Syntrophic Butyrate-Oxidizing Consortium Mitigates Acetate Inhibition through a Shift from Acetoclastic to Hydrogenotrophic Methanogenesis and Alleviates VFA Stress in Thermophilic Anaerobic Digestion

Round 1

Reviewer 1 Report

Shift from acetoclastic methanogenesis to syntrophic acetate oxidation in the process of anaerobic digestion at elevated butyrate concentrations

This study focused on the AD process under high organic load condition, achieved the elevation of VFA concentration and the stabilization of the methanogenic community, meet the need of high-efficient and high-capability waste decomposition. Via the temperature strategy, butyrate concentration and methanogenic available intermediates had increased. Methanogenic pathway and community are verified evolutionarily adaptation. This study is comprehensive in terms of content and microbial analysis, but the logical intention and language expression are not up to the level of publication. The detailed comments are given as follows:

Abstract: Inadequate overview, logical confusing and unspecific key results

1. The significance of the study, key issue, research objectives, solutions and statement of conclusions need to be concisely highlighted in the abstract.

2. The content of abstract was not clear, lack of quantitative description.

3. Why the shift of methanogenesis occurred at high VFA concentration? How to intervene artificially to regulate this adaptive strategy

4. Lack of novelty statement of this research

Introduction: Logical intention, the significance of research and innovation need to revised

5.Why you choose butyrate as methanogenic intermediates? As description, propionate was overloaded which suppressed the methanogensis. In some cases, the butyrate concentration is higher than propionate. However, this case based on you set, without representative. What if the concentration of butyric acid and propionic acid is the same? The formulation now chosen to study methane production from butyric acid is not convincing enough.

6. Lack of description of research progress, which does not reflect the innovation of this research work. In fact, methane production based on butyric acid-based AD has been proposed

Material and Methods:

7. For systems with less performance testing, pH and temperature are all measured values? A description of the repeatability of the experiments, the method of analysis of the data? Community analysis? 16s RNA? Frequency,volume of each sampling?

Results and Discussion:

8. Please show in Figure 1 the concentrations of all the VFAs tested?

9. Part 3.1 can be discussed separately in terms of VFA products, conversion rates, etc. The current discussion is not focused enough and needs to be condensed to be specific and targeted. It is not recommended to analyse each bottle separately

10. Microbial community analysis etc. can be graphically represented for greater visualization. Compared to section 3.1, 3.2 is too brief for microbiological analysis and the explanatory correlation between effectiveness and microorganisms is poor.

11. What is the profile of some VFA product components in Part 3.3? Is it anaerobic digestion with butyric acid as the main component, can be presented graphically

Discussion

14. The rate of degradation of butyrate, `propionate and the methanogenic process can be verified with the aid of thermodynamic interpretations

15. Why the shift of the key pathway of methanogenesis occurred? How to intervene artificially to regulate this adaptive strategy. pH or VFA composition induced this shift?

16 It is recommended that section 4.3 also carries out a microbiological component to test the conjecture.

Conclusion: not concise enough

Author Response

This study focused on the AD process under high organic load condition, achieved the elevation of VFA concentration and the stabilization of the methanogenic community, meet the need of high-efficient and high-capability waste decomposition. Via the temperature strategy, butyrate concentration and methanogenic available intermediates had increased. Methanogenic pathway and community are verified evolutionarily adaptation. This study is comprehensive in terms of content and microbial analysis, but the logical intention and language expression are not up to the level of publication. The detailed comments are given as follows:

Response:  Dear Reviewer, we thank you for the comprehensive and positive review of our manuscript. The comments were very constructive, and we tried to address all of the concerns. Below are the responses point by point.

Abstract: Inadequate overview, logical confusing and unspecific key results

1. The significance of the study, key issue, research objectives, solutions and statement of conclusions need to be concisely highlighted in the abstract.
2. The content of abstract was not clear, lack of quantitative description.
3. Why the shift of methanogenesis occurred at high VFA concentration? How to intervene artificially to regulate this adaptive strategy
4. Lack of novelty statement of this research

Response: The abstract was rewritten, and the title was changed to be more consonant with the results of the work.

Introduction: Logical intention, the significance of research and innovation need to revised

5.Why you choose butyrate as methanogenic intermediates? As description, propionate was overloaded which suppressed the methanogensis. In some cases, the butyrate concentration is higher than propionate. However, this case based on you set, without representative. What if the concentration of butyric acid and propionic acid is the same? The formulation now chosen to study methane production from butyric acid is not convincing enough.

6. Lack of description of research progress, which does not reflect the innovation of this research work. In fact, methane production based on butyric acid-based AD has been proposed

Response: The introduction section has been significantly improved to better show the state of the art and highlight the novelty of this study.

Material and Methods:

7. For systems with less performance testing, pH and temperature are all measured values? A description of the repeatability of the experiments, the method of analysis of the data? Community analysis? 16s RNA? Frequency,volume of each sampling?

Response: Materials and Methods section has been improved by addition of  required information

Results and Discussion:

8. Please show in Figure 1 the concentrations of all the VFAs tested?

Response: Yes, the initial concentrations of butyrate are shown in the caption for Fig.1 (A - 20 mmol/L, B - 50 mmol/L, C - 95 mmol/L, D - 170 mmol/L).

9. Part 3.1 can be discussed separately in terms of VFA products, conversion rates, etc. The current discussion is not focused enough and needs to be condensed to be specific and targeted. It is not recommended to analyse each bottle separately

Response: Section 3.1 is somewhat condensed now. We believe that if VFA production/consumption and methane production are divided into separate subsections, it will be less logical, since each of these processes is interconnected and should be described as a whole for each butyrate concentration experiment.

10. Microbial community analysis etc. can be graphically represented for greater visualization. Compared to section 3.1, 3.2 is too brief for microbiological analysis and the explanatory correlation between effectiveness and microorganisms is poor.

Response: We agree with the reviewer's suggestion and presented microbial diversity in a diagram.

11. What is the profile of some VFA product components in Part 3.3? Is it anaerobic digestion with butyric acid as the main component, can be presented graphically

Response: Unfortunately, we could not collect enough samples to show the VFA dynamics in a graphical form, but some details have been added in section 3.3. In addition, some VFA concentrations were calculated incorrectly.

Discussion

14. The rate of degradation of butyrate, `propionate and the methanogenic process can be verified with the aid of thermodynamic interpretations

Response: The issue of thermodynamics was discussed and equations were added.

15. Why the shift of the key pathway of methanogenesis occurred?

Response: We believe that the shift of the key pathway of methanogenesis was the major mechanism for adaptation of the microbial community to high VFA concentration. Such a shift is probably more important in the case of thermophilic communities, since the SAO process is known to be more efficient under thermophilic conditions, than under mesophilic conditions.

How to intervene artificially to regulate this adaptive strategy. pH or VFA composition induced this shift?

 Response: Elevated acetate concentrations in the medium under thermophilic conditions promoted the shift from acetoclastic to hydrogenotrophic methanogenesis. So, this is the possible way to shift the methanogenesis pathway under thermophilic conditions.

These explanations are given in section 4.2.

16 It is recommended that section 4.3 also carries out a microbiological component to test the conjecture.

Response: Section 4.2 has been strengthened by comparing our results with recent studies. Unfortunately, in this work, we did not analyze the microbial community in bioaugmentation experiment; we will take this into account in future experiments.

Conclusion: not concise enough

Response: The conclusion has been rewritten.

Reviewer 2 Report

Title of the Manuscript: Shift from acetoclastic methanogenesis to syntrophic acetate oxidation in the process of anaerobic digestion at elevated butyrate concentrations

The manuscript is formulated accordingly in accordance with the title given to it. The title of the manuscript is very striking and half of the data presented in the manuscript supports it. But the authors seem to be confused in justifying both the title and manuscript. The aim of the research is fulfilled halfway but the manuscript doesn’t really validate it 100%. The methodology followed for understanding the microbial shifts during the acclimatization of the consortia to various concentrations of VFA’s especially for butyrate and acetate is impressive. A little more of detailed research on the same could be entertained. Also there is a sudden inclusion of the OFMSW in the manuscript without the backdrop anywhere in the MS.

Overall the manuscript lacks scientific depiction of the results. The information regarding the role and importance of various microorganisms is the only information reported in the manuscript and but the interpretation is missing. The structure of the manuscript needs to be changed.

Abstract:The abstract was written well but it doesn’t fully support the manuscript. The abstract is catchy in the reader’s point of view but it is found that the abstract is not representing the work in the manuscript. There is no indication of the use of OFMSW or the parameters like VFA’s of OFMSW affecting the AD process. The whole abstract needs to be modified so that it can justify the data included in the manuscript.

Introduction: The overall introduction broadly gives an idea about the role of VFA’s and their impact on the AD process. Introduction lacks the basic literature that supports the research work presented in the manuscript. There are no inputs regarding the OFMSW used in the experimental studies, existing literature regarding the similar substrates and challenges faced by various researchers during the AD of the OFMSW.

Out of three goals, two goals of the present work mentioned are presented well but the 3^rd goal is not satisfactory. The authors may clearly include information regarding the experiments carried out.

Materials and Methods:

Ø  Physico-Chemical characteristics of both the inoculum and substrate are not mentioned in the whole manuscript.

Ø  The experimental procedure is not clear. Breakdown the acclimatisation strategies to give a clear information how the experiments are carried out.

Ø  Analytical tools used in the experimental studies are not reported.

Ø  Use and details of GC, HPLC etc are not mentioned.

Ø  Phase contrast microscopy is mentioned but the procedure for identification of various bacteria based on the morphology is not reported. It is surprising how the microscopy helped in identifying the desired bacteria in mixed microbial consortia either without any staining or identifying techniques. Literature regarding the similar procedures should be cited for giving clear information about the microscopical techniques in identifying bacteria from a mixed microbial consortium.

Results:

Ø  Section 3.1 and 3.2: Interpretation of results with respect to existing literatureis highly required.

Ø  Section 3.3 is not discussed properly. The data reported in this section is generalised and lacks any scientifically supporting information which justifies the section 3.1 as the experiments are carried out based on the results obtained from it.

Ø  Section 3.3 needs to be rewritten with appropriate supporting literature which supports the impact of butyrate and acetate on the AD process.

Discussion:

Ø  It is suggested that the discussion section is very huge and is irrelevant.

Ø  The some information provided in the discussion can be adjusted in the results section along with the data as it can give strength the results section.

Ø  The information in the discussion section is not relevant unless the same is written in support with the results obtained during the experimentation process.

Conclusions:

Ø  Rewrite the entire section.

Author Response

Title of the Manuscript: Shift from acetoclastic methanogenesis to syntrophic acetate oxidation in the process of anaerobic digestion at elevated butyrate concentrations

The manuscript is formulated accordingly in accordance with the title given to it. The title of the manuscript is very striking and half of the data presented in the manuscript supports it. But the authors seem to be confused in justifying both the title and manuscript. The aim of the research is fulfilled halfway but the manuscript doesn’t really validate it 100%. The methodology followed for understanding the microbial shifts during the acclimatization of the consortia to various concentrations of VFA’s especially for butyrate and acetate is impressive. A little more of detailed research on the same could be entertained. Also there is a sudden inclusion of the OFMSW in the manuscript without the backdrop anywhere in the MS.

Overall the manuscript lacks scientific depiction of the results. The information regarding the role and importance of various microorganisms is the only information reported in the manuscript and but the interpretation is missing. The structure of the manuscript needs to be changed.

Response: Dear Reviewer, we thank you for the comprehensive and positive review of our manuscript. The comments were very constructive and we tried to take into account as many of your comments as possible.Below are the responses point by point.

Abstract:The abstract was written well but it doesn’t fully support the manuscript. The abstract is catchy in the reader’s point of view but it is found that the abstract is not representing the work in the manuscript. There is no indication of the use of OFMSW or the parameters like VFA’s of OFMSW affecting the AD process. The whole abstract needs to be modified so that it can justify the data included in the manuscript.

Response: The abstract was rewritten, and the title was changed to be more consonant with the results of the work.

Introduction: The overall introduction broadly gives an idea about the role of VFA’s and their impact on the AD process. Introduction lacks the basic literature that supports the research work presented in the manuscript. There are no inputs regarding the OFMSW used in the experimental studies, existing literature regarding the similar substrates and challenges faced by various researchers during the AD of the OFMSW.

Response: Actually OFMSW was not the target substrate. It was simply taken as an example of a readily biodegradable substrate, the use of which can lead to destabilization of AD process. Moreover, the introduction section has been significantly improved to better show the state of the art and highlight the novelty of this study.

Out of three goals, two goals of the present work mentioned are presented well but the 3^rd goal is not satisfactory. The authors may clearly include information regarding the experiments carried out.

Response: the goals have been polished

Materials and Methods:

Ø  Physico-Chemical characteristics of both the inoculum and substrate are not mentioned in the whole manuscript.

Response: Some details on characteristics are now given.

Ø  The experimental procedure is not clear. Breakdown the acclimatisation strategies to give a clear information how the experiments are carried out.

Response: The experimental procedure has been improved

Ø  Analytical tools used in the experimental studies are not reported.

Response: Corrected

Ø  Use and details of GC, HPLC etc are not mentioned.

Response: Some details are now given

Ø  Phase contrast microscopy is mentioned but the procedure for identification of various bacteria based on the morphology is not reported. It is surprising how the microscopy helped in identifying the desired bacteria in mixed microbial consortia either without any staining or identifying techniques. Literature regarding the similar procedures should be cited for giving clear information about the microscopical techniques in identifying bacteria from a mixed microbial consortium.

Response: Despite the fact that Methanothrix and Methanosarcina cells are well distinguished by phase contrast microscopy, we agree with the reviewer that this method does not allow them to be identified strictly enough in mixed cultures with bacteria. Microscopy was not used for identification. The identification of microorganisms was performed by analysis of 16S rRNA gene fragments according to the method described in section 2.5. In order not to confuse the reader, the mention of microscopy was removed from the Methods and Results.

Results:

Ø  Section 3.1 and 3.2: Interpretation of results with respect to existing literatureis highly required.

Response: The “Results and Discussion” section of this manuscript has been divided into separate “Results” and “Discussion” sections as required by the MDPI template. That is why we moved the entire discussion and comparison of our results with the literature to the “Discussion” section.

Ø  Section 3.3 is not discussed properly. The data reported in this section is generalised and lacks any scientifically supporting information which justifies the section 3.1 as the experiments are carried out based on the results obtained from it.

Response: Section 3.3 has been improved

Ø  Section 3.3 needs to be rewritten with appropriate supporting literature which supports the impact of butyrate and acetate on the AD process.

Response: The “Results and Discussion” section of this manuscript has been divided into separate “Results” and “Discussion” sections as required by the MDPI template. That is why we moved the entire discussion and comparison of our results with the literature to the “Discussion” section.

Discussion:

Ø  It is suggested that the discussion section is very huge and is irrelevant.

Ø  The some information provided in the discussion can be adjusted in the results section along with the data as it can give strength the results section.

Ø  The information in the discussion section is not relevant unless the same is written in support with the results obtained during the experimentation process.

Response: The “Results and Discussion” section of this manuscript has been divided into separate “Results” and “Discussion” sections as required by the MDPI template. That is why we moved the entire discussion and comparison of our results with the literature to the “Discussion” section.

What has been done to improve the Discussion section:

• The issue of thermodynamics was discussed and equations were added
• The Discussion section has been significantly improved to compare our results with the recent research.

Conclusions:

Ø  Rewrite the entire section.

Response: The conclusion has been rewritten.

Round 2

Reviewer 1 Report

Thank you for the reply.

The authors had revised and added content to the article to make it more readable and persuasive. Further revisions could be demanded to highlight the purpose, significance and conclusions of the study more. The following suggestions are available for further optimization.

1) The title, keywords, abstract and introduction need to be refined and reduced in length. The existing content is sufficient and rich, but the expressions please be further selected, refined and focused.

2) The discussion part which is designed to be irrelevant to the conclusion or only to introduce the method or research progress should be integrated into the introduction, and the discussion part should be direct and focused.

(3) The format descriptions of the charts and graphs, etc. please be unified and corrected according to the standard

Author Response

Thank you for the reply.

The authors had revised and added content to the article to make it more readable and persuasive. Further revisions could be demanded to highlight the purpose, significance and conclusions of the study more. The following suggestions are available for further optimization.

Response: Many thanks to the reviewer! We have tried to take into account all suggestions.

1) The title, keywords, abstract and introduction need to be refined and reduced in length. The existing content is sufficient and rich, but the expressions please be further selected, refined and focused.

Response: Title, keywords, abstract, and introduction have been somewhat condensed.

2) The discussion part which is designed to be irrelevant to the conclusion or only to introduce the method or research progress should be integrated into the introduction, and the discussion part should be direct and focused. Some of the

Response: We did our best to address the reviewers’ concerns. As a part of suggestions raised by another reviewer, the issue of thermodynamics was discussed and equations were added. Also, the Discussion section has been expanded to compare results of the work with the recent research.

Indeed, some parts of Discussion can be also be moved to introduction to better show the state of the art. However, this will inevitably lengthen the introduction, and we believe that the literature used and cited is important and should not be ignored and removed. The "Results and Discussion" section of this manuscript has been deliberately and clearly divided into separate "Results" and "Discussion" sections in order to comply with the requirements of the MDPI template.  That is why we moved the entire discussion and comparison of our results with the literature to the “Discussion” section. We hope that the understanding of the results of the work and its discussion did not suffer much from this. In the Conclusion, we briefly summarized the results of the study, noted the fundamental significance and practical importance of our findings. 

 (3) The format descriptions of the charts and graphs, etc. please be unified and corrected according to the standard

Response: Some changes have been made to make the perception of figures and tables more understandable. The numbering of the figures was also checked.