Dual Strategies for Enriching Electroactive Microorganisms from Anaerobic Digestate: Carbon-Assisted Acclimation and Direct In Situ Enrichment in a Liter-Scale MFC

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a good dataset, but the main data-driven insight is not well-positioned in my analysis. Here's some comment to make it publishable:

1. Results clearly show that raw wastewater solids achieve good COD removal but produce negligible current, whereas carbon-acclimated digestate generates high current (~8.5-10 mA) with comparable COD removal (~67-73%). This directly demonstrates that biodegradation does not imply electroactivity. So I believe this should be the central conclusion, rather than presenting the work as a general MFC performance study.

2. The CH5 and CH7 reactors behave very differently, with power output (333.5 vs 246.8 μW) and internal resistance evolution (76 ohms vs fluctuation up to 411 ohms), and both experienced leakage/operational disturbances. These cannot be treated as true replicates, so claims regarding reproducibility or scale-up reliability are not well supported. This one is my critical concern.

3. Although COD removal exceeds 80% for much of the operation, there are clear instability periods (down to around 49–71% and 63–78%) that coincide with operational issues. This indicates that reactor stability is a major limitation that is not quantitatively addressed in this paper; without this, the contribution becomes incremental.

4. Carbon-assisted acclimation leads to lower residual acetate (~0.098 vs ~0.222 mM) and significantly higher current, supporting enhanced microbial activity. However, the claim of improved electron transfer is not directly demonstrated, as electrochemical analysis remains qualitative (no extraction of onset potentials or kinetic metrics).

5. Power is reported only in μW without normalization, limiting comparison with the literature. In addition, COD loading units appear inconsistent, affecting technical credibility.

6. The microbial community analysis reveals differences, but these are not linked to electrochemical performance, limiting its mechanistic value.

7. Overall, the data are strong, but the manuscript requires clearer data-driven framing, better interpretation, and improved quantitative analysis to support its conclusions.

Author Response

Comments 1: Results clearly show that raw wastewater solids achieve good COD removal but produce negligible current, whereas carbon-acclimated digestate generates high current (~8.5-10 mA) with comparable COD removal (~67-73%). This directly demonstrates that biodegradation does not imply electroactivity. So I believe this should be the central conclusion, rather than presenting the work as a general MFC performance study.

Response 1: Thank you for highlighting the most important data-driven conclusion of this study. We fully agree with this comment. Based on our results, although the raw wastewater solids were able to degrade organic matter and achieve a certain degree of COD removal, they did not establish stable electricity generation in the electrochemical reactors. In contrast, anaerobic digestate acclimated with carbon powder produced clear and reproducible high current under comparable COD removal conditions. These results clearly demonstrate that biodegradation does not necessarily imply electroactivity and also indicate that both inoculum source and acclimation strategy play decisive roles in electricity generation performance. Therefore, we have revised the Abstract and Discussion to refocus the manuscript from the broader topic of “liter-scale MFC operational performance” to the following key points: (1) anaerobic digestate has greater potential than raw wastewater solids for enrichment of electroactive microorganisms; (2) carbon-powder-assisted acclimation significantly improves subsequent electrochemical startup and electricity generation performance; and (3) COD removal alone is not sufficient evidence for the establishment of electroactivity.

Comments 2: The CH5 and CH7 reactors behave very differently, with power output (333.5 vs 246.8 μW) and internal resistance evolution (76 ohms vs fluctuation up to 411 ohms), and both experienced leakage/operational disturbances. These cannot be treated as true replicates, so claims regarding reproducibility or scale-up reliability are not well supported. This one is my critical concern.

Response 2: Thank you for raising this critical issue. We accept this criticism and agree that although CH5 and CH7 were originally designed as parallel reactors, they should not be regarded as true replicates in the strict sense because of reactor leakage, maintenance events, differences in startup conditions, and the marked variation in internal resistance. Therefore, we have revised the wording in the manuscript to avoid overinterpreting these two reactor results as evidence of reproducibility or as sufficient proof of scale-up reliability.

In the revised manuscript, we have made the following changes. First, we replaced the term “replicate reactors” with more appropriate descriptions such as “parallel liter-scale reactors” or “two independently operated reactors.” Second, we deleted or softened statements that overextended the implications regarding scale-up reliability and reproducibility. Third, in both the Results and Discussion sections, we now state more clearly that the differences between CH5 and CH7 may have been related to leakage, local structural differences, biofilm clogging, and insufficient pH control. Finally, we have repositioned this part of the study as a case observation of engineering variability and stability challenges that may arise during scale-up operation, rather than as a strict replicate validation.

We believe these revisions make the manuscript more rigorous in both the strength of the evidence and the wording of the conclusions. The differences between CH5 and CH7 in power output, internal resistance evolution, and operational disturbances are clearly shown in the current dataset, and we have retained these data while interpreting them more conservatively.

Comment 3: Although COD removal exceeds 80% for much of the operation, there are clear instability periods (down to around 49–71% and 63–78%) that coincide with operational issues. This indicates that reactor stability is a major limitation that is not quantitatively addressed in this paper; without this, the contribution becomes incremental.

Response 3: Thank you for reminding us to address the issue of system stability more directly. We agree that reactor stability is one of the important limitations of this study. Although CH5 and CH7 achieved high COD removal during much of the operation, the data did show clear fluctuation periods associated with operational disturbances. For example, during the second continuous-flow phase, COD removal in CH5 decreased to 71%, 62%, 49%, and 70%, while CH7 also showed lower values of 63%, 78%, and 73% on several days. These results indicate that system stability was still affected by reactor integrity and operating conditions. In response to this comment, we have revised both the Results and Discussion sections. In the Results, we now explicitly describe the periods of reduced COD removal and relate them to leakage, maintenance, and operational transition events. In the Discussion, we further acknowledge reactor stability as an important limitation of the present study and clarify that the current results demonstrate the feasibility of electricity generation and wastewater treatment at the liter scale, but do not yet provide sufficient evidence to support claims of stable engineering-scale operation. Further validation using longer-term operation and more structurally robust reactor configurations will be required.

 

Comments 4: Carbon-assisted acclimation leads to lower residual acetate (~0.098 vs ~0.222 mM) and significantly higher current, supporting enhanced microbial activity. However, the claim of improved electron transfer is not directly demonstrated, as electrochemical analysis remains qualitative (no extraction of onset potentials or kinetic metrics).

Response 4: Thank you for this precise and insightful comment. We agree that, based on the current data, it is reasonable to state that carbon-powder-assisted acclimation was associated with faster startup, higher current generation, stronger catalytic current responses in CV, and lower residual acetate. However, if we further claim that these results directly demonstrate improved electron transfer, the current evidence remains indirect. This is because the present study did not further extract onset potential, Tafel slope, charge transfer resistance, or other more explicit kinetic parameters.

Therefore, we have made the following revisions in the manuscript:

We have explicitly clarified in the Discussion that the electrochemical analyses in this study were mainly used for qualitative comparison rather than for a complete kinetic interpretation.

We have revised statements such as “carbon powder enhanced electron transfer” to a more conservative interpretation, namely that carbon powder may have facilitated the enrichment or activation of electrochemically active microbial communities, while the underlying mechanism still requires further verification using more comprehensive electrochemical approaches.

This revision is also consistent with the cautious tone already adopted in the current manuscript. For example, we have stated that no direct SEM evidence of nanowires or other extracellular conductive structures was obtained in this study, and therefore we have avoided making overly strong mechanistic claims.

Comments 5: Power is reported only in μW without normalization, limiting comparison with the literature. In addition, COD loading units appear inconsistent, affecting technical credibility.

Response 5: Thank you for pointing out these two important issues that affect the technical credibility of the manuscript.

First, regarding the presentation of power output, we agree that reporting the maximum power only in μW is not sufficient for direct comparison with the literature. Therefore, in the revised manuscript, we have added normalized power metrics and expressed the data as A m⁻³ and W m⁻³ (as shown in Figs 5-6, 9-11). These normalized values are now presented consistently in both the figures and the main text to improve comparability with previous studies.

Second, regarding the inconsistency in COD loading units, we thank the reviewer for this careful observation. We have re-checked all related units and equation expressions throughout the manuscript and revised them into a uniform format to avoid confusion caused by mixing kg-COD m⁻³ d⁻¹ with other expressions. Where some passages were previously written incorrectly as kg-COD d⁻¹ or lacked a clear volumetric basis, these have also been corrected.

We consider these to be very important technical revisions, and we sincerely thank the reviewer for helping us identify them.

Comments 6: The microbial community analysis reveals differences, but these are not linked to electrochemical performance, limiting its mechanistic value.

Response 6: Thank you for this valuable suggestion. We agree that, at present, the microbial community analysis mainly shows that different inoculum sources and acclimation conditions led to differences in community composition, whereas its relationship with specific electrochemical performance remains largely descriptive and is not yet sufficient to establish a clear mechanistic link. In the current manuscript, we can only state that the community composition of the raw wastewater solids was clearly different from that of the reactor samples and that its electrochemical performance was poorer; likewise, the digestate sludge acclimated with and without carbon powder showed different community compositions, and the carbon-powder-amended group exhibited better electrochemical performance. However, no quantitative linkage has yet been established between specific genera and particular electrochemical metrics.

Therefore, in the revised manuscript, we have repositioned the microbial community analysis as supportive evidence rather than direct mechanistic proof. We have also added a statement in the Discussion noting that further studies combining deeper functional analyses, quantitative electrochemical parameters, and potentially functional gene information will be necessary to more clearly link community structure with electricity-generating performance.

We believe that, with this revision, the strength of the evidence in this section is now more consistent with the conclusions it can reasonably support.

Comments 7: Overall, the data are strong, but the manuscript requires clearer data-driven framing, better interpretation, and improved quantitative analysis to support its conclusions.

Response 7: Thank you for your overall positive evaluation and constructive suggestions. We fully agree that the manuscript required further improvement in both its framing and quantitative presentation. In response to the reviewer’s comments, we have made the following overall revisions: (1) We refocused the main theme of the manuscript by shifting the core conclusion from a general description of MFC performance to the electroactive enrichment potential of anaerobic digestate and the promoting effect of carbon-powder-assisted acclimation on electrochemical startup; (2) we strengthened the interpretation of the results by more clearly distinguishing between organic matter removal and the establishment of electroactivity; (3) we revised the presentation of CH5 and CH7 so that they are no longer overtreated as strict replicates, but rather presented as the operating results of two independently operated liter-scale reactors; (4) we improved the quantitative information and technical consistency by adding normalized power metrics, standardizing COD loading units, and carefully checking the related values and figure descriptions throughout the manuscript; (5) we adopted a more conservative interpretation of the mechanisms related to carbon-powder-enhanced electron transfer and microbial community shifts, so that the wording is better aligned with the actual strength of the current evidence.

We sincerely thank the reviewer for the careful evaluation and valuable suggestions. These comments have substantially improved the rigor and readability of the manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Comment on the paper: “Dual Strategies for Enriching Electroactive Microorganisms from Anaerobic Digestate: Carbon-Assisted Acclimation and Direct In Situ Enrichment in a Liter-Scale MFC” by Shiue-Lin Li et al.

 

 

General comment:

The manuscript deals with investigations of electricity production from livestock wastewater using liter-scale microbial fuel cell system. The effect of inoculum was considered.

The manuscript is suitable to be published in this journal; however, some points should be addressed before publication.

Some minor language mistakes are present that should be corrected.

 

 

1. Introduction

Please highlight the innovation of your work better.

2. Materials and Methods

Please clarify if investigations were carried out in duplicate/triplicate etc.

Please clarify if long-run tests were carried out.

Please clarify if the deactivation/poisoning of microorganisms was investigated.

3. Results and discussion

Please include statistical analysis.

Please clarify deactivation/poisoning of microorganisms.

4. Discussion

Please improve the comparison with the scientific literature.

Author Response

Comments 1: The manuscript deals with investigations of electricity production from livestock wastewater using liter-scale microbial fuel cell system. The effect of inoculum was considered.

The manuscript is suitable to be published in this journal; however, some points should be addressed before publication.

Some minor language mistakes are present that should be corrected.

 Response 1: Thank you for your positive evaluation of the overall value of this study. We are pleased that you consider the manuscript suitable for publication, and we also appreciate your comment that several aspects of the language and content presentation still required improvement. In response to your suggestions, we have carefully reviewed the English throughout the manuscript and revised expressions that were unnatural, repetitive, or insufficiently precise. We have also strengthened the logical flow and technical description across the different sections to improve the overall clarity and rigor of the manuscript.

 

Comments 2: Introduction

Please highlight the innovation of your work better.

Response 2: Thank you for this valuable suggestion. We agree that the innovation of the original manuscript was not highlighted clearly enough in the Introduction. In response, we have revised the final part of the Introduction to more clearly position the novelty of this study. Specifically, we now emphasize that this work not only investigates electricity generation from livestock wastewater in a liter-scale MFC system, but also compares the electroactive enrichment potential of different inoculum sources, including raw livestock wastewater solids and anaerobic digestate. In addition, we clarify that carbon-powder-assisted acclimation was evaluated as a strategy to improve electrochemical startup and electricity generation performance. By integrating small-scale electrochemical reactor tests with liter-scale MFC operation, the revised Introduction now more clearly highlights the novelty and contribution of this study in the context of the existing literature.

Comments 3: Materials and Methods

Please clarify if investigations were carried out in duplicate/triplicate etc.

Response 3: Thank you for this reminder. We have added clarification in the Materials and Methods section regarding the replication of the experiments. Specifically, the liter-scale MFC operation was conducted using two independently operated reactors (CH5 and CH7) in parallel, although in the revised manuscript we have avoided describing them too strongly as strict replicate reactors. The inoculation tests in the electrochemical reactors were also carried out in parallel using two reactors (e.g., CH1 and CH8). In addition, for water quality data with repeated measurements, this has now been indicated in the tables or text where appropriate, and mean values with standard deviations have been provided when applicable. We have incorporated this information into the Methods section to make the experimental design clearer.Comments 4: Please clarify if long-run tests were carried out.

Response 4: We have clarified the durations of the long-run experiments in both the Materials and Methods and Results sections. Specifically, the liter-scale MFCs were operated for 78 days (CH5) and 71 days (CH7), the acclimation experiments for raw wastewater solids and digestate solids were conducted for up to 82 days, and the post-inoculation electrochemical reactor tests were operated for several days to approximately 11 days depending on the experimental condition.

Comments 5: Please clarify if the deactivation/poisoning of microorganisms was investigated.

Response 5: Thank you for your comments. In the revised manuscript, we have clarified in the Methods section that direct analyses of microbial deactivation or poisoning were not included in the experimental design. We have also added a statement in the Discussion to indicate that the temporary declines in performance observed in this study cannot be conclusively attributed to deactivation or poisoning, and are therefore interpreted more conservatively as possible consequences of operational disturbance or environmental variation.

Comments 6: Results and discussion

Please include statistical analysis.

Response 6: Thank you for this reminder. We agree that statistical analysis helps improve the rigor of data presentation, and we have therefore added appropriate statistical information in the revised manuscript. Specifically, for water quality data with repeated measurements, we now report the results as mean values with standard deviations. In addition, the current and power output data are now presented in normalized form based on the anode chamber volume. We have made every effort to provide appropriate statistical and quantitative presentation within the limits of the available dataset, while avoiding interpretations that go beyond what the data can reasonably support.

Comments 7: Please clarify deactivation/poisoning of microorganisms.

Response 7: Thank you for your comments. In the revised manuscript, we have clarified in the Methods section that direct analyses of microbial deactivation or poisoning were not included in the experimental design. We have also added a statement in the Discussion to indicate that the temporary declines in performance observed in this study cannot be conclusively attributed to deactivation or poisoning, and are therefore interpreted more conservatively as possible consequences of operational disturbance or environmental variation.

Comments 8: Discussion

Please improve the comparison with the scientific literature.

Response 8: Thank you for this valuable suggestion. We agree that the comparison with the existing scientific literature in the original Discussion could be further strengthened. Therefore, we have revised the relevant paragraphs and incorporated additional discussion of direct interspecies electron transfer (DIET) in the revised manuscript.

In particular, we now discuss our findings in relation to previous literature suggesting that conductive materials may facilitate DIET-related interactions and thereby influence microbial enrichment and electrochemical performance in mixed-culture systems. This addition allows our Discussion not only to better interpret the present results, but also to more clearly position the contribution of this study within the context of the existing literature.

Reference added:

Cheng, Q.; Call, D. F. Hardwiring microbes via direct interspecies electron transfer: mechanisms and applications. Environ. Sci.: Processes Impacts 2016, 18, 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would not say revisions were fully adequate; still, the manuscript improved a lot.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors revised the manuscript according to the comments/changes suggested. The paper is suitable to be published in this journal in its current form.