and 0.09 g_ethanol·g_{glucose+xylose}⁻¹, respectively. These lower values are likely due to the incomplete assimilation of xylose by Candida tropicalis under the tested conditions, as the yeast preferentially consumes glucose, and xylose metabolism may be repressed in its presence (catabolite repression). Moreover, no xylitol production was observed in these assays, despite the detected activity of xylose reductase. This suggests that the presence of glucose and potential inhibitors in the hydrolysate may have redirected the carbon flux toward ethanol production while limiting the reductive conversion of xylose to xylitol."

 

We are grateful for the reviewer’s thoughtful and constructive feedback, which has undoubtedly contributed to improving the quality and clarity of our manuscript. We carefully considered all suggestions and addressed them to the best of our ability within the scope and limitations of the current study. However, we regret that it was not feasible to fully incorporate some of the proposed additional experiments due to time constraints and the unavailability of certain data from the initial experimental planning. We recognize the scientific value of these suggestions and intend to explore them in future investigations. We sincerely appreciate your understanding.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

• The use of sulfuric acid with autoclaving needs to be justified as it is not safe itself and hazardous to equipment and workers.
• - line 104: remove 20.
• line 118: how to get 3 colonies?
• - 2.4. Influence of Fluid Dynamics and Aeration on the Production of the Xylose Reductase 133 Enzyme and Xylitol: Trial 3 mentioned 4000 mL, but in results, 2000 mL, justify?
• - The C. tropicalis should be italicized ex line 260, 298,....
• Figure 4. SDS-PAGE electrophoresis of xylose reductase: it is not clear and does not provide concise data, it should be redone to get its aim as there are a lot of smear and complex lanes.

Author Response

Dear Editor and Reviewers,

The authors acknowledge the comments and questions raised by your revision of our manuscript entitled “Sustainable Bioconversion of Cashew Apple Bagasse Hemicellulosic Hydrolysate into Xylose Reductase and Xylitol by Candida tropicalis ATCC 750: Aeration and Fluid Dynamics” (Manuscript ID: applmicrobiol-3722629).

We have carefully analyzed and addressed each point raised by the Editor and Reviewers. All questions regarding the study and the manuscript were thoroughly answered, and the suggestions provided were thoughtfully considered and incorporated into the revised version.

We are pleased to submit our revised manuscript and would like to express our sincere gratitude to the Reviewers for their valuable suggestions, which have undoubtedly contributed to improving the quality of our work and will support future developments.

All modifications made in response to the comments have been highlighted in red for clarity.

Please do not hesitate to contact us if any further information is required.

Best Regards,

Authors

Comment 1. The use of sulfuric acid with autoclaving needs to be justified as it is not safe itself and hazardous to equipment and workers.

Response: We thank the reviewer for the comment. Diluted sulfuric acid hydrolysis combined with autoclaving is a widely used method for hemicellulose solubilization from lignocellulosic materials. In our study, we used a low concentration of sulfuric acid (0.6 mol·L⁻¹), which minimizes corrosion risks and safety concerns under controlled laboratory conditions. According to Kumar et al. (2009), dilute acid hydrolysis at concentrations below 1 mol·L⁻¹, followed by autoclaving, is considered relatively safe and effective for hemicellulose breakdown while maintaining manageable operational hazards.
Reference: Kumar, P. et al. (2009). Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial & Engineering Chemistry Research, 48(8), 3713–3729.

 

Comment 2. Line 104: remove 20.

Response: We appreciate the reviewer’s observation. The number “20” corresponds to a reference citation, and the absence of square brackets in the previous version was a formatting error. According to the journal’s author guidelines, we have corrected this by including the appropriate bracket notation. The revised sentence in the manuscript reads: "After, cashew apple bagasse hydrolysate was detoxified using activated carbon (3% w/v), being maintained under agitation at 200 rpm and 30 °C for 2 h [20]." We would also like to clarify that, according to our version of the manuscript, this content appears on lines 369–371. It is possible that formatting or layout changes occurred between submission and review, but we have carefully checked the manuscript and confirmed that the citation has been properly formatted and appears only in its intended context.

 

Comment 3. Line 118: how to get 3 colonies?

Response: Thank you for the question. Prior to inoculum preparation, the microorganism was reactivated on Petri dishes containing YEPD agar to allow for colony separation. Three isolated colonies were then aseptically transferred using an inoculation loop into the cultivation medium. The following revised sentence was included in the manuscript: "For inoculum preparation, the microorganism was inoculated in Petri plates containing YEPD agar and incubated at 30 °C for 48 h. After that, three isolated colonies were transferred to the inoculum medium (CABHM or FM), which was maintained in an orbital shaker at 30 °C and 150 rpm for 24 h." This content appears in lines 384–387 in our version of the manuscript. As previously mentioned, discrepancies in line numbering may result from formatting differences.

 

Comment 4. Trial 3 mentioned 4000 mL, but in results, 2000 mL, justify?

Response: We thank the reviewer for identifying this discrepancy. The correct volume is 2000 mL, as indicated in the Results section. This was a typographical error in the methodology, which we have now corrected. The revised sentence in the manuscript: "…and (3) in 2000-mL Erlenmeyer flasks with 1000 mL of the culture medium,…"

 

Comment 5. The C. tropicalis should be italicized (e.g., line 260, 298, …).

Response: We appreciate the reviewer’s attention to detail. We have carefully reviewed the manuscript and ensured that all instances of C. tropicalis and Candida tropicalis are now properly italicized throughout the text, in accordance with scientific nomenclature.

 

Comment 6. Figure 4. SDS-PAGE electrophoresis of xylose reductase: it is not clear and does not provide concise data, it should be redone to get its aim as there are a lot of smear and complex lanes.

Response: We thank the reviewer for the valuable feedback. Unfortunately, due to time constraints imposed by the revision deadline and the experimental workload required to reproduce all the assays, we are unable to repeat the SDS-PAGE analysis at this time. However, we have enhanced the image quality as much as possible using image optimization tools to improve clarity and contrast. We kindly invite the reviewer to assess the updated figure provided in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This work developed a process about production of xylose reductase and xylitol. The quality of this manuscript need be further improved. Major revision is suggested.

1. In Figure 1, the xylitol changes are missing.
2. In "3.2. The Production of Xylitol and Xylose Reductase Enzyme by CANDIDA Tropicalis", the ethanol and xylitol yield based on the consumption of glucose and/or xylose need be given.
3. Error bars need be provided in all data in Table 1, 2, and 3. Statistical analysis need be implemented, and F value and P value should be calculated.
4. Uncropped SDS-PAGE electrophoresis image need be provided in this manuscript.
5. The captions of Figure 3 need be provided in detail, and the reaction conditions should be given clearly.
6. The advantage and disadvantage of this work need be well summarized by comparing their findings with other works. The novelty of this research should be well highlighted.
7. Mass balance from biomass to xylitol and ethanol need be well calculated, and one related mass balance scheme should be given. The economical evaluation need be implemented.

Author Response

Dear Editor and Reviewers,

The authors acknowledge the comments and questions raised by your revision of our manuscript entitled “Sustainable Bioconversion of Cashew Apple Bagasse Hemicellulosic Hydrolysate into Xylose Reductase and Xylitol by Candida tropicalis ATCC 750: Aeration and Fluid Dynamics” (Manuscript ID: applmicrobiol-3722629).

We have carefully analyzed and addressed each point raised by the Editor and Reviewers. All questions regarding the study and the manuscript were thoroughly answered, and the suggestions provided were thoughtfully considered and incorporated into the revised version.

We are pleased to submit our revised manuscript and would like to express our sincere gratitude to the Reviewers for their valuable suggestions, which have undoubtedly contributed to improving the quality of our work and will support future developments.

All modifications made in response to the comments have been highlighted in red for clarity.

Please do not hesitate to contact us if any further information is required.

Best Regards,

Authors

 

Reviewer #03

This work developed a process about production of xylose reductase and xylitol. The quality of this manuscript need be further improved. Major revision is suggested.

Comment 1. In Figure 1, the xylitol changes are missing.

Response: We thank the reviewer for the observation. The mention of xylitol in the original figure legend was a mistake. As already described in the manuscript, no xylitol was detected under these conditions. This is likely due to the presence of glucose and inhibitory compounds such as acetic and formic acids, which impair xylose metabolism. The legend has been corrected in the revised version as follows: Revised caption of Figure 1: “Figure 1. Effect of temperature on cell growth, carbohydrate consumption, and ethanol production by Candida tropicalis ATCC750 cultivated at 150 rpm in the hemicellulosic hydrolysate from cashew apple bagasse (CABHM). (A) 25 °C, (B) 30 °C, (C) 35 °C, and (D) 40 °C. (■) Biomass (g·L⁻¹); (▲) Xylose (g·L⁻¹); (▼) Ethanol (g·L⁻¹); and (●) Glucose (g·L⁻¹).”

 

Comment 2. In "3.2. The Production of Xylitol and Xylose Reductase Enzyme by CANDIDA Tropicalis", the ethanol and xylitol yield based on the consumption of glucose and/or xylose need be given.

Response: We appreciate the reviewer’s suggestion. Some yield values were already reported in the original version (e.g., Y_P1/S1 in Table 2, referring to xylitol yield from xylose using FM medium). Additionally, we have now calculated the ethanol yield based on glucose and xylose consumption for the experiments using the CABHM medium. The following sentence was added to the manuscript:

New sentence in the manuscript: "The ethanol yield at each temperature was calculated based on both the consumption of glucose alone and the combined consumption of glucose and xylose. When considering only glucose, the ethanol yields were 0.30, 0.35, 0.16, and 0.21 g_ethanol·g_glucose⁻¹ at 25, 30, 35, and 40 °C, respectively. However, when calculated based on the total consumption of glucose and xylose, the yields decreased to 0.15, 0.19, 0.08, and 0.09 g_ethanol·g_{glucose+xylose}⁻¹, respectively. These lower values are likely due to the incomplete assimilation of xylose by Candida tropicalis under the tested conditions, as the yeast preferentially consumes glucose, and xylose metabolism may be repressed in its presence (catabolite repression). Moreover, no xylitol production was observed in these assays, despite the detected activity of xylose reductase. This suggests that the presence of glucose and potential inhibitors in the hydrolysate may have redirected the carbon flux toward ethanol production while limiting the reductive conversion of xylose to xylitol."

 

Comment 3. Error bars need be provided in all data in Table 1, 2, and 3. Statistical analysis need be implemented, and F value and P value should be calculated.

Response: We thank the reviewer for the valuable suggestion. Standard deviation values have been added to Tables 1, 2, and 3. Although the original submission included a general note on standard deviation (Table 3), we have now incorporated specific values for all measurements. Additionally, statistical analysis was performed using Tukey's test to evaluate significant differences among data groups. The F and P values were calculated for each set of variables and added accordingly.

New sentence for statistical analysis has been included in results/discussion: " The influence of temperature on xylose reductase production was statistically evaluated through analysis of variance (ANOVA) for each culture medium. In the formulated medium (FM), temperature significantly affected all evaluated parameters: enzymatic activity expressed as U·mL⁻¹ of extract (F = 22.076; p = 0.000317), U·g⁻¹ of cells (F = 23.544; p = 0.000253), and U·mg⁻¹ of protein (F = 6.109; p = 0.018246). A similar effect was observed in the cashew apple bagasse hydrolysate medium (CABHM), with significant differences also found for enzymatic activity in U·mL⁻¹ of extract (F = 100.935; p = 0.000001) and in U·g⁻¹ of cells (F = 100.937; p = 0.000001). However, the temperature did not significantly affect the enzymatic activity expressed in U·mg⁻¹ of protein (F = 0.85113; p = 0.504039), likely due to the low expression or limited extraction of the enzyme under these conditions, which resulted in minimal variation in specific activity across temperatures. These results confirm the significant role of temperature in modulating enzyme productivity under both nutrient conditions. After, Tukey’s test (p < 0.05) was applied, and statistically homogeneous groups are indicated by identical letters in Table 3.”

New information in the Table 3 legend: "Means followed by the same letter do not differ significantly according to Tukey’s test (p < 0.05)."

 

Comment 4. Uncropped SDS-PAGE electrophoresis image need be provided in this manuscript.

Response: We thank the reviewer for this observation. We have replaced the original image with an enhanced version of the SDS-PAGE electrophoresis to improve its clarity. Unfortunately, it is not feasible to reproduce the entire set of experiments within the current revision deadline. We hope the revised image meets the quality expectations.

 

Comment 5. The captions of Figure 3 need be provided in detail, and the reaction conditions should be given clearly.

Response: We thank the reviewer for the suggestion. The caption for Figure 3 has been revised to provide greater clarity and detail. Also, a topic on materials and methods (new topic 3.7.1) has been written to present more clearly what was accomplished.

Revised Figure 3 caption:“Figure 3. Influence of pH at 25 °C (A) and temperature (B) on the enzymatic activity of xylose reductase produced by Candida tropicalis ATCC750. The enzyme was obtained using hemicellulosic hydrolysate from cashew apple bagasse (CABHM) as substrate, under bioprocess conditions of 25 °C, 150 rpm for 24 h in 250-mL Erlenmeyer flasks containing 100 mL of culture medium.”

 

Comment 6. The advantage and disadvantage of this work need be well summarized by comparing their findings with other works. The novelty of this research should be well highlighted.

Response: We thank the reviewer for this valuable recommendation. The novelty of this study lies in the use of cashew apple bagasse hydrolysate as an alternative and underexplored carbon source for the production of xylose reductase. This enzyme is essential for xylitol synthesis and offers advantages such as fewer by-products, reducing downstream purification costs. Additionally, this approach contributes to circular economy principles by valorizing a widely available agro-industrial residue. The study also explores the effect of fluid dynamics on enzyme production, which is seldom addressed in the literature.

New sentence has been added in the introduction (to highlight relevance and novelty): "The production of value-added biochemicals such as xylose reductase from low-cost agro-industrial residues represents a sustainable alternative for both waste valorization and industrial bioprocess development, with potential to improve xylitol production efficiency and reduce environmental impact."

New sentence for comparison has been added in the Results and Discussion section (Section 2.3): "Compared to conventional synthetic media, the use of cashew apple bagasse hydrolysate demonstrated comparable enzymatic productivity, while promoting waste reduction and adding value to a by-product often discarded by the juice industry.

The main advantage of the present study lies in the use of cashew apple bagasse hydrolysate (CABHM) as a renewable and low-cost carbon source for xylose reductase production, eliminating the need for synthetic media or expensive inducers commonly used in previous studies. While Lugani and Sooch [4], Dasgupta et al. [8], and Zhang et al. [18] reported high enzymatic yields, their systems typically relied on enriched media or controlled induction strategies. In contrast, our process employed a non-detoxified lignocellulosic hydrolysate and a non-genetically modified strain (Candida tropicalis ATCC 750), simplifying implementation and reducing production costs. Notably, other authors, such as Zhang et al. [28], engineered Kluyveromyces marxianus to improve xylose reductase expression, whereas our results were obtained using a wild-type strain under natural fermentation conditions. Additionally, the evaluation of fluid dynamics and aeration effects adds originality to our approach, as these operational parameters are seldom explored in enzyme-focused bioprocess studies. Limitations of this work include the moderate enzyme yields compared to genetically enhanced strains and the reduced xylitol productivity under CABHM conditions, likely due to the presence of inhibitors (as noted by Yablochkova et al., [17]). Nevertheless, the integrated and low-input strategy demonstrated here aligns with biorefinery and circular economy principles, contributing to the valorization of agricultural waste streams through dual production of biocatalysts and biofuels.”

Reference in the manuscript:

[4] Lugani, Y.; Sooch, B.S. Fermentative production of xylitol from a newly isolated xylose reductase producing Pseudomonas putida BSX-46. LWT 2020, 134, 109988. Doi: 10.1016/j.lwt.2020.109988.

[8] Dasgupta, D.; Ghosh, D.; Bandhu, S.; Agrawal, D.; Suman, S.K.; Adhikari, D.K. Purification, characterization and molecular docking study of NADPH dependent xylose reductase from thermotolerant Kluyveromyces sp. IIPE453. Process Biochem. 2016, 51(1), 124-133. Doi: 10.1016/j.procbio.2015.11.007.

[18] Zhang, M.; Puri, A.K.; Wang, Z.; Singh, S.; Permaul, K. A unique xylose reductase from Thermomyces lanuginosus: Effect of lignocellulosic substrates and inhibitors and applicability in lignocellulosic bioconversion. Bioresour. Technol. 2019, 281, 374-381. Doi: 10.1016/j.biortech.2019.02.102.

[28] Zhang, B.; Zhang, L., Wang, D.; Gao, X.; Hong, J. Identification of a xylose reductase gene in the xylose metabolic pathway of Kluyveromyces marxianus NBRC1777. J. Ind. Microbiol. Biotechnol. 2011, 38(12), 2001-2010. Doi: 10.1007/s10295-011-0990-z.

[17] Yablochkova, E.N.; Bolotnikova, O.I.; Mikhailova, N.P.; Nemova, N.N.; Ginak, A.I. The activity of xylose reductase and xyli-tol dehydrogenase in yeasts. Microbiology 2003, 72, 414–417. Doi: 10.1023/A:1025032404238.

 

Comment 7. Mass balance from biomass to xylitol and ethanol need be well calculated, and one related mass balance scheme should be given. The economical evaluation need be implemented.

Response: We thank the reviewer for the suggestion. A simplified mass balance of the process, from biomass to final products (xylitol and ethanol), has been prepared and included in the revised manuscript. However, a detailed economic evaluation was not conducted in this study. This type of analysis requires a broad and robust data set involving capital investment, operational costs, scale-up parameters, and market analysis, factors that are beyond the experimental scope and timeline of the current work. We recognize its importance and suggest this as a valuable subject for future research.

New sentence has been added in the manuscript about Mass Balance (in the Section 2.3): To estimate the product yields at a larger scale, a mass balance was performed considering the use of 1 kg of cashew apple bagasse as the initial raw material. According to the experimental conditions, 200 g of dry bagasse yielded approximately 750 mL of detoxified hemicellulosic hydrolysate (CABHM). Therefore, scaling proportionally, 1 kg of bagasse generated 3.75 L of hydrolysate after pretreatment, separation, and detoxification processes. This hydrolysate contained approximately 85.13 g of glucose and 57.00 g of xylose. When this entire volume was used under the bioprocess conditions described in Experiment 3 (aerobic system at 30 °C for 24 h using 1000 mL of reaction mediun in 2000-mL Erlenmeyer flasks), the bioprocess using Candida tropicalis ATCC 750 resulted in the production of 31.13 g of xylitol, 6.38 g of ethanol, and 5.74 units of xylose reductase activity. These results underscore the biotechnological potential of using cashew apple bagasse as a carbon source for simultaneous enzyme production and generation of value-added bioproducts.

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

This revised version can be accepted.

Author Response

Editor

COMMENT: In the revised version of the manuscript, the authors have addressed the majority of the comments raised, considering time constraints and available resources. Please consider the following modifications as well.

Response: We sincerely appreciate your thorough and constructive review of our manuscript. Your comments have been very helpful in improving the quality and clarity of the work. We also acknowledge and have considered the additional suggestions provided in this step of review and have made the recommended modifications accordingly.

COMMENT 01: Lines 21-23 in the abstract: English modifications are required to better convey the aim of this work.

Response: We appreciate your careful review of our manuscript. We revised the first sentences of the abstract to explicitly state the objective of the study and we also reviewed the English of the final sentence. New abstract: “Abstract: This study aimed to evaluate the production of xylose reductase (XR), an enzyme responsible for converting xylose into xylitol, by Candida tropicalis ATCC 750 using hemicellulosic hydrolysate from cashew apple bagasse (CABHM) as a low-cost carbon source. The effects of temperature, aeration, and fluid dynamics on XR biosynthesis were also investigated. The highest XR production (1.53 U/mL) was achieved at 30 °C, with 8.3 g·L⁻¹ of xylitol produced by the yeast under microaerobic conditions, demonstrating that aeration and fluid dynamics are important factors in this process. Cellular metabolism and enzyme production decreased at temperatures above 35 °C. The maximum enzymatic activity was observed at pH 7.0 and 50 °C. XR is a heterodimeric protein with a molecular mass of approximately 30 kDa. These results indicate that CABHM is a promising substrate for XR production by C. tropicalis, contributing to the development of enzymatic bioprocesses for xylitol production from lignocellulosic biomass. This study also demonstrates the potential of agro-industrial residues as sustainable feedstocks in biorefineries, aligning with the principles of a circular bioeconomy.”

 

COMMENT 02: The authors should be consistent with the abbreviations format; for instance Table 1, reads CABHM in the headline and MCABH in the footnote.

Response: Dear Editor, we apologize for this inconsistency in acronyms. We reviewed the points indicated and the entire manuscript to always present the same acronym for the medium used (CABHM). New footnote for Table 1: *ND: not detected/**CABH: hydrolysate after hydrolysis, CABHM: hydrolysate with pH adjustment, CABHM*: hydrolysate with pH adjustment and treated with activated charcoal.

COMMENT 03: Please modify. 3. English modifications are suggested; for instance Lines 111-113, Line 368 etc.

Response: Thank you for your comment. The sentence in lines 111–113 has been revised for improved clarity and grammar. The updated version reads:

The sentence in lines 111-113: “Furfural and 5-HMF were not detected in the CABH. Regarding acetic acid and formic acid, only a slight reduction in their concentrations was observed after treatments with Ca(OH)₂ and activated carbon. The most suitable detoxification process to improve hemicellulosic hydrolysate fermentation depends on the origin of the raw material.”

The sentence in line 368: “Additionally, the decrease in activity is possibly due to the ionization of functional groups that hinder the binding of the enzyme to its substrate (S) or the formation of the enzyme-coenzyme complex necessary for catalysis [16].”

Additionally, we would like to inform the reviewer that, beyond the specific suggestions received, we are thoroughly reviewing the entire manuscript to enhance the overall quality of the English language and ensure the text is more precise and clearer.

COMMENT 04: The application of formulated medium should be better justified.

Response: We thank the reviewer for the observation. The purpose of applying the formulated medium (FM) was to specifically investigate the influence of glucose on xylitol production, as our previous results using CABHM indicated a repression of xylose metabolism when glucose was present. To clarify this, we have revised the manuscript to expand this justification (Lines 163-167), highlighting that the FM, composed solely of xylose as the carbon source, enabled the evaluation of glucose's inhibitory effect under otherwise similar conditions. Revised sentence in the manuscript: “To better understand the inhibitory role of glucose on xylitol production observed in CABHM, additional bioprocesses were carried out using a formulated medium (FM) with a similar composition to CABHM but containing only xylose as the carbon source. This strategy allowed us to isolate the effect of glucose and assess whether its presence represses xylose metabolism and xylitol biosynthesis.”

Author Response File: Author Response.docx