Substrate Gas Utilization and C3/C4 Metabolic Analysis of Actinobacillus succinogenes: Integration into a Model for Fermentation Prediction in BES

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper conducts a study on the metabolism of Actinobacillus succinogenes, aiming to optimize succinic acid production and establish a metabolic model. This research shows a certain degree of innovation, and the experimental data provide strong support for the research results. However, the article lacks some theoretical data support, and the overall article is slightly lengthy. There are several aspects of the content that need to be modified. After these modifications are made, the manuscript may be accepted. The specific modification suggestions are as follows:

1.Table 1：There is a problem with the decimal point in the yield "0.11" of Saccharomyces cerevisiae (GM).

2.Line 47: According to Table 1, in addition to Mannheimia succiniciproducens MBEL55E (WT) and A. succinogenes Z130 (WT), Basfia succiniciproducens JF4016 (WT) also belongs to the organisms that naturally secrete a large amount of succinic acid, but this sentence does not mention Basfia succiniciproducens JF4016 (WT).

3.The author lacks detailed explanations when elaborating on the NADH regulation strategy (the specific operating conditions for introducing H₂ and the parameter settings for electro-fermentation), making the relevant content incomplete.

4. Figure3:In order to make the article layout more beautiful, the data graph should not be paginated. A page break can be added at the end of the paragraph.

Comments on the Quality of English Language

Some parts of this paper are rather lengthy. The author needs to refine the sentences to make the structure of the article more clear.

Author Response

Comment 1: “Table 1: There is a problem with the decimal point in the yield '0.11' of Saccharomyces cerevisiae (GM).”

Response: Thank you very much for pointing out this formatting issue. We have corrected the value in Table 1 by replacing the comma with a decimal point, ensuring consistency with the rest of the table.

 

Comment 2: “Line 47: According to Table 1, in addition to Mannheimia succiniciproducens MBEL55E (WT) and A. succinogenes Z130 (WT), Basfia succiniciproducens JF4016 (WT) also belongs to the organisms that naturally secrete a large amount of succinic acid, but this sentence does not mention Basfia succiniciproducens JF4016 (WT).”

Response: Thank you for this helpful observation. This particular sentence has been removed as part of a broader editorial revision of the section. Table 1 has been updated to clearly highlight the natural succinic acid producers. We hope this streamlined presentation improves clarity and focus.

 

Comment 3: “The author lacks detailed explanations when elaborating on the NADH regulation strategy (the specific operating conditions for introducing H₂ and the parameter settings for electro-fermentation), making the relevant content incomplete.”

Response: We appreciate this important comment. Starting from line 186, we have added more detailed information on the operating conditions of the bioelectrochemical system (BES), including the gas composition and key parameters relevant to electro-fermentation. Together with the referenced literature, we hope this provides a clearer and more comprehensive description of the NADH regulation strategy.

 

Comment 4: “Figure 3: In order to make the article layout more beautiful, the data graph should not be paginated. A page break can be added at the end of the paragraph.”

Response: Thank you for the suggestion regarding the visual layout. We have adjusted the formatting so that Figure 3 is now displayed on a single page. A page break has been inserted at the end of the preceding paragraph to improve the visual flow of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In this article, Tix et al. investigate the metabolism of Actinobacillus succinogenes to enhance succinate production. A computational model was also developed to predict the effects of metabolic differences. Overall, the scope of the study aligns well with the journal’s interests. However, many conclusions require stronger support through the inclusion of raw data and clearer explanations. The language throughout the manuscript also needs significant improvement. I would recommend publication if the following concerns are addressed.

Major Comments:

1. The Introduction is overly long and tedious. It should be rewritten to present concise background information, clearly define the key research questions, and briefly outline the authors’ approaches.
2. Quantification of key metabolites such as glucose, lactate, and succinate is not well-described. At minimum, HPLC standard curves for each compound should be included in the Supplementary Information to support the reported measurements.
3. The enzymatic parameters used in the modeling are critical for reproducibility but were not provided. Additionally, the authors oversimplify some key metabolic pathways, such as glycolysis. If such simplifications are based on existing studies, relevant references should be cited. Otherwise, more justification is needed to support these assumptions.

Minor Comments:

1. Table 1: add a space between "glucose" and "concentration."
2. Subtitle 3.3: add a space between "succinate" and "production."
3. Line 149–150: Remove the duplicated words.
4. Line 222, Line 330–331, Line 462: Use lowercase for chemical symbols: "N₂," "MgCO₃," and "CO₂."

Author Response

We would like to sincerely thank the Reviewer for the detailed and constructive feedback. We have carefully addressed each of the major and minor comments and revised the manuscript accordingly. Below, we provide a point-by-point response.

 

Major Comments:

1. “The Introduction is overly long and tedious. It should be rewritten to present concise background information, clearly define the key research questions, and briefly outline the authors’ approaches.”

Response:

Thank you for this valuable comment. We have substantially shortened the Introduction to enhance clarity and readability. In doing so, we ensured that the background information is concise, the main research questions are clearly stated, and our methodological approach is briefly outlined.

 

2. “Quantification of key metabolites such as glucose, lactate, and succinate is not well-described. At minimum, HPLC standard curves for each compound should be included in the Supplementary Information to support the reported measurements.”

Response:

We appreciate this important suggestion. A clarifying sentence has been added in line 267 to better explain the quantification of the key metabolites. In addition, we have included the corresponding HPLC standard curves in the Supplementary Information to support the accuracy of our measurements.

 

3. “The enzymatic parameters used in the modeling are critical for reproducibility but were not provided. Additionally, the authors oversimplify some key metabolic pathways, such as glycolysis. If such simplifications are based on existing studies, relevant references should be cited. Otherwise, more justification is needed to support these assumptions.”

Response:

Thank you for highlighting this crucial aspect. We have included a detailed explanation of how the enzymatic parameters were determined starting from line 289. The resulting parameters are now listed in the Supplementary Information. Furthermore, we have expanded the explanation for the simplification of glycolysis in our model beginning at line 570. We have clarified that, in the context of our study, the individual steps of glycolysis are not pivotal for analyzing the C3 and C4 metabolic fluxes. Relevant references and reasoning have been added to justify this approach.

 

Minor Comments:

We are grateful for your careful reading and for pointing out the following issues, which have now all been corrected:

 

Table 1: A space has been added between “glucose” and “concentration.”

Subtitle 3.3: A space has been added between “succinate” and “production.”

Line 149–150: The duplicated words have been removed.

Line 222, Line 330–331, Line 462: The chemical symbols have been corrected to lowercase: “N₂,” “MgCO₃,” and “CO₂.”

Reviewer 3 Report

Comments and Suggestions for Authors

In this research article, the authors explore the role of gaseous substrates (e.g., CO₂) in influencing the carbon flux distribution in A. succinogenes. In detail, they present the study from several perspectives, including the effects of inhibitory substances, the role of CO₂ as a metabolic component, the optimization achieved through a combination of sodium bicarbonate and CO₂, and the use of metabolic modeling.

Overall, this manuscript is suitable for publication in Fermentation, provided that the following concerns and suggestions are carefully addressed:

1. The introduction section contains a significant amount of redundancy. It lacks a clear and progressive structure that moves logically from a broad overview to the specific research question. This section should be thoroughly revised to improve its clarity and logical flow.

2. To make the results and conclusions more convincing, it is important to use well-established and widely accepted methods, such as high-resolution ¹³C metabolic flux analysis. Although such methods may be challenging to implement, the authors should at least acknowledge these advanced techniques in the discussion section and explain how they could contribute to future research.

3. It is unusual to use modeling results to validate experimental data. Typically, models are built prior to experiments to guide and facilitate the experimental design and results. However, in this study, the logic appears reversed. The authors should clarify the rationales of this approach.

4. Some abbreviations (technical terms) should be clearly explained, such as “BES”, which appears in both the title and the main text.

 

Author Response

We would like to sincerely thank the reviewer for the positive evaluation and the valuable suggestions to improve our manuscript. We have carefully addressed each of the comments and revised the manuscript accordingly. Below, we provide a point-by-point response.

 

1. “The introduction section contains a significant amount of redundancy. It lacks a clear and progressive structure that moves logically from a broad overview to the specific research question. This section should be thoroughly revised to improve its clarity and logical flow.”

Response:

Thank you for this helpful comment. We have substantially revised and shortened the introduction. In doing so, we aimed to eliminate redundancies and improve the logical progression from the broader context to the specific research question and methodological approach of this study.

 

2. “To make the results and conclusions more convincing, it is important to use well-established and widely accepted methods, such as high-resolution ¹³C metabolic flux analysis. Although such methods may be challenging to implement, the authors should at least acknowledge these advanced techniques in the discussion section and explain how they could contribute to future research.”

Response:

We appreciate this valuable suggestion. Starting from line 622, we now acknowledge ¹³C metabolic flux analysis as a powerful tool for validating and optimizing kinetic parameters. Although not applied in this study, we emphasize its potential for future research and model refinement in the context of bioelectrochemical systems.

 

3. “It is unusual to use modeling results to validate experimental data. Typically, models are built prior to experiments to guide and facilitate the experimental design and results. However, in this study, the logic appears reversed. The authors should clarify the rationales of this approach.”

Response:

Thank you for pointing out this important issue. We have added clarification both in the Introduction and starting from line 585 to explain our approach. In our case, the model was developed after obtaining experimental data in order to more precisely describe the observed behavior and to enhance the predictive accuracy for future BES setups. We now emphasize that this retrospective modeling strategy aims to build a more robust framework for guiding future experimental designs.

 

4. “Some abbreviations (technical terms) should be clearly explained, such as ‘BES’, which appears in both the title and the main text.”

Response:

Thank you for this remark. The abbreviation "BES" (bioelectrochemical system) is now introduced and defined in the Introduction at line 183 to ensure clarity for all readers.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing my concerns. I have no further comments.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank the authors for their response, it is now appropriate for acceptance.