Caproate Production from Yellow Water Fermentation: The Decisive Roles of Electron Donors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors proposed a two-stage fermentation process to produce caproic acid from yellow water. Several strategies, i.e., ethanol, lactic acid and zero-valent nano-iron supply, were tested to improve carbon conversion into caproic acid. Although at a very small scale and in batch mode, the proposed strategy enabled reaching a concentration of up to 20 g/L of caproic acid with approx. 70% conversion rate. Overall, this article provides important insights into scaling up caproic acid production, exploiting a waste stream such as yellow water. Nevertheless, some aspects, such as materials and methodology description, must be improved. The article can be considered for publication after moderate revisions.

Below are my comments and suggestions.

1. Bacteria names should be italicized in the full manuscript. Please double-check this.
2. Materials and methods. Line 98. The authors report that yellow water has been filtered at 0.45 μm. This is quite an expensive procedure if considered at a larger scale. The authors should clarify the reason behind this step and take into account the costs for that.
3. Materials and methods. Lines 98-102. It is unclear whether the characteristics of yellow water here reported are literature data or actual measurements made on the specific substrate used in this study. Please clarify this aspect.
4. Materials and methods. Line 108. Did the authors perform any pretreatment on the seed sludge to favour hydrogen production? If that is so, this aspect should be included in materials and methods.
5. Materials and methods. The experimental setup must be rewritten entirely. The experimental conditions are not clearly elucidated in this section, but the readers only understand that in results and discussion. In this section, the authors should include aspects such as inoculum-to-substrate ratio, presence of control tests, water addition, and bottles preparation. Also, the first step of the process should be described here.
6. Materials and methods. The authors should describe how the conversion yield was calculated.
7. Materials and methods. The analysis of metals is not mentioned at this point in the manuscript, but results are shown later in the results and discussion section. Please include the applied methodology in materials and methods.
8. Results and discussion. Please improve the comparison with literature, including the current background on yellow water valorization through acidogenic or dark fermentation.
9. Results and discussion. The authors mentioned several times the importance of balancing electron donors and electron acceptors. However, a clear comparison with the available literature on this aspect is missing. Please include that in the revised version of the article.
10. Results and discussion. Ethanol addition. The dilution selected for this set of tests should be clarified at the beginning of this sub-section.
11. Results and discussion. Zero-Valent Nano-Iron Addition. The selected ethanol addition for this set of tests should be clarified at the beginning of this sub-section.
12. The inoculation of Lactobacillus must be clarified. What does 4% mean? What is the origin of Lactobacillus?
13. Figure 6. Why are other metabolites not reported here?
14. The authors are requested to include the pH trend in all graphs.

Author Response

1. Bacteria names should be italicized in the full manuscript. Please double-check this.

Reply: According to this suggestion, we double-checked the Bacteria names and had changed them to italic type.

2. Materials and methods. Line 98. The authors report that yellow water has been filtered at 0.45 μm. This is quite an expensive procedure if considered at a larger scale. The authors should clarify the reason behind this step and take into account the costs for that.

Reply: This process can definitely be omitted in large-scale applications. In this pilot study, in order to ensure high repeatability of experimental data, we used the filtering method to remove the interference of suspended solids. In fact, in large-scale applications, suspended solids can be effectively removed through centrifugation, gravity settling, etc. Moreover, according to this suggestion, we replaced the method of removing suspended solids with the centrifugal method that could be applied on large scales.

3. Materials and methods. Lines 98-102. It is unclear whether the characteristics of yellow water here reported are literature data or actual measurements made on the specific substrate used in this study. Please clarify this aspect.

Reply: The yellow water used in this study was sampled a high-strength wastewater stream of a strong-flavor liquor distillery situated in Wuxi, China and the characteristics mentioned in this paper was obtained by measured the samples. Moreover, according to this suggestion, relevant content has been supplemented in lines 132-138.

4. Materials and methods. Line 108. Did the authors perform any pretreatment on the seed sludge to favor hydrogen production? If that is so, this aspect should be included in materials and methods.

Reply: The microbial agents used in this experiment include the caproic acid producing bacteria and lactic acid producing bacteria. The former comes from a long-term operating caproic acid production reactor in our research group and the latter is a commercial purchase. Moreover, according to this suggestion, relevant content has been supplemented in lines 139-146.

5. Materials and methods. The experimental setup must be rewritten entirely. The experimental conditions are not clearly elucidated in this section, but the readers only understand that in results and discussion. In this section, the authors should include aspects such as inoculum-to-substrate ratio, presence of control tests, water addition, and bottles preparation. Also, the first step of the process should be described here.

Reply: According to this suggestion, we rewrote the experimental setup in the line 147-174.

6. Materials and methods. The authors should describe how the conversion yield was calculated.

Reply: According to this suggestion, the formula for calculating conversion yield was supplemented in line 212-223.

7. Materials and methods. The analysis of metals is not mentioned at this point in the manuscript, but results are shown later in the results and discussion section. Please include the applied methodology in materials and methods.

Reply: According to this suggestion, the methods for metals analysis was supplemented in line 204-211.

8. Results and discussion. Please improve the comparison with literature, including the current background on yellow water valorization through acidogenic or dark fermentation.

Reply: According to this suggestion, relevant content has been supplemented in lines 74-85.

9. Results and discussion. The authors mentioned several times the importance of balancing electron donors and electron acceptors. However, a clear comparison with the available literature on this aspect is missing. Please include that in the revised version of the article.

Reply: According to this suggestion, relevant content has been supplemented in lines 55-71.

10. Results and discussion. Ethanol addition. The dilution selected for this set of tests should be clarified at the beginning of this sub-section.

Reply: According to this suggestion, relevant content has been supplemented in lines 261-262.

11. Results and discussion. Zero-Valent Nano-Iron Addition. The selected ethanol addition for this set of tests should be clarified at the beginning of this sub-section.

Reply: According to this suggestion, relevant content has been supplemented in lines 295-297.

12. The inoculation of Lactobacillusmust be clarified. What does 4% mean? What is the origin of Lactobacillus?

Reply: 4% means that the inoculation amount of Lactobacillus was according to a mass concentrations of 4%. According to this suggestion, relevant content has been revised as that “Figure 6. Lactic acid production at varying Lactobacillus inoculum levels, namely mass concentrations of (a) 0%, (b) 2%, (c) 4% and (d) 6%.”

13. Figure 6. Why are other metabolites not reported here?

Reply: The lactic acid fermentation process is efficient and targeted, with low production of other by-products. To highlight the production and conversion rate of lactic acid, other metabolites were not labeled. The following is a graph of the production of relevant products.

14. The authors are requested to include the pH trend in all graphs.

Reply: pH control is very important for anaerobic fermentation, so we strictly controlled the pH during the experimental process. According to the reviewer's suggestion, we have supplemented the pH for each type of fermentation process in the Materials and Methods section, which would not affect graph clarity and do not interfere with the presentation of key content in each graph.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I reviewed the following manuscript: 'Caproate Production from Yellow Water Fermentation: The Decisive Role of Electron Donors.
In my opinion, it is an interesting topic, but I have some reservations. The results are interesting, but the description of the methodology is the weakest part of the manuscript.
Introduction:
There is no theoretical introduction to the CE process. It is unclear what the process involves, which bacteria are involved, the conditions under which it occurs and how it differs from other anaerobic fermentations.
There is a lack of clearly defined work objectives and an innovation aspect.
Methods
The methodology is not clearly described or explained.
Where do the lactic acid bacteria come from? The results description provides results for different inoculum doses and mentions Lactobacillus, which is not mentioned in the methodology.
How was the multiplication of methanogenic microorganisms prevented, given that they are likely to be found in sewage sludge from sewage treatment plants and would multiply well under the given conditions (lines 105–110)? It is unclear how the fermentation was directed towards chain elongation rather than decomposition in the fermentation process to biogas.
- There is no mention of dilutions in the methodology. What were they used for? This is only mentioned in the conclusions. What was the yellow water diluted with?
The methodology lacks a description of the experiment involving the addition of ethanol, lactic acid, iron and two-stage fermentation. Was diluted or undiluted water used to conduct the experiment involving the addition of ethanol and acid?
The methodology must be improved to ensure that the entire research plan is included and described in detail. It is unacceptable that we only find out about some of the experiments conducted in the conclusions section.
Specific comments:

Author Response

1. Introduction: There is no theoretical introduction to the CE process. It is unclear what the process involves, which bacteria are involved, the conditions under which it occurs and how it differs from other anaerobic fermentations.

Reply: According to this suggestion, relevant content has been supplemented in lines 55-71.

2. There is a lack of clearly defined work objectives and an innovation aspect.

Reply: According to this suggestion, relevant content has been supplemented in lines 121-126 as following.

“This study aims to solve the drawbacks of single-phase systems by enhancing elec-tron availability, stabilizing fermentation dynamics, and improving overall caproate selectivity and yield. Moreover, 16S rRNA sequencing was used to profile the microbi-al community in addition to process optimization to investigate the effects of various supplementation techniques (such as ethanol, lactic acid, NZVI) on microbial structure and function. Crucial information about the CE's metabolic pathways and taxa can be gained by comprehending the microbial shifts under different redox conditions [26, 27]. The key innovation of this work lies in the active integration of endogenous and exogenous electron donors. Unlike previous approaches that relied on the substrate's native composition, our strategy proactively engineers the electron donor pool via lactic acid fermentation and enhances it with NZVI, creating a synergistic effect that significantly boosts caproate yield and provides a novel valorization pathway for yellow water. This study comprehensively evaluates a scalable, economically viable method for converting ethanol-rich industrial effluents like yellow water into high-value MCFAs, thereby contributing to the development of integrated waste-to-resource biotechnologies.”

3. Methods, the methodology is not clearly described or explained. Where do the lactic acid bacteria come from? The results description provides results for different inoculum doses and mentions Lactobacillus, which is not mentioned in the methodology.

Reply: According to this suggestion, relevant content has been supplemented in section 2.2

4. How was the multiplication of methanogenic microorganisms prevented, given that they are likely to be found in sewage sludge from sewage treatment plants and would multiply well under the given conditions (lines 105–110)? It is unclear how the fermentation was directed towards chain elongation rather than decomposition in the fermentation process to biogas.

Reply: In this study, methanogen were inhibited by adding 2-bromoethanesulfonate (BES) with 2 g·L⁻¹, which was supplemented in section 2.3. Actually, the low pH fermentation used in this study is inherently unfavorable for methane production.

5. There is no mention of dilutions in the methodology. What were they used for? This is only mentioned in the conclusions. What was the yellow water diluted with?

 

Reply: According to this suggestion, relevant content has been supplemented in section 2.3.

6. The methodology lacks a description of the experiment involving the addition of ethanol, lactic acid, iron and two-stage fermentation. Was diluted or undiluted water used to conduct the experiment involving the addition of ethanol and acid?

Reply: According to this suggestion, relevant content has been supplemented in section 2.3.

7. The methodology must be improved to ensure that the entire research plan is included and described in detail. It is unacceptable that we only find out about some of the experiments conducted in the conclusions section.

Reply: According to this suggestion, we supplemented the content of the experimental methods section, which should improve the methodology.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Comment #2. 0.45 μm filtering is a much stronger and expensive procedure than centrifugation and settling. Indeed, it can remove large bacteria in addition to suspended solids. The authors must return to their previous methodology, as it reflects the actual procedure followed in this study. Replacing filtering with centrifugation may lead to different results. Aspects related to large-scale applications where filtering at 0.45 μm is not feasible can be included in the discussion chapter or the conclusions, along with suggestions for alternative separation strategies.

Comment #5. The authors improved the quality of information provided in terms of experimental setup and bottle preparation. However, pivotal aspects such as inoculum-to-substrate ratio, presence of endogenous controls, and biological triplicates are still missing. Please improve that.

Comment #8. Comparison with literature is part of the discussion. At the current status, critical discussion of obtained results and their comparison with available literature is still missing.

 

Author Response

Dear Editors,

Thanks for your letter with comments to our manuscript. All comments have been considered. According to these comments, we revised the manuscript which was highlighted by red color. The replies and changes are enclosed below.

Reviewer #1:

Comment #2. 0.45 μm filtering is a much stronger and expensive procedure than centrifugation and settling. Indeed, it can remove large bacteria in addition to suspended solids. The authors must return to their previous methodology, as it reflects the actual procedure followed in this study. Replacing filtering with centrifugation may lead to different results. Aspects related to large-scale applications where filtering at 0.45 μm is not feasible can be included in the discussion chapter or the conclusions, along with suggestions for alternative separation strategies.

Reply: This comment is indeed very crucial. In fact, we have further confirmed that during our experiment, the 0.45 μm filtering was only used for the pre-treatment of the measurements of those dissolved indexes, such as soluble COD, while during the operation of the fermentation reactors, the pre-treatment of yellow water was carried out by centrifugation. We know that the 0.45 μm filtering in laboratory mainly uses the injection tube pressure filtration method. Although the laboratory scale reactor operation also requires the consumption of more than ten to tens of liters of treated water samples per day, it is impossible to obtain them by 0.45 μm filtering. The 0.45 μm filtering mentioned in the first version of the manuscript was originally a writing error, so we have made corresponding corrections.

Comment #5. The authors improved the quality of information provided in terms of experimental setup and bottle preparation. However, pivotal aspects such as inoculum-to-substrate ratio, presence of endogenous controls, and biological triplicates are still missing. Please improve that.

Reply: The inoculum-to-substrate ratio was 10% (v/v), that is, 30 ml enrichment culture was added into 300 ml yellow water (supplemented in section 2.2). All experiments were conducted triplicates (supplemented in section 2.3), which could be reflected in the data graphs through error bars. Then, as shown in the section of 3 results and discussion, endogenous controls in this study were presented by blank controls, such as no dilution for dilution influence, no addition for the influences of Ethanol, Lactic Acid and NZVI, etc.

Comment #8. Comparison with literature is part of the discussion. At the current status, critical discussion of obtained results and their comparison with available literature is still missing.

Reply: According to this suggestion, the best values obtained in this study were compared with the results of relevant literatures (supplemented in section 3.4). Firstly, the obtained caproic acid concentration should belong to one of the highest concentrations reported in relevant literatures. Then, the yield reached 69.50% in this study was also higher than the results of most previous studies.

Please consider the revised manuscript for publication in Fermentation. If you have any suggestion, please let me know.Best Regards,Yours sincerely,Hongbo Liu

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for resposes and changes according with my comments. I do not have any additional comments. I recomment to accept this version of the manuscript.

Author Response

Thank you very much for the valuable suggestions and kind recognition of our paper by the reviewer

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed my concerns and requests to improve the manuscript. The manuscript can be accepted for publication in its present form.