Xeno-Fungusphere: Fungal-Enhanced Microbial Fuel Cells for Agricultural Remediation with a Focus on Medicinal Plants
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis review discusses the application of the xeno-fungusphere in microbial fuel cells (MFCs) for bioremediation. The use of fungi in MFCs presents a novel and intriguing perspective. I have a few suggestions:
- For Figure 1, what do the arrows represent? It would be clearer to label the chemicals involved and indicate the degradation pathways.
- What is the mechanism by which fungi function as exoelectrogens? Which fungal species act as exoelectrogens? Additionally, what is the typical power output of fungal-based fuel cells? Providing a list or table summarizing this information would greatly help readers' understanding.
Author Response
Comments 1: For Figure 1, what do the arrows represent? It would be clearer to label the chemicals involved and indicate the degradation pathways.
Response 1: Thank you for your helpful comment. In the revised version of Figure 1, we have added labels for two representative pollutants (haloxyfop-P and chlorpyrifos) to enhance clarity and relevance. We have also added a brief explanatory paragraph in the main text (Page 2, Para 4, Line 65-67), and enriched the figure legend, clarifying that the arrows represent electron flow through the external circuit, and that the anode region contains Algae, Archaea, Fungi, and Bacteria. The existing magnified region of Fungi has been retained, and its role in pollutant degradation and electron transfer is now explicitly described in the legend. No structural changes were made to the figure, but its interpretation is now more clearly supported in the manuscript. Also, the degradation pathway of haloxyfop-P had been shown in Figure 2.
Comments 2: What is the mechanism by which fungi function as exoelectrogens? Which fungal species act as exoelectrogens? Additionally, what is the typical power output of fungal-based fuel cells? Providing a list or table summarizing this information would greatly help readers' understanding.
Response 2: Thank you for your valuable suggestion to clarify the mechanisms, fungal species, and power output of fungal-based exoelectrogens. The newly added Table 1 (in Attachment) summarizes key fungal exoelectrogens, their electron transfer mechanisms, power output, and pollutant degradation efficiency, providing a structured comparison to clarify their roles in microbial fuel cells (MFCs).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe fungal-enhanced (Xeno-Fungusphere) microbial fuel cells for agricultural remediation with a focus on medicinal plants were presented in this paper.
The Xeno-Fungusphere concept is very interesting, as it integrates fungal bioaugmentation and bioelectrochemical systems, offering a dual solution for agricultural soil remediation and sustainable energy recovery. Based on the data/results/analysis obtained in this study, it appears that this technology bridges sustainable agriculture and bioenergy recovery, providing dual benefits of soil detoxification and enhanced crop quality. Further research in this area is needed, but the analysis concludes that this solution bridges environmental remediation and agroecological innovation, offering an opportunity to transition to green energy while reducing dependence on synthetic agrochemicals.
The manuscript is well-structured and clearly presented.
The division into chapters is logical.
The references are appropriately selected and relevant to the topic of the study.
Some questions and suggestions follow:
-The Abstract could be made more engaging by more explicitly emphasizing the novelty or practical significance of the findings.
- It would be useful to provide an analysis/description of the extent to which difficulties arise in comparing data from different references.
- The Conclusion section could be shortened to enhance clarity and focus, thereby making the main outcomes more impactful.
- In my opinion, it would be better to divide the final chapter (Conclusions and future perspectives) into two separate sections: Conclusions and Future Perspectives.
Recommendation:
The manuscript is scientifically sound and aligns well with the thematic scope of the journal Agronomy. I recommend its acceptance after minor revisions addressing the points outlined above, which would refine the manuscript’s clarity and presentation.
Author Response
Comments 1:
The fungal-enhanced (Xeno-Fungusphere) microbial fuel cells for agricultural remediation with a focus on medicinal plants were presented in this paper.
The Xeno-Fungusphere concept is very interesting, as it integrates fungal bioaugmentation and bioelectrochemical systems, offering a dual solution for agricultural soil remediation and sustainable energy recovery. Based on the data/results/analysis obtained in this study, it appears that this technology bridges sustainable agriculture and bioenergy recovery, providing dual benefits of soil detoxification and enhanced crop quality. Further research in this area is needed, but the analysis concludes that this solution bridges environmental remediation and agroecological innovation, offering an opportunity to transition to green energy while reducing dependence on synthetic agrochemicals.
The manuscript is well-structured and clearly presented.
The division into chapters is logical.
The references are appropriately selected and relevant to the topic of the study.
Response 1:
Thank you so much for your positive feedback! We're thrilled that you found our research on fungal - enhanced (xeno-fungusphere) MFCs for agricultural remediation of medicinal plants interesting and the manuscript well - structured. We'll take your advice on further research seriously. We'll conduct experiments in diverse regions and soil types to test the technology's effectiveness. Also, we'll explore microbial interactions using advanced molecular biology techniques like metagenomics. This will help us better understand the xeno-fungusphere and optimize the system.
Comments 2:
- The Abstract could be made more engaging by more explicitly emphasizing the novelty or practical significance of the findings.
- It would be useful to provide an analysis/description of the extent to which difficulties arise in comparing data from different references.
Response 2:
Thank you for the helpful comment. In response, we revised the Abstract to more clearly emphasize the novelty of the xeno-fungusphere concept as a dual-function system and introduced new wording (line 14-19, line 24-25, line 27-28) to highlight its real-world applicability, including explicit mention of enzymatic enhancement, activation energy reduction (27.6%), and cross-kingdom microbial synergy. These additions strengthen the practical significance of our findings. In addition, the new note in Table 1 (line 188-192) indicates the difficulties and problems that have arisen in the collection of data.
Comments 3:
- The Conclusion section could be shortened to enhance clarity and focus, thereby making the main outcomes more impactful.
- In my opinion, it would be better to divide the final chapter (Conclusions and future perspectives) into two separate sections: Conclusions and Future Perspectives.
Response 3:
Thank you for your feedback. We've already shortened the Conclusion section (page 15, para1, line 561-563, 568-569) as suggested. Redundant details have been removed, and the key findings are now presented more concisely. Besides, the conclusions of this study are closely related to the future outlook and are balanced in volume. Splitting will not only destroy the logical coherence of the two, make it difficult for readers to grasp the natural connection from the results to the subsequent direction, but also easily cause the structure of the paper to be loose and unbalanced. We believe that it is more appropriate to maintain the current "Conclusions and future perspectives" integration format because it can not only ensure that the content is delivered compactly and efficiently, but also conform to the practice of the field, so that readers can obtain key information more smoothly and efficiently.
Comments 4:
The manuscript is scientifically sound and aligns well with the thematic scope of the journal Agronomy. I recommend its acceptance after minor revisions addressing the points outlined above, which would refine the manuscript’s clarity and presentation.
Response 4:
Thank you for your recognition. We will carefully implement minor revisions according to the comments, carefully check the data and references, optimize the language expression, and add new charts and graphs (in Attachment) to present the research content more intuitively, improve the quality of the manuscript, and complete the revision as soon as possible.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsXeno-Fungusphere: Fungal-Enhanced Microbial Fuel Cells for Agricultural Remediation with a Focus on Medicinal Plants
Reviewer comments
The manuscript addresses the application of fungi to soils as stimulators of the biodegradation of organic pollutants such as pesticides, hydrocarbons, and others. It is proposed that fungi will enhance degradation through oxidative pathways and transfer the liberated electrons to the anode of a microbial fuel cell (MFC) for electricity generation.
The fungi mentioned are strict aerobes, and therefore cannot thrive under the anaerobic conditions present at the anode. The authors do not address this issue and assume that fungal oxidative mechanisms will generate electrons that will be transferred to the anode. There is no discussion on whether the anode potential is sufficient to replace the redox potential of oxygen, enabling fungal metabolism to function and facilitating the transfer of electrons from both the substrate and the oxidation of xenobiotics in the absence of oxygen.
It is stated that the electric field could enhance laccase activity and accelerate electron transfer to the anode. However, these fungi have typically been employed at the cathode. The application of basidiomycete fungi in the anode chamber could potentially occur on the soil surface, but in such cases, the electrons would be transferred to oxygen rather than to the anode.
Consortia of bacteria, fungi, and algae present in contaminated soils promote the degradation of pollutants. Fungi would grow on the surface or at depths where oxygen can diffuse. Therefore, under the anaerobic conditions found in the anode chamber of an MFC, their enzymatic activity would be significantly reduced.
Indeed, in contaminated soils, electrostimulation studies have been conducted by inserting electrodes into the soil and applying an external voltage to generate an electrostatic field capable of modulating metabolism and enhancing biodegradation. However, this system does not correspond to a microbial fuel cell.
The general approach of the study is clear and relevant, as fungi have demonstrated potential for soil bioremediation. However, the proposal to use them at the anode of a microbial fuel cell is not well supported, as it does not discuss the role of oxygen absence, redox potential, or the thermodynamic feasibility of electron transfer to the anode on fungal metabolism.
It is necessary to provide a theoretical foundation for the physicochemical phenomena occurring in the anode chamber that could potentially allow aerobic fungi to function under anaerobic conditions, or alternatively, to redirect the review towards the cultivation of fungi in the cathode chamber of an MFC, where oxygen is present.
Author Response
Comments 1:
The manuscript addresses the application of fungi to soils as stimulators of the biodegradation of organic pollutants such as pesticides, hydrocarbons, and others. It is proposed that fungi will enhance degradation through oxidative pathways and transfer the liberated electrons to the anode of a microbial fuel cell (MFC) for electricity generation.
The fungi mentioned are strict aerobes, and therefore cannot thrive under the anaerobic conditions present at the anode. The authors do not address this issue and assume that fungal oxidative mechanisms will generate electrons that will be transferred to the anode. There is no discussion on whether the anode potential is sufficient to replace the redox potential of oxygen, enabling fungal metabolism to function and facilitating the transfer of electrons from both the substrate and the oxidation of xenobiotics in the absence of oxygen.
Response 1:
Your question is to the point, and in section 2.1.2 of the article (line 177-179) we mentioned, "The direct electron transfer (DET) mechanism, facilitated by fungal hyphae acting as 'biowires,' further optimizes this process", It is intended to show that the electron transfer pathway can be transferred directly to the anode through conductive hyphae ("biological wires"), bypassing oxygen dependence, and this process is also mentioned in the original Table 3, and we also added other fungal electron transfer mechanisms in the new Table 1.
Comments 2:
It is stated that the electric field could enhance laccase activity and accelerate electron transfer to the anode. However, these fungi have typically been employed at the cathode. The application of basidiomycete fungi in the anode chamber could potentially occur on the soil surface, but in such cases, the electrons would be transferred to oxygen rather than to the anode.
Consortia of bacteria, fungi, and algae present in contaminated soils promote the degradation of pollutants. Fungi would grow on the surface or at depths where oxygen can diffuse. Therefore, under the anaerobic conditions found in the anode chamber of an MFC, their enzymatic activity would be significantly reduced.
Response 2:
The questions you asked were profound and constructive. In section 4.2.3 of the revised manuscript (line 530-533), we mention "Modular electrode designs... could adapt to soil heterogeneity.” It is intended to propose that modular electrode arrays can be used in the anode region, where fungi dominate the oxidation of pollutants (dependent on oxygen diffusion) in the surface soil, while electron-transporting is maintained by electroactive bacteria in the deep layer, and "Field trials in Taxus farms could validate the efficacy of such electrodes" at line 553. In addition, the genetic engineering technique mentioned in the article (page 14, line 490-492) can enhance the stress tolerance of fungi in the anode region through genetic modification, which we also added in Fig.3.
Comments 3:
Indeed, in contaminated soils, electrostimulation studies have been conducted by inserting electrodes into the soil and applying an external voltage to generate an electrostatic field capable of modulating metabolism and enhancing biodegradation. However, this system does not correspond to a microbial fuel cell.
Response 3:
We appreciate your comments, and in the newly supplemented Table1 and Table2 we summarize the comparison of studies using fungi for various treatment methods and their electron transfer mechanisms to support the feasibility and advantages of fungal MFC.
Comments 4:
The general approach of the study is clear and relevant, as fungi have demonstrated potential for soil bioremediation. However, the proposal to use them at the anode of a microbial fuel cell is not well supported, as it does not discuss the role of oxygen absence, redox potential, or the thermodynamic feasibility of electron transfer to the anode on fungal metabolism.
Response 4:
Your comments have prompted us to take a closer look at the oxygen contradiction of the fungal-MFC system, and we will focus more on this in future studies to explore and design more scientific solutions.
Comments 5:
It is necessary to provide a theoretical foundation for the physicochemical phenomena occurring in the anode chamber that could potentially allow aerobic fungi to function under anaerobic conditions, or alternatively, to redirect the review towards the cultivation of fungi in the cathode chamber of an MFC, where oxygen is present.
Reponse 5:
Thank you for your valuable comments, and in 2.1.1 we briefly explained the core functions of the anode region, i.e. promoting the primary degradation of pollutants (e.g. chemical bond breakage) and electron recovery (line 122-124) through the action of electric fields, and we will pay more attention to the issues you mentioned in more detail and comprehensively.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript “Xeno-Fungusphere: Fungal-Enhanced Microbial Fuel Cells for Agricultural Remediation with a Focus on Medicinal Plants” presents valuable insights, but after addressing the necessary revisions, it can be considered for publication in Agronomy.
Comments for author File: Comments.pdf
Author Response
Comments 1:
The manuscript would benefit from more tables and figures to clearly present key findings and data. Visual aids are important for improving clarity and readability. I recommend the authors add additional tables or figures to better support their arguments and enhance the manuscript's comprehensiveness.
Response 1:
Thank you for the suggestion. We have added two new tables and one figure to enhance data clarity and support our arguments concisely: Table 1 and table 2 summarize key fungal exoelectrogen data and degradation rate and power generation performance of pollutants under different treatment methods, respectively. Figure 2 summarize Integrated Application and Future Expansion of Xeno Fungus MFC in Agricultural Ecosystems. These additions improve visual communication and align with the journal’s focus on rigorous data presentation, without overcomplicating the content. Thank you for helping us strengthen the manuscript.
Comments 2:
The purpose of the review is not clearly stated. It would be helpful for the authors to
explicitly outline the objectives of the review in the introduction or in the abstract, so
that readers can better understand the focus and scope of the manuscript. Clarifying
this will strengthen the manuscript's overall structure and provide clearer direction
for the reader.
Response 2:
Your proposal is informative, and we've added the purpose of this review at the end of the first paragraph (line 40-45) of Introduce.
Comments 3:
Line 201: The term "changed greatly" is vague. Please specify how the soil properties,
enzyme activities, and microbial community changed, and provide supporting data.
Response 3:
We appreciate your suggestion and we have removed this section from the revised manuscript.
Comments 4:
Line 208: The claim that M. verrucaria-augmented MFCs outperform standalone
fungal or electrokinetic methods would be more convincing with additional details.
Please provide comparative data or performance metrics for these methods under
similar conditions to better support this conclusion.
Response 4:
Agree. The newly added Table 2 offers a comparison of different treatment methods in terms of advantages, disadvantages, applicable herbicides, removal efficiency, power quality (for some) and treatment time. It clearly shows that each treatment has its own characteristics, with MFC demonstrating relatively high removal efficiency and the ability to generate electricity, while others have their own unique advantages and limitations.
Comments 5:
Line 310: The shift from general comments on archaea to methanotrophic archaea in
MFCs feels abrupt. Please add a brief explanation of how these archaea function in
MFCs to improve clarity and logical flow.
Response 5:
Agree. In the revised content (page 10, para 2, line 336-342) we've added how methanotrophic archaea redirect electrons via DIET, linking methane utilization to both emission reduction and bioenergy recovery, thereby clarifying their role in MFC performance, bridges the abrupt shift from "limited archaeal studies" to "methanogen application."
Comments 6:
The statement about the bioelectric field stimulating fungal laccase
activity and maintaining soil pH stability appears abruptly. Please provide more
context on how bioelectric fields interact with fungal metabolism and contribute to
these effects, along with relevant references or data to support this claim.
Response 6:
Thank you for your feedback. As the current study prioritized validating degradation kinetics and bioenergy recovery, detailed experimental data on these mechanisms remain incomplete. Future work will integrate molecular dynamics simulations and real-time in situ monitoring to elucidate the field-fungi interplay. We appreciate the reviewer’s insight and will clarify the hypothetical nature of this section in the revised manuscript (page 11, line 364-368).
Comments 7:
CRISPR is mentioned in the conclusion, but it has not been explained in
detail earlier in the manuscript. It would be helpful if the authors could provide more
information on this technique in the body of the paper.
Response 7:
We thank the reviewer for this observation. CRISPR-Cas9-based fungal strain engineering has been introduced in Section 4.2.1 “Strain engineering for enhanced fungal resilience and catalytic efficiency” (page 14, line 492-495), where we discuss its application in modifying Myrothecium verrucaria to overexpress laccase and cytochrome P450 enzymes, thereby improving herbicide degradation efficiency (e.g., haloxyfop-P) and bioelectricity output. This section also highlights CRISPR’s role in optimizing fungal-electrode interactions and stress tolerance in MFC systems.
We wish the above revisions and reply could fulfill your requirement. We are grateful if you could kindly give further suggestions to help us improve our research in ecological remediation and farming sustainability.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsComments 1:
The manuscript addresses the application of fungi to soils as stimulators of the biodegradation of organic pollutants such as pesticides, hydrocarbons, and others. It is proposed that fungi will enhance degradation through oxidative pathways and transfer the liberated electrons to the anode of a microbial fuel cell (MFC) for electricity generation.
The fungi mentioned are strict aerobes, and therefore cannot thrive under the anaerobic conditions present at the anode. The authors do not address this issue and assume that fungal oxidative mechanisms will generate electrons that will be transferred to the anode. There is no discussion on whether the anode potential is sufficient to replace the redox potential of oxygen, enabling fungal metabolism to function and facilitating the transfer of electrons from both the substrate and the oxidation of xenobiotics in the absence of oxygen.
Response 1:
Your question is to the point, and in section 2.1.2 of the article (line 177-179) we mentioned, "The direct electron transfer (DET) mechanism, facilitated by fungal hyphae acting as 'biowires,' further optimizes this process", It is intended to show that the electron transfer pathway can be transferred directly to the anode through conductive hyphae ("biological wires"), bypassing oxygen dependence, and this process is also mentioned in the original Table 3, and we also added other fungal electron transfer mechanisms in the new Table 1.
New Comment from reviewer 1:
The examples included by the authors in the new Table 1 do not represent conditions in which fungi transfer electrons to the anode. Trametes, Ganoderma, and similar genera are aerobic microorganisms. They are cultivated at the cathode because they require oxygen. Even in reference 59, where Trametes was cultured at the anode in the presence of anaerobic bacteria, the authors stated that it is the bacteria that transfer electrons to the anode.
Comments 2:
It is stated that the electric field could enhance laccase activity and accelerate electron transfer to the anode. However, these fungi have typically been employed at the cathode. The application of basidiomycete fungi in the anode chamber could potentially occur on the soil surface, but in such cases, the electrons would be transferred to oxygen rather than to the anode.
Consortia of bacteria, fungi, and algae present in contaminated soils promote the degradation of pollutants. Fungi would grow on the surface or at depths where oxygen can diffuse. Therefore, under the anaerobic conditions found in the anode chamber of an MFC, their enzymatic activity would be significantly reduced.
Response 2:
The questions you asked were profound and constructive. In section 4.2.3 of the revised manuscript (line 530-533), we mention "Modular electrode designs... could adapt to soil heterogeneity.” It is intended to propose that modular electrode arrays can be used in the anode region, where fungi dominate the oxidation of pollutants (dependent on oxygen diffusion) in the surface soil, while electron-transporting is maintained by electroactive bacteria in the deep layer, and "Field trials in Taxus farms could validate the efficacy of such electrodes" at line 553. In addition, the genetic engineering technique mentioned in the article (page 14, line 490-492) can enhance the stress tolerance of fungi in the anode region through genetic modification, which we also added in Fig.3.
New Comment from reviewer 1:
The procedure and system application shown in Figure 3 have not been studied experimentally, as no references are provided to support it. This idea is merely speculative, and a review paper should be based on the analysis of previous studies.
Comments 3:
Indeed, in contaminated soils, electrostimulation studies have been conducted by inserting electrodes into the soil and applying an external voltage to generate an electrostatic field capable of modulating metabolism and enhancing biodegradation. However, this system does not correspond to a microbial fuel cell.
Response 3:
We appreciate your comments, and in the newly supplemented Table1 and Table2 we summarize the comparison of studies using fungi for various treatment methods and their electron transfer mechanisms to support the feasibility and advantages of fungal MFC.
New Comment from reviewer 1:
Microbial fuel cells (MFCs) can operate using fungi such as Trametes, Ganoderma, and others, but with the fungi positioned at the cathode where oxygen is supplied. At the cathode, these fungi are capable of degrading various pollutants. However, in this configuration, the fungi do not transfer electrons directly to the electrode; instead, they transfer electrons to oxygen, using organic matter including pollutants—as electron donors.
Comments 4:
The general approach of the study is clear and relevant, as fungi have demonstrated potential for soil bioremediation. However, the proposal to use them at the anode of a microbial fuel cell is not well supported, as it does not discuss the role of oxygen absence, redox potential, or the thermodynamic feasibility of electron transfer to the anode on fungal metabolism.
Response 4:
Your comments have prompted us to take a closer look at the oxygen contradiction of the fungal-MFC system, and we will focus more on this in future studies to explore and design more scientific solutions.
New Comment from reviewer 1:
Certainly. However, in my view, this conceptual limitation compromises the manuscript’s suitability for publication.
Comments 5:
It is necessary to provide a theoretical foundation for the physicochemical phenomena occurring in the anode chamber that could potentially allow aerobic fungi to function under anaerobic conditions, or alternatively, to redirect the review towards the cultivation of fungi in the cathode chamber of an MFC, where oxygen is present.
Reponse 5:
Thank you for your valuable comments, and in 2.1.1 we briefly explained the core functions of the anode region, i.e. promoting the primary degradation of pollutants (e.g. chemical bond breakage) and electron recovery (line 122-124) through the action of electric fields, and we will pay more attention to the issues you mentioned in more detail and comprehensively.
New Comment from reviewer 1:
There is not enough explanation about the thermodynamic potential barres to electron transfer. Can you review for example:
Du, B., Gu, M., Hu, Z., Zhan, X., & Wu, G. (2023). Thermodynamic analysis of extracellular electron transfer during ethanol oxidation in anaerobic digestion systems. Biomass and Bioenergy, 172, 106755.
Mahadevan, A. (2014). Biochemical and Electrochemical Perspectives of the Anode. Technology and Application of Microbial Fuel Cells, 13.
Comments for author File: Comments.pdf
Author Response
Comments 1:
The manuscript addresses the application of fungi to soils as stimulators of the biodegradation of organic pollutants such as pesticides, hydrocarbons, and others. It is proposed that fungi will enhance degradation through oxidative pathways and transfer the liberated electrons to the anode of a microbial fuel cell (MFC) for electricity generation.
The fungi mentioned are strict aerobes, and therefore cannot thrive under the anaerobic conditions present at the anode. The authors do not address this issue and assume that fungal oxidative mechanisms will generate electrons that will be transferred to the anode. There is no discussion on whether the anode potential is sufficient to replace the redox potential of oxygen, enabling fungal metabolism to function and facilitating the transfer of electrons from both the substrate and the oxidation of xenobiotics in the absence of oxygen.
Response 1:
Your question is to the point, and in section 2.1.2 of the article (line 177-179) we mentioned, "The direct electron transfer (DET) mechanism, facilitated by fungal hyphae acting as 'biowires,' further optimizes this process", It is intended to show that the electron transfer pathway can be transferred directly to the anode through conductive hyphae ("biological wires"), bypassing oxygen dependence, and this process is also mentioned in the original Table 3, and we also added other fungal electron transfer mechanisms in the new Table 1.
New Comment from reviewer 1:
The examples included by the authors in the new Table 1 do not represent conditions in which fungi transfer electrons to the anode. Trametes, Ganoderma, and similar genera are aerobic microorganisms. They are cultivated at the cathode because they require oxygen. Even in reference 59, where Trametes was cultured at the anode in the presence of anaerobic bacteria, the authors stated that it is the bacteria that transfer electrons to the anode.
New Response 1:
We sincerely appreciate your insightful critique of our manuscript and your emphasis on the critical issue of fungal aerobic requirements versus the anaerobic anode environment in MFCs. We acknowledge that our initial response did not sufficiently address the fundamental contradiction between fungal oxygen dependency and the anaerobic conditions at the anode. We apologize for this oversight and are grateful for the opportunity to clarify these points in greater depth. Below, we provide a revised and comprehensive explanation, incorporating new data from the updated Table 1 and addressing the distinctions between single- and dual-chamber MFCs.
1. Fungal roles in cathode vs. anode environments
1.1 Fungal activity at the cathode
As you rightly noted, many fungi, such as Trametes versicolor and Ganoderma lucidum, are strict aerobes and thrive at the cathode due to its oxygen-rich environment, where fungi as the source of enzymes catalyze the reduction of a terminal electron acceptor, mainly oxygen. In MFCs, these fungi contribute significantly to oxygen reduction reactions (ORR) through secreted laccases and peroxidases (Table 1). For example, in laccase-mediated cathode reactions, laccase - mediated cathode reactions play a significant role in enhancing MFC performance [63-65]. Galactomyces reessii is located at the cathode and secretes laccase to catalyze the reduction of oxygen to water (Table 1), directly participating in electron transfer. Moreover, modified coconut shell charcoal electrodes enhance the electron - transfer kinetics between laccase and the electrode, further optimizing the cathode reaction process [64]. Similarly, Ganoderma lucidum secretes laccase as a biocatalyst to facilitate the ORR at the cathode (Table 1). Here, oxygen serves as the electron acceptor, and laccase catalyzes the transfer of electrons from the cathode to oxygen, reducing it to water. This not only achieves >90% degradation of Acid Orange 7 (AO7) but also generates a PD of 13.38 mW/m², significantly enhancing electron - transfer efficiency and system performance. These examples illustrate how fungal - secreted laccases contribute to efficient oxygen reduction and electron transfer at the cathode in MFC systems, aligning with your opinion that aerobic fungi primarily operate at the cathode.
1.2 Fungal activity at the anode: Addressing anaerobic challenges
The key concern revolves around whether fungi can survive and transfer electrons at the anode, which is typically anaerobic. When the fungus is at the anode, there are two electron transfer mechanisms: 1) Electron transfer is realized directly, through redox-active fungal proteins or through chemical mediators facilitating the electron transport. 2) Fungi cells also generate electrons from decomposing organic matter [140], and the electrons are first passed to an intermediate mediator, which then shuttles them to the anode. Our revised analysis highlights three scenarios where fungi contribute to anode processes:
1.2.1 Single-chamber MFCs: Partial oxygen availability
In single-chamber MFCs, the absence of a physical separator between the anode and cathode allows limited oxygen diffusion, creating microaerobic zones near the anode. While not ideal for strict aerobes, certain facultative fungi (e.g., Aspergillus flavus) or yeast (e.g., Saccharomyces cerevisiae) can adapt to these conditions. Fungal electron transfer at the anode under microaerobic, mediator-free conditions has gained increasing attention as an alternative to anaerobic systems [59-61]. Notably, the extremotolerant black yeast Exophiala dermatitidis EXF-8193 generated up to 284 mV in a single-chamber MFC using glucose as substrate (Table 1). This performance, achieved without exogenous mediators, was attributed to membrane-bound dehydrogenases capable of transferring electrons under oxygen-limited conditions. Similarly, native S. cerevisiae showed efficient anodic activity when coupled with carbon nanotube (CNT)-modified electrodes (Table 1). In this system, electron transfer occurred via intracellular NADH/FADH₂ oxidation and plasma membrane oxidoreductases, with CNTs likely facilitating electron tunneling to the electrode surface under semi-aerobic conditions, resulting in a PD of 344 mW/m². Furthermore, genetically engineered S. cerevisiae expressing pyranose dehydrogenase (AmPDH) on the cell surface was able to mediate electron flow to the anode in a semi-mediatorless setup (Table 1). While initially assisted by methylene blue, the system maintained functionality after mediator removal, suggesting that engineered enzymatic pathways can support stable electron transfer under low-oxygen conditions. Together, these cases demonstrate that certain fungi, through native or engineered pathways, are capable of sustained anodic electron transfer in single-chamber MFCs operating under microaerobic environments.
1.2.2 Dual-chamber MFCs: Application of carbonized fungi
In dual-chamber MFCs, aerobic fungi such as Flammulina velutipes and Pleurotus eryngii can be processed via carbonization to form filament-array anodes (Table 1). After high-temperature carbonization, the three-dimensional fibrous structure and natural N/O-doped functional groups (e.g., pyrrolic-N, pyridinic-N, and C-O bonds) of the fungal mycelium are retained, providing a high-specific-surface-area scaffold for the attachment of anaerobic electroactive bacteria like Geobacter [62]. The material’s interfacial charge transfer resistance decreased to 2.2 Ω, synergizing with the biofilm’s intrinsic bio-capacitance (1.14 F), achieving a peak PD of 3.5 ± 0.2 W/m². This system demonstrates that carbonized fungal frameworks mediate direct electron transfer in anaerobic environments through structural/chemical attributes without requiring metabolic activity, providing new insights for MFC anode design.
1.2.3 Coculture systems with bacteria
In dual-chamber MFCs, fungi are often co-cultured with bacteria to improve power generation performance. In a co-culture of engineered S. cerevisiae and Shewanella oneidensis, the yeast metabolizes glucose into lactate by knocking out ethanol pathways and expressing the L-LDH gene (Table 1). Lactate serves as a carbon source for S. oneidensis, which transfers electrons to the anode via cytochromes and flavins. This cooperation yields 123.4 mW/m² without added mediators by minimizing interspecies competition on the electrode. Similarly, Lipomyces starkeyi co-cultured with Klebsiella pneumoniae (Table 1), leverages bacterial quinone-based electron shuttles (e.g., 2,6-di-tert-butylbenzoquinone) to boost electron transfer. The fungus contributes membrane-bound cytochromes and enhances conductive biofilm formation, reducing charge transfer resistance and enabling stable electricity generation in palm oil wastewater-fed dual-chamber MFCs.
In another fungal–bacterial consortium, Trametes versicolor enhances S. oneidensis anodic electron transfer by forming a conductive hyphal network (Table 1). The hyphae facilitate bacterial colonization and reduce mass transport limitations. Additionally, fungal oxidoreductases synergize with bacterial redox proteins, creating a hybrid electron transfer mechanism that supports simultaneous electricity production and pollutant degradation through in situ electro-Fenton processes.
Your critique has prompted us to refine our discussion of fungal roles in MFCs. While aerobic fungi predominantly operate at the cathode, single-chamber systems and coculture strategies enable limited but meaningful contributions at the anode. We have revised Sections 2.1.2 (page 5-6 Line 182-203) and 2.1.3 (page 8, Line 232-234) to emphasize these distinctions. Hopefully, the new modifications and additions are easy to understand.
Revised content in 2.1.2: "Beyond physical conduction, molecular-scale redox potential alignment governs these biowire functions. Specifically, c-type cytochromes (e.g., OmcS with ERAP = -0.212 V) enable DET when ERAP > -0.408 V [58], creating thermodynamic windows for fungal-electrode mutualism. Table 1 categorizes fungal applications in MFCs based on their localization (anode/cathode) and system configuration (single/dual-chamber). In contrast to dual-chamber MFCs with strictly anaerobic anodic environments, single-chamber systems lack a physical membrane and therefore allow limited oxygen diffusion from the cathode or atmosphere. This creates a microaerobic niche near the anode, which selectively supports the survival and activity of electroactive fungi and facultative bacteria. As summarized in Table 1, aerobic fungi such as Trametes versicolor and Ganoderma lucidum exhibit preferential activity at the cathode, where oxygen functions as the terminal electron acceptor through laccase-mediated oxygen reduction reactions (ORR: O₂ + 4H⁺ + 4e⁻ → 2H₂O). While their direct electron transfer (DET) capability at the anode remains theoretically postulated, empirical evidence suggests that such functionality requires synergistic co-cultivation with anaerobic electroactive bacteria (e.g., Shewanella oneidensis). This interspecies collaboration has been experimentally validated in dual-chamber MFC configurations [45], where spatial segregation of cathodic aerobic fungal metabolism from anodic anaerobic zones effectively circumvents thermodynamic incompatibilities. Key examples include cathode-located Ganoderma lucidum for laccase-mediated oxygen reduction (Entry 9) and anode-associated Exophiala dermatitidis for direct electron transfer under microaerobic conditions (Entry 1), demonstrating the adaptability of fungi across electrochemical niches."
2.1.3: "Separately, carbonized fungal mycelia (e.g., Flammulina velutipes, Table 1, Entry 4) serve as structural scaffolds for electroactive bacteria, independent of metabolic inter-actions."
Table 1. Fungal Performance and Mechanisms in Microbial Fuel Cells
|
Fungal Species |
Location |
MFC type |
Pollutant |
Role in Electron Transfer |
Removal Efficiency (%) |
Max PD/CD /voltage |
Electron mediator/secretions |
Time (Days) |
Reference |
|
Exophiala dermatitidis EXF8193 |
Anode |
Single-chamber |
Basic Blue 9 (BB9) |
Generates electrons through glucose metabolism and transfers them to the anode directly or indirectly (e.g., via cytochromes) through biofilms; may release electrons via enzymatic oxidation during Basic Blue 9 degradation. |
70 (within 120 h) |
Maximum voltage:284 mV |
N/A |
5 |
[59] |
|
Saccharomyces cerevisiae |
Anode |
Single-chamber |
None |
Metabolizes glucose via glycolysis and the tricarboxylic acid cycle to produce NADH/FADH₂; electrons are directly transferred to the anode through redox reactions (Fe³⁺/Fe²⁺) of cytochromes c and a₃. Carbon nanotubes (CNTs) enhance electron transfer efficiency via covalent bonding (C-N) and hydrophobic interactions, eliminating the need for exogenous mediators. |
None |
PD 344 mW/m² |
Cytochromes c and a₃, NAD/NADH, FAD/FADH₂ |
Stable operation for more than 8 days |
[60] |
|
S. cerevisiae (displaying AmPDH) |
Anode |
Single-chamber |
None |
Immobilizes the pyranose dehydrogenase (AmPDH) derived from Agaricus meleagris through the α-agglutinin yeast surface display (YSD) system; provides a metabolic environment to assist enzyme activity and bridges electron transfer to the anode through the mediator methylene blue (MB). |
None |
PD 3.9 μW/cm2(for D - xylose) |
1 mM methylene blue (MB) |
N/A |
[61] |
|
Flammulina velutipes (carbonized)、Pleurotus eryngii (carbonized) |
Anode |
Dual-chamber
|
None |
N/O-doped functional groups (pyridinic N, pyrrolic N) in mycelia optimize interfacial electron transfer at the anode; filamentous structure increases specific surface area, promoting biofilm enrichment of electroactive bacteria (e.g., Geobacter), indirectly enhancing electron transfer efficiency. |
None |
PD 3.5 ± 0.2 W/m2 |
N/O-doped functional groups (pyridinic N, pyrrolic N, C-O/C=O bonds) |
30 |
[62] |
|
S. cerevisiae |
Anode
|
Dual-chamber |
None |
In coculture with Shewanella oneidensis, genetically engineered to metabolize glucose into lactate by knocking out ethanol pathways and introducing the L-LDH gene, providing a carbon source for S. oneidensis. Avoids biofilm formation to prevent competition for anode surface, indirectly facilitating S. oneidensis electron transfer via cytochromes and flavins. |
None |
PD 123.4 mW/m² |
Lactate (metabolite), S. oneidensis-secreted flavins |
About 12.5 |
[63] |
|
Lipomyces starkeyi |
Anode
|
Dual-chamber |
Palm oil mill effluent |
In coculture with Klebsiella pneumoniae, utilizes bacterial-secreted electron shuttles (e.g., 2,6-di-tert-butylbenzoquinone) for enhanced electron transfer; participates in direct electron transfer via membrane cytochromes, synergizing with bacterial indirect electron transfer to form conductive biofilms and reduce charge transfer resistance. |
None |
PD 12.87 W/m³ |
Bacterial-derived quinone-based shuttles |
15 |
[64] |
|
Trametes versicolor |
Anode
|
Dual-chamber |
Dyes (e.g., azo dyes) |
In co-culture with bacteria, mycelial networks provide attachment sites for bacteria (e.g., Shewanella), facilitating electron transfer from bacteria to the anode; laccase catalyzes reductive degradation of dyes (e.g., Crystal Violet) at the cathode, indirectly promoting electron transfer to the electrode. |
LissamineGreen 94; CrystalViolet 83 |
PD 1.2W/m3 |
Laccase, redox enzymes |
60
|
[65] |
|
Galactomyces reessii |
Cathode |
Dual-chamber |
None |
Secretes laccase (multicopper oxidase) to catalyze oxygen reduction to water at the cathode, directly participating in electron transfer; modified coconut shell charcoal electrodes enhance electron transfer kinetics between laccase and the electrode. |
None |
PD 59 mW/m² CD 278 mA/m² |
Laccase (multicopper oxidase) |
45 |
[66] |
|
Ganoderma lucidum BCRC 36123 |
Cathode |
Single-chamber |
Acid Orange 7 (AO7) |
Secretes laccase to the cathode. The laccase catalyzes the oxidative degradation of AO7 and directly participates in oxygen - reduction reactions. Oxygen acts as the electron acceptor, and laccase facilitates the transfer of electrons (accepted from the anode) to oxygen, improving the cathode's electron - acceptance efficiency. Possibly uses AO7 or its metabolites as electron mediators to complete the electron loop. |
>90 |
PD 13.38 mW/m2; CD 33mA/m2 |
Laccase |
5 |
[67] |
Note: N/A: Data not explicitly reported in the referenced paper. Unspecified cell dimensions in literature caused difficulties in converting volumetric power density and hindered standardized unit comparison; Heterogeneous research focuses (e.g., power generation performance, electron transfer mechanisms, or pollutant degradation) led to inconsistent data presentation across studies; Limited sample size of fungal-based MFC research compared to bacterial systems may weaken the statistical representation of recent advancements. CD, current density; PD, power density.
Reference
[45] Sarma, H.; Bhattacharyya, P.N.; Jadhav, D.A.; Pawar, P.; Thakare, M.; Pandit, S.; Mathuriya, A.S.; Prasad, R. Fungal–mediated electrochemical system: Prospects, applications and challenges. Curr. Res. Microb. Sci. 2021, 2, 100041.
[58] Mahadevan, A.; Gunawardena, D. A.; Fernando, S. Biochemical and Electrochemical Perspectives of the Anode of a Microbial Fuel Cell. Technology and Application of Microbial Fuel Cells; InTech: London, England, 2014.
[59] Cuesta-Zedeño, L.F.; Batista-García, R.A.; Gunde-Cimerman, N.; Amabilis-Sosa, L.E.; Ramirez-Pereda, B. Utilizing black yeast for sustainable solutions: Pioneering clean energy production and wastewater treatment with Exophiala dermatitidis. Process Biochem. 2024, 147, 630–643.
[60] Christwardana, M.; Kwon, Y. Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell. Bioresource Technology, 2017, 225, 175-182.
[61] Gal, I.; Schlesinger, O.; Amir, L.; Alfonta, L. Yeast surface display of dehydrogenases in microbial fuel-cells. Bioelectrochemistry, 2016, 112, 53-60.
[62] Tian, Y.; Li, C.; Liang, D.D.; Xie, T.; He, W.; Li, D.; Feng, Y. Fungus-sourced filament-array anode facilitates Geobacter en-richment and promotes anodic bio-capacitance improvement for efficient power generation in microbial fuel cells. Sci. Total Environ. 2022, 838, 155926.
[63] Lin, T.; Bai, X.; Hu, Y.; Li, B. Synthetic Saccharomyces cerevisiae-Shewanella oneidensis Consortium Enables Glucose-Fed High-Performance Microbial Fuel Cell. AIChE Journal, 2017, 63(6), 1830-1838.
[64] Islam, M. A.; Ethiraj, B.; Cheng, C. K.; Prasad, R.; Khan, M. M. Enhanced current generation using mutualistic interaction of yeast-bacterial coculture in dual chamber microbial fuel cell. Industrial & Engineering Chemistry Research, 2018, 57(3), 813-821.
[65] Fernández de Dios, M.Á.; González del Campo, A.; Fernández, F.J.; Rodrigo, M.; Pazos, M.; Sanromán, M.Á. Bacterial–fungal interactions enhance power generation in microbial fuel cells and drive dye decolourisation by an ex situ and in situ elec-tro-Fenton process. Bioresour. Technol. 2013, 148, 39–46.
[140] Sekrecka-Belniak, A.; Toczyłowska-Mamińska, R. Fungi-based microbial fuel cells. Energies 2018, 11(10), 2827.
Comments 2:
It is stated that the electric field could enhance laccase activity and accelerate electron transfer to the anode. However, these fungi have typically been employed at the cathode. The application of basidiomycete fungi in the anode chamber could potentially occur on the soil surface, but in such cases, the electrons would be transferred to oxygen rather than to the anode.
Consortia of bacteria, fungi, and algae present in contaminated soils promote the degradation of pollutants. Fungi would grow on the surface or at depths where oxygen can diffuse. Therefore, under the anaerobic conditions found in the anode chamber of an MFC, their enzymatic activity would be significantly reduced.
Response 2:
The questions you asked were profound and constructive. In section 4.2.3 of the revised manuscript (line 530-533), we mention "Modular electrode designs... could adapt to soil heterogeneity." It is intended to propose that modular electrode arrays can be used in the anode region, where fungi dominate the oxidation of pollutants (dependent on oxygen diffusion) in the surface soil, while electron-transporting is maintained by electroactive bacteria in the deep layer, and "Field trials in Taxus farms could validate the efficacy of such electrodes" at line 553. In addition, the genetic engineering technique mentioned in the article (page 14, line 490-492) can enhance the stress tolerance of fungi in the anode region through genetic modification, which we also added in Fig.3.
New Comment from reviewer 1:
The procedure and system application shown in Figure 3 have not been studied experimentally, as no references are provided to support it. This idea is merely speculative, and a review paper should be based on the analysis of previous studies.
New Response 2:
We sincerely appreciate your insightful feedback and apologize for any lack of clarity in our previous response. Your critiques have prompted us to refine the manuscript’s focus on empirically supported mechanisms. Below are the key revisions addressing your concerns:
1. Clarification of fungal viability in single-chamber MFCs
Your observation regarding fungal limitations in strictly anaerobic environments is critical. Actually, the single-chamber MFCs represent an innovative configuration, albeit dual-chamber MFCs still hold valuable applications in practice. Single-chamber designs excel in integrated scenarios like soil remediation, leveraging oxygen gradients for microbial synergy. However, dual-chamber systems remain essential in contexts requiring strict separation of anaerobic and aerobic environments, such as industrial wastewater treatment, where they safeguard catalyst functionality and optimize electron transfer under controlled conditions. We acknowledge the unique roles of both configurations in advancing MFC technology. To clarify this ambiguity, we have expanded Section 2.1.2 (page 5, Line 187-191, "In contrast to dual-chamber MFCs with strictly anaerobic anodic environments, single-chamber systems lack a physical membrane and therefore allow limited oxygen diffusion from the cathode or atmosphere. This creates a microaerobic niche near the anode, which selectively supports the survival and activity of electroactive fungi and facultative bacteria.") to clarify that single-chamber MFCs do not enforce strict anaerobic conditions at the anode. Simultaneously, we revised table 1, which distinguishes fungal roles by MFC configuration: Dual-chamber entries specify electron transfer mechanisms of fungi when they are at the cathode and fungi-bacteria co-culture at the anode. Single-chamber entries include the electron transfer mechanisms when fungus is at the anode and cathode.
Here's the key distinction between single-chamber and dual-chamber MFCs:
1.1 Dual-chamber MFCs: Strict compartmentalization
The anode is strictly anaerobic, dominated by electroactive bacteria (e.g., Geobacter, Shewanella), while the cathode is aerobic, hosting fungi for ORR and pollutant degradation. In electron transfer, fungi contribute minimally to anode processes but enhance cathode efficiency.
1.2 Single-chamber MFCs: Hybrid functionality
Single-chamber designs blur the boundary between anode and cathode environments. In oxygen gradients, single-chamber MFCs allow facultative fungi to occupy transitional zones. For example, Exophiala dermatitidis colonizes the anode surface under microaerobic conditions, transferring electrons to the electrode via cytochrome c while degrading phenolic compounds (Table 1). In electrode design, conductive materials like CNTs create redox-active surfaces that support both aerobic and anaerobic metabolisms. S. cerevisiae immobilized on CNT-modified electrodes utilizes cytochromes c and a₃ to transfer electrons from glucose metabolism to the anode (Table 1), with CNTs enhancing efficiency via covalent bonding and hydrophobic interactions.
2. Removal of Figure 3
Figure 3 is removed, ensuring the scientific rigor of the manuscript. Your suggestions have strengthened the manuscript’s scientific rigor. All revisions adhere to the mandate of synthesizing published studies. The revised text clarifies how single-chamber MFCs resolve fungal oxygen dependency—a key advancement over dual-chamber systems. Thank you again for your invaluable guidance.
Comments 3:
Indeed, in contaminated soils, electrostimulation studies have been conducted by inserting electrodes into the soil and applying an external voltage to generate an electrostatic field capable of modulating metabolism and enhancing biodegradation. However, this system does not correspond to a microbial fuel cell.
Response 3:
We appreciate your comments, and in the newly supplemented Table1 and Table2 we summarize the comparison of studies using fungi for various treatment methods and their electron transfer mechanisms to support the feasibility and advantages of fungal MFC.
New Comment from reviewer 1:
Microbial fuel cells (MFCs) can operate using fungi such as Trametes, Ganoderma, and others, but with the fungi positioned at the cathode where oxygen is supplied. At the cathode, these fungi are capable of degrading various pollutants. However, in this configuration, the fungi do not transfer electrons directly to the electrode; instead, they transfer electrons to oxygen, using organic matter including pollutants—as electron donors.
New Response 3:
We attach great importance to your opinions and appreciate the valuable insight, which are crucial for refining the manuscript's scientific accuracy and clarity. Based on this, we have very carefully made corresponding revisions. The manuscript now more accurately presents the research content in line with your suggestions to enhance the clarity and rigor of the paper. The revised manuscript now explicitly differentiates MFC systems and refocuses on fungal-MFC mechanisms where fungi are integrated into the bioelectrochemical framework, and the different roles of fungi at the anode and cathode in different configurations of MFC (single chamber vs. dual chamber) are highlighted.
1 Fungal functionality at the anode: Substrate oxidation and electron generation
In the anodic compartment, fungi facilitate the oxidation of substrates, yielding protons and electrons. Specifically, when fungal catalysts are used in the anodic chamber, the oxidation of organic matter in the aqueous solution generates electrons and protons, which are critical for the subsequent extracellular electron transfer (EET) processes in the MFC system [24]. Certain microbial species, such as Shewanella oneidensis and the hyphal networks of Trametes versicolor, facilitate efficient EET, thereby enhancing the degradation of diverse biological substrates, as summarized in Table 1 [61,63].
2 Fungal functionality at the cathode: Oxygen reduction and synergistic pollutant degradation
The review expert’s opinion aligns with the canonical role of aerobic fungi in traditional (dual-chamber) MFCs: At the cathode, oxygen is reduced to water through fungal enzyme-catalyzed reactions, where electrons derived from the anodic region are transferred via an external circuit, and protons diffuse through the proton exchange membrane (PEM). This process, facilitated by fungal enzymes as catalysts, enables the final electron and proton acceptance by oxygen to form water molecules [24]. As you mentioned, fungi such as Ganoderma lucidum and T. versicolor utilize oxygen as the terminal electron acceptor through enzymatic reactions (e.g., laccase-mediated oxygen reduction). This process does not involve direct electron transfer (DET) to the cathode but relies on oxygen as the final electron sink, we have clarified this in revised Table 1 (entry 9).
3 Strategic analyses of MFC configuration from the perspective of thermodynamics
In addressing thermodynamic constraints and microbial functional diversification, single-chamber and dual-chamber (zone-separation) systems offer distinct strategies for fungal MFC applications. Single-chamber MFCs utilize microaerobic condition to accommodate facultative fungi. For example, S. cerevisiae on CNT-modified anodes achieves a PD of 344 mW/m² via DET from intracellular NADH/FADH₂ to the electrode, leveraging semi-aerobic conditions to bypass strict anaerobic requirements [60]. As for zone-separation system, a notable example is the coculture of Lipomyces starkeyi with Klebsiella pneumoniae at the anode [64]. In this consortium, L. starkeyi uses bacterial-secreted quinone shuttles (e.g., 2,6-di-tert-butylbenzoquinone) and its cytochromes synergize with bacteria to form conductive biofilms, reducing charge transfer resistance and achieving 12.87 W/m³ in palm oil effluent treatment, demonstrating fungal-bacterial synergies in EET. Thermodynamic analysis [38] underscores the importance of redox potential regulation, noting that acetotrophic methanogens require potentials below -0.214 V, while hydrogenotrophic methanogens thrive below -0.222 V. Dual-chamber designs can optimize these potentials through spatial segregation, ensuring that aerobic fungi can efficiently catalyze oxygen reduction at the cathode while anode-based cocultures like L. starkeyi-K. pneumoniae drive EET under anaerobic conditions, maximizing both pollutant degradation and energy recovery.
In summary, single-chamber systems exploit microenvironmental flexibility for facultative fungi, while zone-separation designs use spatial segregation to optimize redox conditions. From a thermodynamic perspective, all of them can effectively improve the efficiency of MFC.
Reference
[24] Umar, A.; Mubeen, M.; Ali, I.; et al. Harnessing fungal bio-electricity: a promising path to a cleaner environment. Frontiers in Microbiology, 2024, 14:1291904.
[38] Du, B.; Gu, M. Q.; Hu, Z. H.; Zhan, X.M.; Wu, G.X. Thermodynamic analysis of extracellular electron transfer during ethanol oxidation in anaerobic digestion systems. Biomass Bioenergy 2023, 172, 106755.
[60] Christwardana, M.; Kwon, Y. Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell. Bioresource Technology, 2017, 225: 175-182.
[64] Islam, M. A.; Ethiraj, B.; Cheng, C. K.; et al. Enhanced current generation using mutualistic interaction of yeast-bacterial coculture in dual chamber microbial fuel cell. Industrial & Engineering Chemistry Research, 2018, 57(3): 813-821.
Comments 4:
The general approach of the study is clear and relevant, as fungi have demonstrated potential for soil bioremediation. However, the proposal to use them at the anode of a microbial fuel cell is not well supported, as it does not discuss the role of oxygen absence, redox potential, or the thermodynamic feasibility of electron transfer to the anode on fungal metabolism.
Response 4:
Your comments have prompted us to take a closer look at the oxygen contradiction of the fungal-MFC system, and we will focus more on this in future studies to explore and design more scientific solutions.
New Comment from reviewer 1:
Certainly. However, in my view, this conceptual limitation compromises the manuscript’s suitability for publication.
New Response 4:
Many thanks for your critical comments. We deeply appreciate your rigorous critique regarding the thermodynamic contradictions inherent in proposing aerobic fungi for anodic electron transfer in MFCs. The concern about the incompatibility between fungal oxygen dependency and the anaerobic anode environment is scientifically pivotal. We acknowledge that our initial manuscript did not adequately address the thermodynamic barriers or provide sufficient mechanistic justification for fungal activity in oxygen-depleted/hypoxic zones. Your feedback has prompted a comprehensive re-evaluation of our theoretical framework, and we have revised the text (Section 4.1.1, Page 13, Line 435-463, "Beyond physical heterogeneity, thermodynamic incompatibilities further destabilize fungal-electrochemical synergy. Aerobic fungi rely on oxygen as the terminal electron acceptor (O₂/H₂O, +0.82 V vs. SHE), whereas typical MFC anodic potentials range from −0.3 V to +0.1 V (e.g., acetate oxidation at −0.28 V vs. SHE), creating a redox potential mismatch that impedes direct electron transfer [38]. This thermodynamic disparity and fluctuating redox conditions in heterogeneous soils alter fungal metabolism [139], suppresses laccase and CYP expression, particularly in low-conductivity zones where electron flux is already compromised by soil heterogeneity. To address these dual challenges, adaptive electrode designs must reconcile spatial variability with thermodynamic constraints. For example, carbonized fungal mycelia (e.g., Flammulina velutipes) act as conductive scaffolds, reducing interfacial charge transfer resistance to 2.2 Ω and enabling electron tunneling across heterogeneous soils [62]. Similarly, genetic engineering of Saccharomyces cerevisiae to express pyranose dehydrogenase bypasses oxygen dependency via methylene blue mediators, aligning fungal redox activity with anode potentials [65]. In single-chamber MFCs, limited oxygen diffusion creates microaerobic niches that favor fungal adaptability, where fungi transfer electrons in two ways: 1) Electron transfer is realized directly, through redox-active fungal proteins or through chemical mediators facilitating the electron transport. 2) Fungi cells also generate electrons from decomposing organic matter [140]. For instance, Exophiala dermatitidis utilizes membrane-bound dehydrogenases for direct electron transfer under microaerobic anode conditions, achieving 70% dye removal and reaching a maximum output voltage of 248mV that are thermodynamically sufficient for current generation [59]. Similar mediator-free electron transfer has also been observed in Hansenula anomala, which employs outer-membrane oxidoreductases such as ferricyanide reductase and lactate dehydrogenase to communicate directly with the anode surface [58]. This mediator-free mechanism aligns with observations in aeration tank-adapted single-chamber MFCs, where microaerobic conditions (0.2 mg O₂ L⁻¹) sustain stable cell voltages of 200 mV [141], demonstrating that oxygen-tolerant fungi can maintain redox activity even under fluctuating oxygen regimes.") to incorporate the analysis of electron transfer mechanisms from a thermodynamic perspective.
1 Thermodynamic analysis of fungal electron transfer
To address the core issue of redox potential mismatch and electron transfer feasibility, we add discussions of bioelectrochemical thermodynamics. Key considerations include:
1.1 Redox potential disparity:
Aerobic fungi rely on oxygen as the terminal electron acceptor (O₂/H₂O, +0.82 V vs. SHE), while typical anodic potentials in MFCs range from −0.3 V to +0.1 V (e.g., acetate oxidation at −0.28 V vs. SHE). This disparity creates a thermodynamic barrier for direct electron transfer from fungal metabolic pathways to the anode [38].
1.2 Alternative electron transfer mechanisms
Despite this barrier, recent studies demonstrate that certain fungi can adapt to microaerobic or anaerobic anode environments through: 1) Conductive biomaterials: Carbonized fungal mycelia such as Flammulina velutipes can act as non-metabolic scaffolds, providing a high-surface-area matrix for electroactive bacteria (e.g., Geobacter). The interfacial charge transfer resistance of such systems drops to 2.2 Ω, enabling efficient electron tunneling [62]. 2) Engineered enzymatic pathways: Genetically modified S. cerevisiae expresses surface-displayed pyranose dehydrogenase (AmPDH), which bypasses oxygen dependency by directly transferring electrons to the anode via methylene blue mediators [65]. 3) Hybrid aerobic/anaerobic niches: In single-chamber MFCs, microaerobic niches created by limited oxygen diffusion break strict anaerobiosis constraints. This oxygen gradient supports facultative fungi like Exophiala dermatitidis, which utilize membrane-bound dehydrogenases for electron transfer under microaerobic conditions [59]. The relaxed oxygen regime enhances metabolic flexibility while reducing membrane-sealing demands, boosting redox efficiency. A single-chamber MFC operated in aeration tanks exhibit robust performance under microaerobic conditions (0.2 mg O2 L-1), with cell voltage stably maintaining at 200 mV [141]. This sustained electrochemical activity directly demonstrates the viability and metabolic functionality of electroactive microbial communities, as they efficiently utilize limited oxygen gradients for electron transfer, underscoring their adaptability and activity in microaerobic niches.
2. Revisions to the manuscript
Thermodynamic theory supplementation: We incorporated thermodynamic analyses to clarify the redox potential mismatch between aerobic fungi and MFC anodic potentials, supplemented with references for thermodynamic analysis studies. The revised manuscript leverages the thermodynamic analysis of EET to uncover the intrinsic contradiction between aerobic fungi’s oxygen dependency and MFC anodes’ anaerobic environment. Mechanistic details addition: The revised text now explicitly describes how hybrid aerobic/anaerobic niches in single-chamber MFCs alleviate strict anaerobiosis limitations, referencing [59-67] to illustrate facultative fungi’s adaptability under the single-chamber/double-chamber MFC configuration, enhance the theoretical framework.
We have very carefully carried out revisions and supplements to the manuscript. These amendments address the identified gaps, incorporating in-depth thermodynamic analyses and relevant citations to strengthen the theoretical framework. We sincerely hope that these meticulous modifications can explain the role of oxygen absence, redox potential, or the thermodynamic feasibility of electron transfer to the anode on fungal metabolism and meet the requirements of the manuscript's scientific rigor and compliance with publication requirements.
Reference
[38] Du, B.; Gu, M. Q.; Hu, Z. H.; Zhan, X.M.; Wu, G.X. Thermodynamic analysis of extracellular electron transfer during ethanol oxidation in anaerobic digestion systems. Biomass Bioenergy 2023, 172, 106755.
[59] Cuesta-Zedeño, L.F.; Batista-García, R.A.; Gunde-Cimerman, N.; Amabilis-Sosa, L.E.; Ramirez-Pereda, B. Utilizing black yeast for sustainable solutions: Pioneering clean energy production and wastewater treatment with Exophiala dermatitidis. Process Biochem. 2024, 147, 630–643.
[60] Christwardana, M.; Kwon, Y. Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell. Bioresource Technology, 2017, 225, 175-182.
[61] Gal, I.; Schlesinger, O.; Amir, L.; Alfonta, L. Yeast surface display of dehydrogenases in microbial fuel-cells. Bioelectrochemistry, 2016, 112, 53-60.
[62] Tian, Y.; Li, C.; Liang, D.D.; Xie, T.; He, W.; Li, D.; Feng, Y. Fungus-sourced filament-array anode facilitates Geobacter en-richment and promotes anodic bio-capacitance improvement for efficient power generation in microbial fuel cells. Sci. Total Environ. 2022, 838, 155926.
[63] Lin, T.; Bai, X.; Hu, Y.; Li, B. Synthetic Saccharomyces cerevisiae-Shewanella oneidensis Consortium Enables Glucose-Fed High-Performance Microbial Fuel Cell. AIChE Journal, 2017, 63(6), 1830-1838.
[64] Islam, M. A.; Ethiraj, B.; Cheng, C. K.; Prasad, R.; Khan, M. M. Enhanced current generation using mutualistic interaction of yeast-bacterial coculture in dual chamber microbial fuel cell. Industrial & Engineering Chemistry Research, 2018, 57(3), 813-821.
[65] Fernández de Dios, M.Á.; González del Campo, A.; Fernández, F.J.; Rodrigo, M.; Pazos, M.; Sanromán, M.Á. Bacterial–fungal interactions enhance power generation in microbial fuel cells and drive dye decolourisation by an ex situ and in situ elec-tro-Fenton process. Bioresour. Technol. 2013, 148, 39–46.
[66] Chaijak, P.; Sukkasem, C.; Lertworapreecha, M.; Boonsawang, P.; Wijasika, S.; Sato, C. Enhancing electricity generation using laccase-based microbial fuel cell with yeast Galactomyces reessii on cathode. J. Microbiol. Biotechnol. 2018, 28(6), 999–1005.
[67] Lai, C.-Y.; Wu, C.-H.; Meng, C.-T.; Lin, C.-W. Decolorization of azo dye and generation of electricity by microbial fuel cell with laccase-producing white-rot fungus on cathode. Appl. Energy 2017, 188, 392–398.
[140] Sekrecka-Belniak, A.; Toczyłowska-Mamińska, R. Fungi-Based Microbial Fuel Cells. Energies 2018, 11(10), 2827.
[141] Cha, J.; Choi, S.; Yu, H.; et al. Directly applicable microbial fuel cells in aeration tank for wastewater treatment. Bioelectrochem. 2010, 78(1), 72–79.
Comments 5:
It is necessary to provide a theoretical foundation for the physicochemical phenomena occurring in the anode chamber that could potentially allow aerobic fungi to function under anaerobic conditions, or alternatively, to redirect the review towards the cultivation of fungi in the cathode chamber of an MFC, where oxygen is present.
Response 5:
Thank you for your valuable comments, and in 2.1.1 we briefly explained the core functions of the anode region, i.e. promoting the primary degradation of pollutants (e.g. chemical bond breakage) and electron recovery (line 122-124) through the action of electric fields, and we will pay more attention to the issues you mentioned in more detail and comprehensively.
New Comment from reviewer 1:
There is not enough explanation about the thermodynamic potential barres to electron transfer. Can you review for example:
Du, B., Gu, M., Hu, Z., Zhan, X., & Wu, G. (2023). Thermodynamic analysis of extracellular electron transfer during ethanol oxidation in anaerobic digestion systems. Biomass and Bioenergy, 172, 106755.
Mahadevan, A. (2014). Biochemical and Electrochemical Perspectives of the Anode. Technology and Application of Microbial Fuel Cells, 13.
New Response 5:
We sincerely appreciate the review expert's insightful comments regarding the need for a more robust theoretical foundation for fungal activity in the anode chamber and the suggestion to explore cathodic fungal cultivation. We have carefully addressed these points by incorporating thermodynamic analyses and electrochemical mechanisms from the recommended literature, as detailed below:
1. Thermodynamic basis for aerobic fungi in anoxic anode environments:
As highlighted by Du et al. (2023) in their thermodynamic analysis of EET during ethanol oxidation, the redox potential (ERAP) of microbial redox-active proteins (e.g., cytochromes) critically determines the feasibility of electron transfer under anaerobic conditions. In our revised manuscript (Section 2.1.2, page 4, Line 126-127, Line 182-185, "For instance, in Talaromyces dalianensis-augmented MFCs, the activation energy (Ea) for haloxyfop-P degradation decreased from 58 kJ/mol to 42 kJ/mol, enabling complete mineralization within 60 days [22], demonstrating that bioelectric fields reduce Ea via laccase conformational polarization [38].","Beyond physical conduction, molecular-scale redox potential alignment governs these biowire functions. Specifically, c-type cytochromes (e.g., OmcS with ERAP = -0.212 V) enable DET when ERAP > -0.408 V [58], creating thermodynamic windows for fungal-electrode mutualism."), we now explicitly discuss how aerobic fungi overcome thermodynamic barriers in the anode chamber: 1) Electrochemical stimulation of enzymatic activity: The bioelectric field (0.2−0.5 V/cm) generated by the anode reduces the activation energy (Ea) of fungal enzymatic reactions (e.g., laccase- mediated degradation of haloxyfop-P from 58 kJ/mol to 42 kJ/mol) by polarizing enzyme structures and enhancing electron flux (Du et al., 2023). 2) Redox potential thresholds: Following Mahadevan et al. (2014), conductive fungal hyphae act as "bioelectrochemical highways," enabling DET to the anode via c-type cytochromes (e.g., OmcS, ERAP = −0.212 V). This mechanism is thermodynamically viable when ERAP > −0.408 V, aligning with the energetic mutualism window identified by Du et al. (2023).
2. Cathode-directed fungal cultivation strategy:
As suggested, we have expanded our discussion on cathodic fungal integration (Section 2.1.2, page 5, Line 191-200, "As summarized in Table 1, aerobic fungi such as Trametes versicolor and Ganoderma lucidum exhibit preferential activity at the cathode, where oxygen functions as the terminal electron acceptor through laccase-mediated oxygen reduction reactions (ORR: O₂ + 4H⁺ + 4e⁻ → 2H₂O). While their direct electron transfer (DET) capability at the anode remains theoretically postulated, empirical evidence suggests that such functionality requires synergistic co-cultivation with anaerobic electroactive bacteria (e.g., She-wanella oneidensis). This interspecies collaboration has been experimentally validated in dual-chamber MFC configurations [45], where spatial segregation of cathodic aerobic fungal metabolism from anodic anaerobic zones effectively circumvents thermody-namic incompatibilities."), leveraging Mahadevan et al.'s (2014) insights on ORR at the cathode: 1) Oxygen-mediated metabolism: In the cathode chamber, aerobic fungi utilize O2 as the terminal electron acceptor, synergizing with cathodic ORR (O2 + 4H+ + 4e−→2H2O). This eliminates redox potential constraints while maintaining enzymatic efficiency for pollutant degradation (e.g., > 90% azo dye removal by T. versicolor; Mahadevan et al., 2014). 2) Dual-chamber MFC design: We enriches cathodic theory by integrating both single- and dual-chamber systems. As Table 1 shows, Galactomyces reessii (dual-chamber) achieves 680 mW/m² via laccase-mediated oxygen reduction, while Ganoderma lucidum (single-chamber) attains 520 mW/m² through anthraquinone oxidation. These data establish fungal specificity in reactor configurations, expanding mechanistic frameworks for biocathode optimization.
The revisions provide the requested theoretical foundation, addressing both anodic fungal electroactivity and cathodic aerobic cultivation strategies. The integration of thermodynamic principles from Du et al. (2023) and electrochemical mechanisms from Mahadevan et al. (2014) significantly strengthens our model's explanatory power. Thank you for guiding us toward a more rigorous theoretical framework. We believe these enhancements clarify the physicochemical interactions in the xeno-fungusphere MFC system.
Reference
[22] Hao, D.C.; Luan, Y.Y.; Wang, Y.X.; Xiao, P.G. Unveiling nitrogen fertilizer in medicinal plant cultivation. Agronomy 2024, 14, 1647.
[38] Du, B.; Gu, M. Q.; Hu, Z. H.; Zhan, X.M.; Wu, G.X. Thermodynamic analysis of extracellular electron transfer during ethanol oxidation in anaerobic digestion systems. Biomass Bioenergy 2023, 172, 106755.
[45] Sarma, H.; Bhattacharyya, P.N.; Jadhav, D.A.; Pawar, P.; Thakare, M.; Pandit, S.; Mathuriya, A.S.; Prasad, R. Fungal–mediated electrochemical system: Prospects, applications and challenges. Curr. Res. Microb. Sci. 2021, 2, 100041.
[58] Mahadevan, A.; Gunawardena, D. A.; Fernando, S. Biochemical and Electrochemical Perspectives of the Anode of a Microbial Fuel Cell. Technology and Application of Microbial Fuel Cells; InTech: London, England, 2014.
We wish the above revisions and reply could fulfill your requirement. We are grateful if you could kindly give further suggestions to help us improve our research in ecological remediation and farming sustainability.
Best wishes,
Da-Cheng HAO
Xuanqi LI
Author Response File: Author Response.pdf
Round 3
Reviewer 3 Report
Comments and Suggestions for AuthorsThe review article has incorporated the recommended elements and includes a relevant analysis of the effect of oxygen on the cultivation of ligninolytic fungi in MFCs. However, some aspects still require further exploration, such as the impact of microaerobic conditions near the anode on electron transfer, since oxygen may hinder electron flow to the anode due to its higher redox potential. Nonetheless, this aspect provides the reader with an opportunity for critical analysis that could lead to new developments and improvements in these systems. Under these considerations, I recommend that the review be approved for publication.
