Peer review of: Single cell electron collectors for highly efficient wiring-up electronic abiotic/biotic interfaces (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The aim of this study was to propose a new concept of single cell electron collector, which was in-situ 
built up with an intact conductive layer on and cross the individual cell membrane. The authors used 
this method to achieve record-high interfacial electron transfer efficiency and bioelectrochemical 
systems performance. However, the authors fail to distinguish this work from previous publications. 
Therefore, I recommend a major revision of the manuscript as indicated. 

1. How to identify the interfacial electron transfer efficiency of single cell? In BES, please provide the 
CLSM or SEM of the bacteria on the carbon electrode. Was the biofilms formed? The interfacial 
electron transfer of single cell or biofilms is different (Torres CI et al., Fems Microbiol Rev, 2010, 34: 
3-17). Electrochemical workstation and in-situ micro imaging and micro fabrication technology might 
help to conduct this experiment (please refer to Jiang et al, P Natl Acad Sci USA, 2010, 107 (39), 
16806-16810; Jiang et al., Nat Commun 2013, 4, e2751). 

2. As widely reported, many research groups have used the similar ways to achieve record-high 
interfacial electron transfer efficiency. For instance, Du et al., Environ. Sci. Technol. Lett. 2017, 4 (8), 
345-349, intact conductive layer on the individual cell membrane; Wu X et al., Angew. Chem. Int. 
Edit., 2011, 50 (2), 427-430, intact conductive layer cross the individual cell membrane. The authors 
should clearly elaborate the significant improvement of this work. 

3. Electron shuttle is well known to efficiently mediate microbial EET for Shewanella oneidensis MR-1. 
This point should be added into the introduction. Also, more discussion about this issue should also be 
added. Was this intact conductive layer a type of solid electron shuttle? 

4. In order to exclude other reasons to improve the interfacial electron transfer efficiency, the 
interfacial electron transfer efficiency between the native Shewanella oneidensis MR-1 and the 
electrode modified with PDA or FeS2 should be examined. 

5. Considering that an intact conductive layer was on and cross the individual cell membrane, could 
the interfacial electron transfer efficiency maintain at a high level during the bacterial cultivation 
period? 




Reviewer #2 (Remarks to the Author): 

This submitted work attempts to maximise the interfacial electron transfer and bioelectrochemical 
systems (BES) performance between living electroactive cells and conductive abiotic surfaces. This 
engineering attempt is termed as wiring to enhance intercellular connection because close surface 
contact is essential for maximal electrical output. The author justified the work in reference to current 
methods that lack in improving the cellular properties for efficient electron transfer, but only focusing 
on the properties of the conductive abiotic surfaces. For electron transfer improvement, the work 
targeted two parts of the model electroactive organism, Shewanella oneidensis MR-1: (1) “idling” 
transmembrane electron conduits and (2) “dead” periplasmic electron conduits, both of which are not 
in contact with electrode surface. To engage these inactive electron conduits, the electroactive 
bacteria were encapsulated with either polydopamine (PDA) or anchoring FeS nanoparticles in the 
periplasmic and on outer membrane surface, to act as electron collectors that was shown to increase 
the BES performance by 1.4- to 3.2-fold, respectively, in comparison to the intact cells. This work 
demonstrates a proof-of-concept for optimizing BES performance by modifying cell surface chemistry 
towards contact enhancement with a conductive surface, of which is an attractive strategy to be 
exploited for potential bioelectronic applications. This is a very interesting paper and a good read. 

Overall, the results are quite impressive but there are some concerns that need to be resolved: 

1. Introduction of PDA and FeS as nanomaterials were rather abrupt. How did the author decide on 
the materials to be use as electron collectors? Was there a panel of materials compared or at least 
considered to give the desired pooling of electrons like PDA? 

2. In reference to Figs 2 and 3, the TEM image of native SW surface (2d) displayed homogenous 
distribution of its cytoplasmic content. However, the TEM images of SW@S (2e & 2f) and SW@SP (3c) 
displayed significant clusters of its cytoplasmic content. It seems to concentrate at the outer 
membrane in SW@S, while its clumps in SW@SP. Is there any explanation on these observations? 

3. The authors must briefly describe the Live/dead staining method, for example, how long it was 
incubated post staining, dyes used and excitation/emission wavelengths of the respective dyes. The 
live/dead stained images, (Fig 2g and Fig 3f), shows all cells to be live. Just wondering how come 
there is not even a single dead/membrane compromised cell. It would be good to quantify the amount 
of live/dead cells, post encapsulation (S and SP), from multiple images and present the percentage of 
live and dead cells. 

4. Live/Dead stains gave a reasonable idea on their viability but do these cell surface modifications 
have any effect on their growth? The surface modifications are not of genetic engineering nature. 
Hence, it is not a feature that can be inherited for the following generations. Encapsulation/FeS 
anchoring would be diluted while cells grow. This aspect needs to be explained/discussed. 

5. The work discussed on the possibility of networking as an electroconductive biofilm. Technically, 
biofilms form through their embodiment in a self-secreted biological matrix (extracellular polymeric 
substances, EPS). In relation to my concern #4, if the surface modification is not inherited, the 
mentioned biofilm could possibly consist of both surface modified and native cells as well as large 
amount of EPS. In this case, the discussed “construction of artificial conductive network” (page 13, 
line 252) may still be possible but dampened with presence of unmodified cells and EPS. It would be 
good if the authors could provide some biofilm (including EPS) images of S or SP capsulated cells. The 
effect of EPS should be discussed. 

6. The process of S collector assembly has been explained on page 14 (line 289- 294). Process needs 
more details for the reproducibility. How much time was required for polydopamine coating on the 
surface of bacteria? How thick is the PDA layer and how does the thickness affect the EET 
performance? Similarly, since S collector and SP collector are based on nanostructures, how does the 
size of these nanostructures affect the EET performance? 

7. The effect of PCP on the current output has been shown on Page 9 (line 181-187). However, 
authors did not mentioned the concentration of PCP used in the methods section (page 17, line 338-
340). The concentration of inhibitor is critical as we do not know if the same concentration of inhibitor 
was used for all 3 types of cells or not. 

8. Bioelectronic setup (page 16, line 322-333) needs more details. The authors mentioned that in-situ 
assembly of SP collector was done by adding precursors in same ratio (line 328-330). However, the 
time required for in-situ assembly should also be mentioned here. Was it the same as that required for 
general process of SP collector assembly on the cells? 

9. It was mentioned earlier (page 15, line 296) that anaerobic conditions are necessary for FeS based 
SP collector assembly. How anaerobic conditions were maintained during in-situ assembly (page 16, 
line 322-333)? 

10. Figure legend 2g and 3f: Please change “strained” to “stained”. 
11. Supplementary Figure S2: HRTEM image of FeS nanoparticles collected from SW@SP, not clear. 
Please replace the image.