A Hybrid Microbial–Enzymatic Fuel Cell Cathode Overcomes Enzyme Inactivation Limits in Biological Fuel Cells

Reviewer Comment or Suggestion

Author Correction or Rebuttal

I read with attention the manuscript "A Hybrid Microbial-Enzymatic Fuel Cell Cathode Overcomes Enzyme Inactivation Limits in Biological Fuel Cells" from J. Parker Evans et. al. The manuscript deals about oxygen reduction reaction catalytic properties of Laccases enzymes: to overcome the short enzyme lifetimes associated with pure laccase the authors introduced, over the cathodes, several types of fungi that secrete laccases enzymes to provide a continous regeneration of the enzymes over the cathodes. The idea, applied to microbial fuel cell cathode for oxygen reduction reaction purpose, is original and promising. Moreover there are several serious issues in this manunscript that restrain me to approve it. 

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1) Introduction is not extensively detailed: more references can be added. Other articles that deals same topic must be discussed in lights of their respective findings and why this manuscript can perform an outbreak in the actual the state-of-art. See [doi.org/10.1016/S1369-7021(12)70070-6]; and [Tarasevich, M. R., V. A. Bogdanovskaya, and L. N. Kuznetsova. "Bioelectrocatalytic reduction of oxygen in the presence of laccase adsorbed on carbon electrodes." Russian journal of electrochemistry 37.8 (2001): 833-837.] as an example.

The introduction has been rewritten to add additional literature review and establish problems with the state of the art. Additional citations have been added to give detail to the introduction. The reviewer’s suggested references have been added where appropriate (lines 40, 54, and 220).

2) Introduction: The authors select carbon nanotubes as conductive carbon scaffold to allow electronic transfer from laccases. Moreover the authors have to further explain the importance of carbon nanotubes as electron acceptors: why they are preferred among other carbon nanostructures that are more sustainable in terms of costs and toxicity? See for example: [K. Kostarelos, The long and short of carbon nanotube toxicity, Nat. Biotechnol., 26 (2008) 774-776.] [S.F. Hansen, A. Lennquist, Carbon nanotubes added to the SIN List as a nanomaterial of Very High Concern, Nat. Nanotechnol., 15 (2020) 3-4.]
[D.A. Heller, P.V. Jena, M. Pasquali, K. Kostarelos, L.G. Delogu, R.E. Meidl, S.V. Rotkin, D.A. Scheinberg, R.E. Schwartz, M. Terrones, Y. Wang, A. Bianco, A.A. Boghossian, S. Cambré, L. Cognet, S.R. Corrie, P. Demokritou, S. Giordani, T. Hertel, T. Ignatova, M.F. Islam, N.M. Iverson, A. Jagota, D. Janas, J. Kono, S. Kruss, M.P. Landry, Y. Li, R. Martel, S. Maruyama, A.V. Naumov, M. Prato, S.J. Quinn, D. Roxbury, M.S. Strano, J.M. Tour, R.B. Weisman, W. Wenseleers, M. Yudasaka, Banning carbon nanotubes would be scientifically unjustified and damaging to innovation, Nat. Nanotechnol., 15 (2020) 164-166.].

The coating part of the materials and methods section was rewritten to highlight  the importance of CNTs as electron acceptors in this and other systems. Additionally, two sentences and citations were added justifying the selection of CNTs (lines 539-544)

3) Figure 2: it seems that the picture has low resolution. Please check it 

Figure 2 has been reuploaded into the document provided by the journal in a scalable vector graphics format to preserve the resolution. The colors have been modified slightly to ensure readability. X axis labels had to be rotated to -60 degrees to accommodate font changes. Legend was moved below the figure to accommodate font changes.

3) Figure 3: units on y axes are missing. The picture has a frame around, the overall layout (e.g. font size) is completely different from previous one. Please unform it.

The frame around figure 3 was covering the y axis labels. The frame has been removed and figure 3 has been reuploaded into the document provided by the journal in a scalable vector graphics format to preserve the resolution. The colors have been modified slightly to ensure readability. The font style and size was normalized to the other figures in the manuscript. Legend was moved below the figure to for consistency.

4) Figure 4: same comments as before. 

Figure 4 has been reuploaded into the document provided by the journal in a scalable vector graphics format to preserve the resolution. The colors have been modified slightly to ensure readability. Legend was moved below the figure to for consistency.

Figure 5 was also modified for consistency: legend was moved below the figure, font was adjusted to match other figures, and the colors were modified to for legibility.

5) paragraph 2.2: "Laccases facilitate the four-electron reduction of oxygen to water while peroxidases facilitate the two-electron reduction of oxygen to peroxide which poisons the cell and can kill the fungus and inactivate functional laccases." Please insert reference for this statement: lack of references is one of the main issue of this paper. e.g. doi.org/10.1021/cs200527c and others that the authors will consider suitable. 

This sentence in the first paragraph of section 2.2 was rewritten and three citations were added to support its claims.

Parimi, N. S., Umasankar, Y., Atanassov, P., & Ramasamy, R. P. (2012). Kinetic and mechanistic parameters of laccase catalyzed direct electrochemical oxygen reduction reaction. Acs Catalysis, 2(1), 38-44.

Kataoka, K., Sugiyama, R., Hirota, S., Inoue, M., Urata, K., Minagawa, Y., ... & Sakurai, T. (2009). Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center. Journal of Biological Chemistry, 284(21), 14405-14413.

6) Table 1 and Figure 6: why water is added to the characterization? The authors have to describe in deeper detail this. 

Line 229 was rewritten to read: Water was used as a negative control to account for any structural changes to the electrodes during the coating process”

7) Figure 6 : J on y and E on  x axes are missing. 

The image has been reuploaded to the revised document to ensure axis labels are visible.

8) Figure 7: the SEM picture is just introduced and not described at all. It seems that it does not provide more details to text discussion. The authors have to decide if they want to describe this or remove from text. Furthermore, the picture seems to belong to paragraph 2.3 but it is inserted at the beginning of paragraph 2.4

The image has been moved to its correct position and a paragraph describing the SEM image has been added to lines 381-386. The SEM image was also annotated at the request of another reviewer.

9) The authors show linear sweep voltammetries (pictures 6 and 8) but they discuss in the text about open circuit potential and power density performances of the electrodes. I would recommend to couple linear sweep voltammetries with chronopotentiometry experiments and polarization curves to better describe the cells performances at open circuit potential and during their operation. see e.g. doi.org/10.1002/cplu.201500347 

The authors agree that additional electrochemical characterization of the system, especially chronopotentiometry and polarization curves, is desirable. However, this manuscript is written to give electrochemists in our group and other groups the biological footing to start the more detailed electrochemical characterization of the system while providing initial electrochemical validation via linear sweep voltammetry.

10) Average and replicates: the authors declare that they performed measures in replicates but standard deviations are sometimes missing. See, for example, values reported in Table 1: uncertainties are missing. About Linear Sweep Voltammetries: the authors have to provide replicates to demonstrate the reproducibility of results. 

The values in table 1 were modified to the open circuit potential and current density of the coatings during linear sweep voltammetry.

Replicates of Linear Sweep Voltammetries were not performed due to the high activity of pure laccase trials and expense incurred by the large volumes of electrolyte used in all trials.

11) Conclusion session is missing

A conclusions section has been added.

12) Materials and methods session: This session is poorly detailed (e.g. Linear sweep voltammetries: the scan rate, start potential, stop potential and numbers of scans are missing in the description of the experiment). Some repetitions (see lines 228-233 and 267-274: they are basically identical and they can be merged). Some concentrations are expressed in mol and others in g/L (see line 270) please use mol for all concentrations. I would recommend to the authors to rewrite this session with particular care. 

The materials and methods section was restructured and rewritten after consulting other publications in Catalysis. Repetitious sections were combined and concentrations of reagents are expressed in mol when the molecular weight of the compound is known. When the molecular weight of the compound is unknown (media reagents) the concentration was converted to weight-percent of reagent to solvent volume. 

13) Some spelling mistakes: see line 150, 170, 281 (mulipore?). 

Milipure water is the manufacturer’s term for 18MΩ deionionized water. To clarify this, the authors have replaced all instances of the Milipure term with “18MΩ deionionized water”

14) TiO2 nanoparticle synthesis: is this a novel sinthesis procedure or is it already published? Please justify or add reference. 

In the rewritten materials and methods section a reference to Wen, Z., Ci, S., Mao, S., Cui, S., Lu, G., Yu, K., ... & Chen, J. (2013). TiO2 nanoparticles-decorated carbon nanotubes for significantly improved bioelectricity generation in microbial fuel cells. Journal of Power Sources, 234, 100-106.

Was added as this is not a novel synthesis procedure.

Reviewer Comment or Suggestion

Author Correction or Rebuttal

The manuscript lacks quantitative comparison of CV performance with previous papers on laccase in MFC. A thorough table is needed.

The authors agree that a quantitative comparator is needed to give the linear sweep voltammetries context. To this end the results and discussion sections have been amended to give specific numbers from Sané, S., Jolivalt, C., Mittler, G., Nielsen, P. J., Rubenwolf, S., Zengerle, R., & Kerzenmacher, S. (2013). Overcoming bottlenecks of enzymatic biofuel cell cathodes: crude fungal culture supernatant can help to extend lifetime and reduce cost. ChemSusChem, 6(7), 1209-1215.

However, this comment also highlights a gap in the literature that is outside the scope of this article. A comprehensive review of microbial and enzymatic cathodes is needed to assemble a though table quatitatively comparing CV performance in differing architectures over the past 5-10 years. Differences between full and half cells, characterization factors, reference electrodes, and fabrication techniques make this table a large undertaking and one deserving of a separate review article.

"However, at higher CNT loading the nanotubes form aggregates significantly diminishing the effective surface area of the coating."  in SEM Fig. 7- Where are the aggregates?

Figure 7 is a scanning electron micrograph of the optimized coating without significant CNT aggregation. A reference was added supporting the phenomena of CNT aggregation in the coating.

"nanotubes form aggregates significantly diminishing the effective surface area of the coating" No surface area measurements were carried out.

References to Koh, B., & Cheng, W. (2014). Mechanisms of carbon nanotube aggregation and the reversion of carbon nanotube aggregates in aqueous medium. Langmuir, 30(36), 10899-10909.

Yang, Q. S., He, X. Q., Liu, X., Leng, F. F., & Mai, Y. W. (2012). The effective properties and local aggregation effect of CNT/SMP composites. Composites Part B: Engineering, 43(1), 33-38.

Mathieu, B., Anthony, C., Arnaud, A., & Lionel, F. (2015). CNT aggregation mechanisms probed by electrical and dielectric measurements. Journal of Materials Chemistry C, 3(22), 5769-5774.

Were added to cite the phenomenon. Since this is a common phenomenon we did not measure it directly.

Write the chemical reaction equations with their stoichiometry.

Chemical reaction equations have been added to the introduction for the substrate oxidation reaction and the oxygen reduction reaction on lines 56 - 59.

How were the coating mass and coating thickness determined?

The values in table 1 were modified to the open circuit potential and current density of the coatings during linear sweep voltammetry at the request of another reviewer.

This remves mass and thickness from the paper. Mass was measured using an analytical balance and thickness calculated using the density, mass , and electrode surface area..

Role of TiO2 is not clear

The text of section 2.3 was modified extensively to make the role of Tio2 clear and reference to Makhdoomi, H., Moghadam, H. M., & Zabihi, O. (2015). Effect of different conditions on the size and quality of titanium dioxide nanoparticles synthesized by a reflux process. Research on Chemical Intermediates, 41(3), 1777-1788.

Was added to further support the claim.  

"power density of pure laccase in sterile electrolyte was 20% higher than that of the crude laccase from the T. versicolor culture" Why?

Lines 381 - 384 and a reference to Rubenwolf, S., Sané, S., Hussein, L., Kestel, J., von Stetten, F., Urban, G., ... & Kerzenmacher, S. (2012). Prolongation of electrode lifetime in biofuel cells by periodic enzyme renewal. Applied microbiology and biotechnology, 96(3), 841-849.

were added to address this comment.

“This discrepancy is supported by the proteomics data (Figure 5) which confirms the presence of electrochemically inert enzymes in the crude filtrate. Inert enzymes like glucose oxidase and manganese peroxidase can occupy direct electron transfer sites that are otherwise used by laccase to produce current.”

Fig. 3 - where is the y-axis?

The frame around figure 3 was covering the y axis labels. The frame has been removed and figure 3 has been reuploaded into the document provided by the journal in a scalable vector graphics format to preserve the resolution. The colors have been modified slightly to ensure readability. The font style and size was normalized to the other figures in the manuscript. Legend was moved below the figure to for consistency.

"The high activities of extracellular laccases produced by F. oxysporum are likely due to F.oxysporum’s role as a plant pathogen while the high activity of cellular laccases produced by the T.90 versicolor and the P. ostreatus strains can be attributed to their roles as a lignin degrading saprotrophs." citation?? Check elsewhere for such instances.

The role of laccases in plant pathogens and saprotrophs is well documented. References to Patel, H., Gupte, S., Gahlout, M., & Gupte, A. (2014). Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1. 3 Biotech, 4(1), 77-84.

Canero, D. C., & Roncero, M. I. G. (2008). Functional analyses of laccase genes from Fusarium oxysporum. Phytopathology, 98(5), 509-518.

Thangadurai, D., Sangeetha, J., & David, M. (2016). Fundamentals of molecular mycology. CRC Press.

Were added to support the claims in this section.

"Laccases facilitate the four-electron reduction of oxygen to water while peroxidases facilitate the two-electron reduction of oxygen to peroxide which poisons the cell and can kill the fungus and inactivate functional laccases." Where is the citation????

Citations to Parimi, N. S., Umasankar, Y., Atanassov, P., & Ramasamy, R. P. (2012). Kinetic and mechanistic parameters of laccase catalyzed direct electrochemical oxygen reduction reaction. Acs Catalysis, 2(1), 38-44.

Kataoka, K., Sugiyama, R., Hirota, S., Inoue, M., Urata, K., Minagawa, Y., ... & Sakurai, T. (2009). Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center. Journal of Biological Chemistry, 284(21), 14405-14413.

Were added to support the authors claims.

"Figure 7. Scanning electron micrograph of the optimized coating with T. versicolor growing on top of the coating." label fungus and CNT

The fungus and the coating were labeled in figure 7.

"optimized coasting is depicted in Figure 7"

The typo on line 159 was rectified to “…optimized coating is depicted in figure 7.”

Reviewer Comment or Suggestion

Author Correction or Rebuttal

2: Line 51. The authors discuss faster reaction velocities at the laccase cathodes and additional work is required to address losses from other factors. I believe mentioning briefly, what these losses are could improve the quality of the content.

The authors clarified that

Kataoka, K., Sugiyama, R., Hirota, S., Inoue, M., Urata, K., Minagawa, Y., ... & Sakurai, T. (2009). Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center. Journal of Biological Chemistry, 284(21), 14405-14413.

Parimi, N. S., Umasankar, Y., Atanassov, P., & Ramasamy, R. P. (2012). Kinetic and mechanistic parameters of laccase catalyzed direct electrochemical oxygen reduction reaction. Acs Catalysis, 2(1), 38-44.

Refer to current density loses.

Results section: 2.1 subsection line 78 on Pg.2, I believe there is a typo. The current version reads produced 54 less than……Please confirm.

Typo on line 78 is confirmed. The typo has been rectified to read “…produced less than 10 U*mL^-1 of laccase.”

Figure 2 caption has an extra period at the end of the sentence.

This seems to be an artifact of converting the PDF to a word document. All extra periods at the end of all captions have been removed.

Figure 3, x-axis label is missing, font size is different than figure 2 and the plot has an extra shaded background. Please keep all the plots of same fonts and types.

The frame around figure 3 was covering the axis labels. The frame has been removed and figure 3 has been reuploaded into the document provided by the journal in a scalable vector graphics format to preserve the resolution. The bar and background colors have been modified slightly to ensure readability. The font style and size were normalized to the other figures in the manuscript. Legend was moved below the figure to for consistency.

Line 112 on pg.4 has an extra period at the end of the sentence.

The extra period has been removed. Additional changes to the manuscript resulted in this becoming line 120.

The authors mention, “The use of copper sulfate as an inducer leads to concerns of heavy metal sequestration by T. versicolor precluding its use for medicinal purposes. We explored Tween 20 as an alternative laccase inducer as the detergent causes oxidative stress to the fungus and is non-toxic to humans and animals.” And also,” However, the small reduction in electrical production could be offset by the benefits of a valuable byproduct in edible or medicinal biomass that is not contaminated with heavy metal inducers.” What is the criteria for choosing CuSO4 as an inducer over the mixture or Tween20 if the there was no significant differences between biomass from any of the treatments and clearly less to none heavy metal contamination? Activity of Fo, N001 looks similar for mixture and CuSO4 except for Tv which is slightly lesser for mixture. Is that significant to overrule the use of mixture as an inducer?

This was a difficult choice to make during the study, but eventually copper sulfate was chosen as the inducer for the remaining studies due to the requirements of the proteomics study. The authors’ desire to characterize the secretome of the fungus in the cathode meant that an inducer compatible with proteomics methods was required.

Lines 160-164 were changed to address this comment to read: Despite the absence of heavy metal contamination in Twwen20-induced cultures, copper sulfate was chosen as the inducer for subsequent studies because proteomics is needed to provide a deeper insight into the mechanistic differences between crude filtrate and pure laccases in fuel cell cathodes. Tween20 can cause signal loss in proteomic characterization and contaminates mass spectrometry instruments used in proteomics.”

Table 1 talks only about composition, avg. coating mass and thickness. Subsection 2.3, line 143 figure 6 need to be added in addition to the mention of table 1.

Additional references to figure 6 were added in section 2.3 to address this comment.

Subsection 2.4, line 170, space need to be provided between was and performed.

Additional revisions moved this to line 194. The additional space was added.

Extended spacing is provided in discussion 2^nd para line 186 on Pg.8.

Additional revisions moved this to line 210. The extended spacing was an artifact of converting the PDF to a word document and has been corrected.

Statistical significance need to be calculated and plotted wherever necessary.

ANOVA and a multiple comparison mean tests were performed in MATLAB 2020b (Mathworks, Natick, MA) according using Sídák's multiple comparisons test for one way ANOVA (figures 2 and 4) and two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for two-way ANOVA (figure 3). In figure 4 Means that do not differ from P. ostreatus N001 induced with CuSO4 by a p-value of 0.05 are annotated with “a”; Means that do not differ from P. ostreatus Blue induced with CuSO4 by a p-value of 0.05 are annotated with “b”; Means that do not differ from P. ostreatus N001 induced with water by a p-value of 0.05 are annotated with “c”. In figure 3 Means that do not differ from T. versicolor induced with CuSO4 grown in 20 mL reaction vessels by a p-value of 0.05 are annotated with “a”; Means that do not differ from T. versicolor induced with CuSO4 grown in 200 mL reaction vessels by a p-value of 0.05 are annotated with “b”; Means that do not differ from T. versicolor induced with water grown in 200 mL reaction vessels by a p-value of 0.05 are annotated with “c”. In figure 2 Means that do not differ from P. ostreatus White grown for 4 days after induction with CuSO4 by a p-value of 0.05 are annotated with “a”; Means that do not differ from P. ostreatus Blue grown for 4 days after induction with CuSO4 by a p-value of 0.05 are annotated with “b”; Means that do not differ from P. ostreatus Blue grown for 2 days after induction with CuSO4 by a p-value of 0.05 are annotated with “c”. Means that do not differ from P. ostreatus Blue measured immediately after induction with CuSO4 by a p-value of 0.05 are annotated with “d”.

ANOVA results are given with the figure data.

The biggest concern with using laccase as an enzyme at cathode is its instability and operation conditions. Laccase is known to operate at acidic pH and researchers also explore bilirubin oxidase as a potential replacement. As the authors have comprehensively characterized these cathodes, what is their thought on the operating pH and temperature for 5 fungi based cathodes. I would suggest the authors perform variable pH and temperature studies as they would complete the characterization and make a complete story.

Effects of temperature and pH have been extensively studied on laccase activity.

Mazlan, S. Z., & Hanifah, S. A. (2017). Effects of temperature and pH on immobilized laccase activity in conjugated methacrylate-acrylate microspheres. International Journal of Polymer Science, 2017.

Kim, K., Kim, E., & Lee, S. J. (2012). New enzymatic time–temperature integrator (TTI) that uses laccase. Journal of Food Engineering, 113(1), 118-123.

Ghosh, P., & Ghosh, U. (2017). Statistical optimization of laccase production by Aspergillus flavus PUF5 through submerged fermentation using agro-waste as cheap substrate. Acta Biologica Szegediensis, 61(1), 25-33.

Mazlan, S. Z., & Hanifah, S. A. (2017). Effects of temperature and pH on immobilized laccase activity in conjugated methacrylate-acrylate microspheres. International Journal of Polymer Science, 2017.

Zhang, R., Wang, L., Han, J., Wu, J., Li, C., Ni, L., & Wang, Y. (2020). Improving laccase activity and stability by HKUST-1 with cofactor via one-pot encapsulation and its application for degradation of bisphenol A. Journal of hazardous materials, 383, 121130.

The authors are interested in the effects of temperature and pH on the fungi-based cathodes but feel this is outside the scope of this paper. It is the hope of the authors that the characterization of this system will be continued by the authors and other research groups to show the reproducibility of the system.

The authors agree that temperature and pH studies would help complete the story and a statement to this effect has been added in the discussion section.

Same should be discussed in the discussions section as well.

The authors agree that temperature and pH studies would help complete the story and a statement to this effect has been added in the discussion section.

Smaller reaction vessels yielded highest activity. Possible ideas behind such observation needs to be discussed in discussion section.

The statement “While the mechanism for this is unclear, it may be related to the ability of laccases to degrade phenolic compounds that arrest fungal growth.” Was added, with support from Baldrian, P. (2006). Fungal laccases–occurrence and properties. FEMS microbiology reviews, 30(2), 215-242.

 Cited to add depth to the discussion of this finding.

Statistical significance calculation and the method used to do so need to be mentioned in methods section as a subsection.

ANOVA and a multiple comparison mean tests were performed in MATLAB 2020b (Mathworks, Natick, MA) according using Sídák's multiple comparisons test for one way ANOVA (figures 2 and 4) and two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for two-way ANOVA (figure 3). In figure 4 Means that do not differ from P. ostreatus N001 induced with CuSO4 by a p-value of 0.05 are annotated with “a”; Means that do not differ from P. ostreatus Blue induced with CuSO4 by a p-value of 0.05 are annotated with “b”; Means that do not differ from P. ostreatus N001 induced with water by a p-value of 0.05 are annotated with “c”. In figure 3 Means that do not differ from T. versicolor induced with CuSO4 grown in 20 mL reaction vessels by a p-value of 0.05 are annotated with “a”; Means that do not differ from T. versicolor induced with CuSO4 grown in 200 mL reaction vessels by a p-value of 0.05 are annotated with “b”; Means that do not differ from T. versicolor induced with water grown in 200 mL reaction vessels by a p-value of 0.05 are annotated with “c”. In figure 2 Means that do not differ from P. ostreatus White grown for 4 days after induction with CuSO4 by a p-value of 0.05 are annotated with “a”; Means that do not differ from P. ostreatus Blue grown for 4 days after induction with CuSO4 by a p-value of 0.05 are annotated with “b”; Means that do not differ from P. ostreatus Blue grown for 2 days after induction with CuSO4 by a p-value of 0.05 are annotated with “c”. Means that do not differ from P. ostreatus Blue measured immediately after induction with CuSO4 by a p-value of 0.05 are annotated with “d”.

ANOVA results are given with the figure data.

Conflicts of interest: line 312, I believe there is a typo in”…..in the decision to 264 publish the results.”

The section has been corrected to read: “The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.”

Conclusion is missing. Need to incorporate it in the manuscript.

Conclusion section has been added to the manuscript.

Reviewer Comment or Suggestion

Author Correction or Rebuttal

The manunscript has been improved after the first revision. Moreover there are still some points that remian unclear:

N/A

1) the authors stress the point about the suitability of their enzymatic cathodes for Microbial Fuel Cells, moreover the Fuel Cell design details as well as polarization and power density curves in Microbial Fuel Cells prototypes are not presented in the paper. These measures and details are required and they will strongly support the author's conclusions.

The authors added the fuel cell design details in the materials and methods section on lines 372 -377.

The authors agree that polarization curves would support the suitability of the hybrid microbial-enzymatic cathode for microbial fuel cells. Statements in the results section (lines 250 – 252) and the discussion section (lines 291 – 295) were added to acknowledge this weakness and suggest that future research designs include polarization experiments.

The authors have added power density plots to the manuscript as supplemental figures 1 – 5.

2) In the conclusions the authors stressed the self regeneration of their proposed cathode, thus suggesting long life cathode: I will therefore reccomend to the authors to perform galvanostatic experiments, as reported in doi.org/10.1021/cs2003142, to effectively demonstrate their cathode stability. See also doi.org/10.3390/en3040803 for the stability tests experiments timescale. 

The authors agree that quantifying the long-term stability using galvanostatic experiments is the best method to demonstrate cathode stability. This study was designed to focus on screening fungi for suitability in the proposed cathode, the proteomic differences between suitable and unsuitable fungi, and the novelty of incorporating the laccase producing fungus at a direct electron transfer cathode. The authors have acknowledged the lack of quantification of cathode lifetime (lines 250 – 252) and suggested that future research designs be modified with galvanostatic experiments in the Discussion section of the revised manuscript (lines 291 – 295). 

3) lines 329-335 should not be presented in materials and methods but in introduction.

Lines 339 – 335 were moved to new lines 43 – 49. An additional sentence was added on lines 52 and 53 to help transition between introducing CNTs and laccases. The sentence on lines 52 and 53 references

Zhang, Y., Lv, Z., Zhou, J., Xin, F., Ma, J., Wu, H. & Dong, W. (2018). Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects. Applied microbiology and biotechnology, 102(24), 10409-10423.