Enterococcus faecalis NADH Peroxidase-Defective Mutants Stain Falsely in Colony Zymogram Assay for Extracellular Electron Transfer to Ferric Ions
Round 1
Reviewer 1 Report
It’s an interesting paper but confusing at points. I think the results could be reframed to enhance readability. The stated goal of the paper is to identify mutants with enhanced ferric reductases activity in the presence of heme but then most of the discussion is of a mutant that has reduced activity in the absence of heme. The author may be better served but just focusing on the apparent loss of EET phenotypes.
Specific points
The author might think about changing the title. It might confuse some readers to have "the role of npr in EET" in the title when the conclusion is that it doesn't have a role in EET.
The abstract is confusing, doesn’t fully convey the findings and conclusions, and should be further edited.
Line 52: I find this statement confusing “Desired mutants with increased EET were not found…” since you’ve yet to explain that you think the npr mutants are an artifact.
Line 114: The description of the screen is confusing.
Line 133: I’m confused about the point being made.
Line 169: I’m a little confused. The screen was done in the presence of heme with the goal of identifying mutants with enhanced EET activity. That is how npr was identified. Why then is this section focused on the no heme condition in which npr was observed to have less apparent activity?
Line 198 refers to “WY85. ” Is WY84 meant? If not, what is WY85?
Line 228: The author infers that hydrogen peroxide accumulation inhibits the ferrozine assay. While this interpretation is consistent with the results, more could be done to test this hypothesis. For example, a simple assay would be to test whether culture supernatant from npr cells inhibits activity and whether this inhibition is relieved by the addition of catalase.
Author Response
Response provided in attached file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
EET review ​ Anode biofilm review paper: review ​ Anode is a very important factor in MFC performance. It looks interesting, but it needs improvement before publication. ​ 1. A number of figures is very small and their qualities are not good. Please make more necessary figures and increase their quality. ​ 2. These papers compared electrochemistry of Geobacter and Shewanella in MFC. These two bacterial species are very important biocatalysts in MFC. Please include them in your introduction and discussion. - Impedance Analysis of Geobacter sulfurreducens PCA, Shewanella oneidensis MR-1, and their Coculture in Bioeletrochemical Systems. Int J Electrochem Sci 2012 Nov; 7(11): 11091-11100 - Shewanella secretes flavins that mediate extracellular electron transfer. Proc Natl Acad Sci U S A. 2008 Mar; 105(10): 3968-73 - Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms. App Environ Microb 2008 Dec; 74(23): 7239-7337 ​ 3. Overall, there are unnecessary and confusing statements throughout the manuscript in the abstract and the introduction. Please rewrite and emphasize key findings. ​ 4. EET can be used in microbial elecrtrochemical systems such MFC, MEC, MDC, MRC. etc. Please introduce them by citing related references like below: MEC (methane) - Microbial electrolysis cells for electromethanogenesis: Materials, configurations and operations. Environ Eng Res 2022 Feb; 27(1): 200484 MEC (hydrogen) - Comparison of hydrogen production and system performance in a microbial electrolysis cell containing cathodes made of non-platinum catalysts and binders. J Water Process Eng 2021 Apr; 40: 101844 MDC - Microbial desalination cell: Desalination through conserving energy. Desalination 2022 Jan; 521: 115381 MRC - Influence of flowrates to a reverse electro-dialysis (RED) stack on performance and electrochemistry of a microbial reverse electrodialysis cell (MRC). Int J Hydrogen Energ 2017 Nov 9; 42(45): 27685-27692 MESC - Valorization of CO2 into value-added products via microbial electrosynthesis (MES) and electro-fermentation technology. Fermentation 2021 Nov; 7(4): 291 ​ 5. Please reorganize tables, which is hardly readable ​ 6 There is no critical discussion and key findings in this paper. Please provide them.
7. There is no impact of highlights. Please modify them so that the key findings are clearly shown.​
​ 8. Please introduce limiting factors which enhance EET in the biological anode. Please improve your manuscript by citing relevant articles like below: (1) Anode biofilm and mediators - Impedance and Thermodynamic Analysis of Bioanode, Abiotic Anode, and Riboflavin-Amended Anode in Microbial Fuel Cells. B Korean Chem Soc 2012 Oct; 33(10): 3349-3354 B Korean Chem Soc 2012 Oct; 33(10): 3349-3354 (2) Anode current collectors - Effects of wire-type and mesh-type anode current collectors on performance and electrochemistry of microbial fuel cells. Chemosphere 2018 Oct; 209: 542-550 (3) Anode structure and materials
- Comparative evaluation of performance and electrochemistry of microbial fuel cells with different anode structures and materials. Int J Hydrogen Energ 2017 Nov; 42(45): 27677-27684
(4) Anode configurations: - Effects of brush-anode configurations on performance and electrochemistry of microbial fuel cells. Int J Hydrogen Energ 2017 Nov; 42(45): 27693-27700 (5) Anode number
- Effects of vertical and horizontal configurations of different numbers of brush anodes on performance and electrochemistry of microbial fuel cells. J Clean Prod 2020 Dec; 277: 124125
(6) medium pH - Impedance and polarization behavior were tested in MFCs in different medium pH (Environ Sci Technol 2011 Oct 15; 45(20): 9069–9074). (7) external resistance - Influence of external resistance on electrogenesis, methanogenesis, and anode communities were tested in MFCs (Appl Environ Microb 2011 Jan 15; 77(2): 564-571) (8) substrate concentration: Effects of substrate concentrations on performance of serially connected MFCs were tested in a continuous mode (Biotechnol Lett 2012 Oct; 34(10): 1833-1839) (9) anode biofilm maturation - Anode biofilm maturation time, stable cell performance time, and time-course electrochemistry were tested in a single-chamber MFCs (J Ind Eng Chem 2022 Feb; 106: 269-278) (10) anode biocatalysts - Impedance of anodes inoculated with Geobacter sulfurreducens, Shewanella oneidensis, and their Coculture were tested in MECs (Int J Electrochem Sci 2012 Nov; 7(11): 11091-11100 ) (11) anode mediators - Impedance and polarization of bioanode, abiotic anode, and riboflavin-amended anode were tested in MFCs (B Korean Chem Soc 2012 Oct; 33(10): 3349-3354)
Author Response
Response provided in attached file.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
They have not done the necessary corrections as suggested.
Author Response
The original submitted manuscript has been revised based on the comments of both reviewers and according to the responses to the comments. In the revised manuscript the title is changed, the abstract rewritten, and there are substantial changes in the text.
In the comments to the first version, Reviewer 2 indicated that the manuscript can be improved and provided comments. Now, regarding the revised manuscript, it is indicated that the manuscript must be improved but no specific comments are presented concerning what and why matters must be improved.
