Enhanced Anaerobic Digestion Performance Through Acyl Homoserine Lactone-Mediated Quorum Sensing and Supplemental Voltage Regulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is well-structured and engaging, requiring only minor revisions. I recommend refining the methodology section by adding more details and including relevant citations for clarity in all parts if possible.

Introduction part>

1. There are so many anaerobic digestion (AD) in the text; one time is ok, then it is ok to use AD. Check that. 
2. I want to be conscious of the application of this novel approach, which could be here or at the end of the manuscript (Briefly review at the end of the discussion part). I invite you to add potential strategies for scaling reactors using signaling molecules.
3. Define low-quality biomass.

Methods part>

1. I suggest adding citations in lines 122 and 124.
2. Add the moisture% (humedity%) in the characterization
3. Add the operation mode of the reactor and the time of sample collection (gas/liquid).
4. Add the wires (SS, titanium, ???)
5. Section> 2.3.2. Electrochemical and morphological characteristics of electrodes: Add the time of the operation you did CV. 
6. Cite line 138.
7. In line 140, Data analysis was carried out following the procedures de-140 described in our previous study [18]. Give a general context of the analysis; please mention the analysis; then, you can end with your line.

Discussion part>

1. Section 3.5.1 (Electrode Biofilm) could strengthen the text by incorporating additional studies to support the claim. For instance, in lines 347–348, the statement, "This indicates that the cathode environment is more conducive to the growth and metabolism of methanogenic archaea [43]," would benefit from further evidence. For example, these findings align with the observed higher relative abundances of methanogenic genera on the cathode in a single-chamber system where different ranges of applied voltage have been used (0.3-1.5?)/ potentials, combined or not with other electron transfer inducers?, particularly the enhanced growth of hydrogenotrophic methanogens such as Methanobacterium and Methanobrevibacter (More references, 43 ok, 2or 3, more maybe). This further supports the conclusion that the cathode environment is more suitable for their growth and metabolism. This is just an example for the author, but I would like to suggest supporting a little more in this paragraph. Some suggestions, please Check them out >10.3390/ijms18040874 , https://www.nature.com/articles/s41598-020-76229-5 ,  https://doi.org/10.1016/j.biortech.2019.122624

Comments on the Quality of English Language

I suggest to review the grammar. 

Author Response

Comments and Suggestions for Authors

The paper is well-structured and engaging, requiring only minor revisions. I recommend refining the methodology section by adding more details and including relevant citations for clarity in all parts if possible.

Response: We sincerely thank you for taking the time to review our manuscript and for your valuable feedback. We have carefully considered all of your insightful comments and suggestions, and have made revisions accordingly. The changes in the manuscript are highlighted in red. Below, we provide detailed responses to both your major and specific comments.

Introduction part>

1. There are so many anaerobic digestion (AD) in the text; one time is ok, then it is ok to use AD. Check that. 

Response: Thank you very much for your careful review. In the manuscript, we have checked and made revisions to use the abbreviation "AD" for anaerobic digestion from the second occurrence onwards, in order to improve the manuscript's conciseness.

 

2. I want to be conscious of the application of this novel approach, which could be here or at the end of the manuscript (Briefly review at the end of the discussion part). I invite you to add potential strategies for scaling reactors using signaling molecules.

Response: Thank you for your suggestion regarding the potential applications and signaling molecules. To enhance the practical application of this strategy, cost-effective alternatives are essential. Wastewater sludge is a low-cost and abundant source of AHLs, containing various signaling molecules (such as C7-HSL, 3OC6-HSL, and C4-HSL) that can enhance quorum sensing in mixed culture systems. The sentence has been revised according to your suggestions (see Lines 476-482).

3. Define low-quality biomass.

Response: Thank you very much for your constructive suggestions. The phrase "low-quality biomass" refers to biomass materials that have limited economic or practical value in their raw form and often require specific processes or treatments to be used effectively. In this context, straw and livestock manure are considered low-quality biomass because, without proper treatment or conversion, they cannot be efficiently utilized as energy sources or in other applications, and their improper disposal can contribute to environmental pollution. The key issue lies in their inefficient use and potential harm to the environment when not disposed of properly. The sentence has been revised according to your suggestions (see Lines 32-35).

Methods part>

1. I suggest adding citations in lines 122 and 124.

Response: Thank you for your valuable suggestion. We have added citations on lines 122 to 124 (see Line 144).

 

2. Add the moisture% (humidity %) in the characterization

Response: Thank you very much for your suggestion regarding the addition of the moisture%（humidity %）. In this study, we used dried cow manure and straw powder, the inoculum with an inoculum dose of 30% (w/w), the physicochemical properties of the substrates and inoculum see Table 2 (see Table 2).

 

3. Add the operation mode of the reactor and the time of sample collection (gas/liquid).

Response: We sincerely appreciate your suggestion. In this experiment, a batch-type feeding system was used with an AD bottle (working volume: 400 mL) as the reactor. Every day, 1 mL of gas was sampled to determine the gas content. For experimental groups with applied voltage, potential and voltage were measured at fixed times daily. Every 5 days, a 10 mL sample of the digestate was taken to measure pH, COD, NH₄⁺-N, VFAs concentrations, and coenzyme F₄₂₀ activity, among other chemical properties. Following your advice, we have added the operating mode of the reactor and the time of sample collection to provide better insight into the reactor's operation and the related sample information (see Lines 105-111).

 

4. Add the wires (SS, titanium, ???)

Response: Thank you very much for your suggestion regarding the electrode wire material. We have specified in the manuscript that the electrode wires are made of titanium (see Line 117).

 

5. Section> 2.3.2. Electrochemical and morphological characteristics of electrodes: Add the time of the operation you did CV. 

Response: Thank you very much for your suggestion. The anode electrode was subjected to cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) scans using an electrochemical workstation (CHI660E, Shanghai, China) at 0 d, 5 d, and 30 d during the operation of the AD system. We have added the measurement times for the electrochemical parameters in section 2.3.2, making the article more professional and rigorous (see Lines 105-111).

 

6. Cite line 138.

Response: Thank you very much for your suggestion. We have added the reference on line 161(see Line 161).

 

7. In line 140, Data analysis was carried out following the procedures de-140 described in our previous study [18]. Give a general context of the analysis; please mention the analysis; then, you can end with your line.

Response: Please allow us to express our sincerest gratitude. The raw data were analyzed using QIIME software and the UPARSE pipeline. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE. The taxonomy of each 16S rRNA gene sequence was analyzed using the RDP Classifier algorithm. The relevant information has been supplemented in line 161-164 (see Lines 161-164).

Discussion part>

1. Section 3.5.1 (Electrode Biofilm) could strengthen the text by incorporating additional studies to support the claim. For instance, in lines 347–348, the statement, "This indicates that the cathode environment is more conducive to the growth and metabolism of methanogenic archaea [43]," would benefit from further evidence. For example, these findings align with the observed higher relative abundances of methanogenic genera on the cathode in a single-chamber system where different ranges of applied voltage have been used (0.3-1.5?)/ potentials, combined or not with other electron transfer inducers?, particularly the enhanced growth of hydrogenotrophic methanogens such as Methanobacteriumand Methanobrevibacter (More references, 43 ok, 2or 3, more maybe). This further supports the conclusion that the cathode environment is more suitable for their growth and metabolism. This is just an example for the author, but I would like to suggest supporting a little more in this paragraph. Some suggestions, please Check them out >10.3390/ijms18040874, https://www.nature.com/articles/s41598-020-76229-5, https://doi.org/10.1016/j.biortech.2019.122624.

Response: Thank you very much for your detailed and professional suggestions. We fully agree with your advice and examples, and have incorporated them into the manuscript. Additionally, following your recommendation, we have added more references to further support and enrich the content of the manuscript (see Lines 366-369).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors The authors set the stage to investigate the effects of combined regulation of exogenous N-Hexanoyl-L-Homoserine lactone (C6-HSL) signaling molecule in concert with an applied external voltage of 0.8 V on methane production and microbial community succession in an anaerobic digestion (AD) system using corn straw and cow manure as substrates. The results revealed that the combined regulation significantly increased methane production by (29.74 %) and substrate rate (by 74.73 %), while preventing acid inhibition and ammonia nitrogen inhibition. Mechanism analysis showed that the external voltage enhanced the system's electrocatalytic activity, while the C6-HSL signaling molecule further facilitated the electron transfer efficiency of the biofilm on the electrode. This combination also enriched hydrogenotrophic methanogens establishing a stable metabolic cooperative network on both the electrode and in the digestate, optimizing the hydrogenotrophic methanogenesis pathway, and enhanced the synergistic effects among microbial communities and system robustness. This research study is interesting since it uncovers the synergistic enhancement mechanism of C6-HSL and external voltage, providing new methane generation pathways for efficient conversion of low-quality biomass resources. However, there are some issues that have to be addressed.   1) In Introduction the authors should mention additional studies based on microbial electrolysis, exogenous AHLs and on the coupled phenomenon. Table 2 could be moved on introduction as a justification of their study.   2) In introduction, the authors should mention the difference between this study and their previous research work (reference 13).    

Author Response

Comments and Suggestions for Authors：

The authors set the stage to investigate the effects of combined regulation of exogenous N-Hexanoyl-L-Homoserine lactone (C6-HSL) signaling molecule in concert with an applied external voltage of 0.8 V on methane production and microbial community succession in an anaerobic digestion (AD) system using corn straw and cow manure as substrates. The results revealed that the combined regulation significantly increased methane production by (29.74 %) and substrate rate (by 74.73 %), while preventing acid inhibition and ammonia nitrogen inhibition. Mechanism analysis showed that the external voltage enhanced the system's electrocatalytic activity, while the C6-HSL signaling molecule further facilitated the electron transfer efficiency of the biofilm on the electrode. This combination also enriched hydrogenotrophic methanogens establishing a stable metabolic cooperative network on both the electrode and in the digestate, optimizing the hydrogenotrophic methanogenesis pathway, and enhanced the synergistic effects among microbial communities and system robustness. This research study is interesting since it uncovers the synergistic enhancement mechanism of C6-HSL and external voltage, providing new methane generation pathways for efficient conversion of low-quality biomass resources.

However, there are some issues that have to be addressed. 

Response: We sincerely appreciate you for your time in reviewing the manuscript and giving your valuable comments. We have thoroughly considered your precious comments and suggestions, and carefully addressed every one of them as best as we can. Revisions in the manuscript were highlighted in red. Here, we provide a point-to-point response to your major and specific comments below.

1) In Introduction the authors should mention additional studies based on microbial electrolysis, exogenous AHLs and on the coupled phenomenon. Table 2 could be moved on introduction as a justification of their study.  

Response: We feel great thanks for your valuable suggestions. Following your helpful suggestions, We have added additional relevant studies on microbial electrolysis, exogenous AHLs, and the coupled phenomenon. Additionally, we agree that moving Table 2 to the introduction to better support the content of the manuscript is a valuable suggestion, and we have made the corresponding revisions to improve the paper (see Lines 72-78).

2) In introduction, the authors should mention the difference between this study and their previous research work (reference 13).    

Response: We sincerely appreciate your valuable suggestions, which have helped highlight the innovation of our research. In the introduction, we have clarified the distinctions between our study and previous work. Additionally, we have made appropriate revisions to the reference list (see Lines 71-72).

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors studied the enhancement of anaerobic co-digestion performance of corn straw and cattle manure through Acyl Homoserine Lactone-Mediated Quorum Sensing and supplemental voltage regulation.

The experimental design was correctly performed and necessary parameters were measured. The results were analysed for physicochemical and biological parameters following standard methods. Microbial communities were analyzed to highlight variations in microbial community population and diversity. Microbial community analysis was sufficient for this type of laboratory work. The results obtained were well discussed and compared with previous studies. The conclusions matched the data presentation and discussion of results.

Comments to authors:

• It would have been more advantageous if the authors had conducted the experiment in a continuous anaerobic co-digestion process for at least three HRTs of 30 days to clearly visualizse the key variations between the experimental conditions investigated. Batch tests are less sensitive to variations in digestion processes and it is not possible to detect key variations.

• The results are well presented but need more attractive figures to show the results effectively.

• Better screening of the microbial analysis is required to clearly show microbial diversity between the experimental conditions.

   

ض

 

Author Response

Comments and Suggestions for Authors

The authors studied the enhancement of anaerobic co-digestion performance of corn straw and cattle manure through Acyl Homoserine Lactone-Mediated Quorum Sensing and supplemental voltage regulation.

The experimental design was correctly performed and necessary parameters were measured. The results were analysed for physicochemical and biological parameters following standard methods. Microbial communities were analyzed to highlight variations in microbial community population and diversity. Microbial community analysis was sufficient for this type of laboratory work. The results obtained were well discussed and compared with previous studies. The conclusions matched the data presentation and discussion of results.

Response: We sincerely thank you for taking the time to review our manuscript and provide your valuable feedback. We have carefully considered all of your insightful comments and suggestions, and made every effort to address each one. The revisions in the manuscript are highlighted in red. Below, we provide a detailed, point-by-point response to your major and specific comments.

Comments to authors:

• It would have been more advantageous if the authors had conducted the experiment in a continuous anaerobic co-digestion process for at least three HRTs of 30 days to clearly visualize the key variations between the experimental conditions investigated. Batch tests are less sensitive to variations in digestion processes and it is not possible to detect key variations.

Response: We greatly appreciate your professional comments on our manuscript. Considering that our study is a preliminary investigation into the coupling effect of microbial electrolysis and exogenous signaling molecule AHLs, we chose to use batch anaerobic fermentation rather than continuous fermentation. Due to the extended monitoring period and the numerous parameters involved, the results provide insight into the key changes occurring during the fermentation process. In the future, we will focus on further exploring the effects in continuous anaerobic fermentation.

 

• The results are well presented but need more attractive figures to show the results effectively.

Response: We sincerely appreciate your valuable suggestions. We have more attractive figures (Fig.1d) to better present the results (see Lines 201-204).

 

• Better screening of the microbial analysis is required to clearly show microbial diversity between the experimental conditions.

Response: Thank you very much for your valuable suggestions. We have added relevant data to strengthen the microbial analysis, providing a clearer presentation of the microbial diversity between the experimental conditions (see Lines 412-417).

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

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