Specific Desulfuromonas Strains Can Determine Startup Times of Microbial Fuel Cells
Round 1
Reviewer 1 Report
Specific Desulfuromonas strains can determine the time required to reach steady-state operation in microbial fuel cells:
The biggest issue is with the title: Most microbiologists would agree that it is impossible to achieve a steady state in batch culture. What you describe as steady state is simply the point when deceleration phase of growth gives way to the stationary phase of the batch cycle which stays constant for a period of time until nutrient limitation or product inhibition gives way to the decline phase.
The aims are briefly mentioned at the end of the introduction, where the authors state (lines 79-84): “Here, we analyze patterns of the MFC startup phase across three separate experiments to further investigate the microbial dynamics proposed to affect startup times, focusing on Desulfuromonadaceae strains using a specific Desulfuromonas-targeted PCR 81 primer set. Furthermore, direct application of an Desulfuromonas-enriched consortium was attempted in order to substantially reduce startup times relative to those observed by inoculating MFCs with a wastewater sample alone”.
For MFC that are designed to work over long periods of time (e.g over ten years in one experiment), the start up time is such a small part of its operation it can easily be ignored. Whether it takes a day or two or a week or two does not really matter. Your system is batch culture recycle, keeping the biofilm alive throughout the cycles. i.e. Once a fully mature biofilm is formed it stays much the same thereafter.
The whole approach has errors:
- Batch culture even when repeated in cycles with the same biofilm electrode, as a closed system can never reach a true dynamic steady state. What you refer to as steady state is a pseudo- or quasi- steady state which simply means a period of time where measured outputs appear stable. It does not mean balanced dynamic growth; indeed the growth rate is very low to non-existent during this period (stationary phase). This is in complete contrast to a true steady state in a continuous flow system, where cells continue to grow at a set growth rate during steady state and it is truly dynamic.
- I notice that your experiments are conducted in relatively large volume MFC. You refer to previous work when describing the MFC and should always include basic features like size and volume and whether operated in batch fed repeat cycling mode. I found it difficult to source the journal where you have set out the details. Since you are dealing with a Desulfuromonas, what was the sulphur or sulphate levels in your system?
Yanuka-Golub K, Reshef L, Rishpon J, Gophna U. 2016. Community structure dynamics during startup in microbial fuel cells - The effect of phosphate concentrations. Bioresour Technol 212:151–159.
Firstly, MFC designs vary enormously from microfluidic to 20L in size, batch or continuous flow.
In a closed batch system the physicochemical environment is constantly changing, cells and products accumulate during growth. Microcosms that can abstract electrons from the carbon energy source and use the anode as the terminal electron acceptor can do this by more than one mechanism (e.g. soluble mediator, sulphate reduction into H2S, direct conduction via cytochromes to name but three). For large scale (> 20 ml) MFC the combination of low power density, types of electrode, and zero rate of flow of liquid make it very likely that conditions favour the formation of thick diffusion-limiting biofilms at low growth rate. This, plus the increasing concentration of planktonic as well as biofilm populations favours the case that soluble mediators will be playing an important role in power production. Your distribution of planktonic cells outnumbers the biofilm population (also favouring soluble mediators). The contribution by species that can use soluble mediators is located anywhere in the anode chamber; planktonic just as much as biofilm. The contribution of soluble mediators drops to practically zero with continuous flow systems at high flow rate, because mediator washes away. If species are inoculated in conditions of flow, they become more selective for species with high affinity binding to the electrode. Most exoelectrogenic conductive types of species express high affinity binding to the electrode. These are selected and enriched for during early colonisation. Once a thin biofilm type of electrode is matured, its ecological profile is fairly “fixed” providing the physicochemical conditions do not change to any great degree and that C/E supply rate determines the cell growth rate (as it does in a chemostat). The liquid flow rate produces a shear rate that removes new progeny, so the biofilm remains “thin” and the cells grow quickly, providing the C/E supply rate is limiting growth. The distribution of growth rates is tightly clustered around the mean (i.e. cells are fairly homogeneous).
Secondly, the most important measure of fitness and adaptation is growth rates of the colonising species, yet you have not used quantitative methods to count the cells (both on the biofilm and appearing in the planktonic phase) and calculate growth rate. There are many methods to sample and enumerate cells, none of which were used in your research. The growth rate of attached cells in a thick biofilm (>50 micron) is doomed to be relatively slow because of diffusion limitation and slow transport and penetration of nutrients. Growth rates are very heterogeneous. In large scale MFC the planktonic biomass can be greater in total than the biomass on the biofilm electrode. This ratio changes for small scale MFC because the surface area to volume ratio (SA:V) increases the smaller the scale. At small scale (e.g. <20ml) the main contribution to electricity generation is the biofilm population much more so than the planktonic cells.
Batch culture will probably never be used in manufactured MFC of the future, because it is far less efficient than continuous flow and more difficult to control the ever-changing physicochemical environment. Your model is definitely different to continuous flow small scale MFC, so you cannot generalise from your model to that of a different type of MFC.
Different species have different phenotypes and different growth rates. So, it is not possible to compare different species since the physicochemical environment that you set for both, may be closer to the optimum for one species than it is for the other, e.g. marine species prefer higher salinity than terrestrial species. So, what concentration should you use? The physicochemical setting is more important than the species difference for exoelectrogens. To compare mixed species (e.g. Shewanella, Geobacter and Desulfuromonas) requires data from a wider range of environmental settings, not just one or two.
Page 130: 2.5. Electroactive consortium enrichment on agar plates: Authors describe viable recovery and enrichment using samples plated on to BCM-Sodium Fumarate plates. This would have been a good opportunity to carry out viable counts of one type or another to give you a quantitative approach. Otherwise you are left with DNA analysis to describe abundance, which is not the same as a quantitative assessment of viable populations.
Community and phylogenetic analysis: I assume abundance difference shows as brightness of the PCR-line. If this is the case, then abundance is always greater in the biofilm than in planktonic for the efficient MFC, but the other way round for the inefficient MFC. Is this the case for the rest (when comparisons of anode biofilm and planktonic cells have been made. Why did you not co-sample?
The key to understanding ecology comes from knowing total population levels and distribution (biofilm or planktonic), species composition and growth rates, but only one of these has been accessed in this study.
Otherwise the findings are of minor interest; some ecologies are quicker at starting up than others in your system but nearly all ended up with similar power output levels in fullness of time as shown in supplementary material, S2. I do not know why the data is not expressed in power output rather than voltage. Electricity production depends on metabolic power, not a voltage. Growth rate is proportional to metabolic reducing power of the cell which is measured in Watts (or microwatts). This is proportional to the power output of the MFC in watts not volts. There were only a few examples where power output remained low even after numerous cycles.
On line 197 you state “even MFCs that had a non-efficient startup phase yielded a PCR product with the Desulfuromonas dechlorinator-targeted primers at later stages of the MFC operation (Figure 4).
Clearly this species or very closely related species or strain is strongly associated with the anodic microflora. Is it classed as a soluble mediator type, or a direct conductor to the anode type or has it not been established?
Isolation of putative strain that reduces start up time should be grown in monoculture for testing, using sterile input of medium and pure culture.
Additional experiments are required.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript “Specific Desulfuromonas strains can determine the time required to reach steady-state operation in microbial fuel cells” by K. Yanuka-Golub and colleagues is interesting to the journal readers but requires improvement.
General comments. The introduction is adequate; material and methods must be improved and the other sections could be improved in order to improve clarity.
The names of the phylogenetic categories must be in italic.
The figures should be improved because its interpretation is not straightforward.
Specific comments:
Lines 12-17. I suggest that the abstract appears in first place. The “Featured Application” if could not be included in the abstract section could be described in the introduction section for instance in the end of this section together with the main goal of the work.
Lines 33-34. I suggest that either all or none of the keyword start with capital letters.
Line 85. Materials and methods section. At some point in this section, the procedure for DNA electrophoresis must be described.
Line 97. Specify the culture media used
Line 101. Specify the trace elements
Lines 132-133. “Sample was vortexed at half of maximal speed, on the 132 flat-bed adaptor, for 2.5 minutes.” This sentence does not give any concrete information since we do not know which vortex was used and its maximum speed. I suggest the introduction of the number of rpm.
Lines 133-134. “At this point anode "dust" was observed but most of the anode remained whole.” Explain the meaning of this sentence. It is not clear.
Lines 134-135. “100μl of each crude sample and of a x10 dilution (in BCM) were plated (using a plastic Dregalski stick) on BCM-Sodium Fumarate plates (Supplementary Table 1).” This is also not clear. Did the authors meant that 100μl crude sample and of a 10-fold dilution were plated? If yes, how they were plated? The plating method and not the tool used to do it should be described.
Lines 135-136. “Plates were kept 135 in an anaerobic jar at room temperature in the dark.” It was used an anaerobic jar only or an anaerobic atmosphere. If it was the second, the apparatus used to produce the anaerobic atmosphere should be mentioned.
Lines 138-140. Refer when the analysed results were considered significantly different.
Line 159. This is a scientific study so wished is not an adequate word. Suggestion: In this study, our main goal was to ....
Line 183. Table 2. Either the unit of the abundance is introduced or being undimensional the authors should describe which abundances were used to compare the ratio.
Line 187. Figure 3. The figure should be modified in order to increase clarity. I suggest that the lanes in which no samples were loaded should be removed. The numbers under the wells are the samples ID number or the abundance in %. Was the Desulfuromonas' pure DNA used as control? Why is the band so weak? Specify the amount of DNA loaded per well in the material and methods section.
Line 201. Figure 34. This figure needs improvement. Check if Desulforomonas pure DNA is a non-efficient sample or just a control.
The third and fourth lanes (planktonic) are both from day 39? Is this a mistake? If not explain why a duplicate is used only here or was DNA from different planktonic cultures used only for this day? The lane (from the right) is a DNA ladder but this is not said by the authors.
Line 218. Figure 5. I suggest the sequential numbering of the lanes. It is weird that we have numbers from 1-6 and then letters. How does the authors explain the weak bands with the same molecular weight in the lane loaded with DDW (negative control) and in lane S with a lower molecular weight?
The authors should consider using another image without overexposure of the gel.
Lines 229-230. “Figure 6 compares the lag time and time to reach steady-state current density for all experiment sets conducted in this study.” This might not be true since the figure legend sends the reader to table 5.
Line 240. Figure 6. Check the Y- axis labels. In both graphs in this axis, the different experiment batches are represented. If my interpretation is wrong then a legend for the different colours is missing.
Lines 251-254. Consider re-arrange the text because it is not clear.
Lines 261-264. “The Desulfuromonas dechlorinator primer set has previously been shown to be specific towards D. acetexigens ,D. chloroethenica, and Desulfuromonas sp. strain BB1 but other Desulfuromonas strains tested, including D. thiophila, Desulfuromonas palmitatis, D. acetoxidans, D. succinoxidans, P. acetylenicus, and G. metallireducens, did not result in amplification (23). “
Consider revising because it looks meaningless as it is. Does the author meant Pelobacter acetylenicus and Geobacter metallireducens? Since the bacteria used/ referred in the manuscript are not trivial I suggest that the first time they appear the author write their full names (genus and species).
Line 288. “…specific Desulfuromonas strain…” Clarify if it is strain or species specific.
Line 292. Check if the “5” is required.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript describes experimental work concerning Microbial Fuel Cell (MFC) operation, particularly observation of forming a potential biomarker for monitoring the formation of an efficient anodic biofilm to analyze patterns of the MFC startup phase and investigate the microbial dynamics that affect startup times.
The abstract clearly states the scope of the experiment, providing information about the work's main findings. The introduction explains the principles of MFC operation and is well integrated with the main objectives. The authors provide numerous significant and well-selected references.
The experimental set up was well designed and comprehensively described. Authors provide an in-depth analysis of obtained results, developing evidence that may be utilized in having additional control over MFC systems and expand the understanding of its operation.
I recommend the manuscript for publication, and I encourage authors to consider the following suggestions:
- In line 92, the authors mention the timing for collecting the wastewater sample for the experiment 2015 and 2016. There is no mention of timing for experiment 2016-2, or it is not stated very clearly.
- The manuscript might benefit from providing the separate “Conclusions” section at the end of the text.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors have responded positively to my previous review comments and have produced a much-improved manuscript. The authors have changed the most problematic areas, including corrections to the title and the abstract. They have robustly defended some of the less important points that I made. All-in-all, I am now happy for the manuscript to be accepted for publication.
Author Response
Thank you for your important points you raised.
Reviewer 2 Report
In this version, the content of the manuscript was significantly improved. Nevertheless, the following points should be addressed:
- Line 126. Consider "..was prepared and immediately sterilized" instead of was prepared and was….
- Lines 129-130. For the trace elements, it would be more informative to give for each element (and not its source) the final concentration in the media.
- Line 139. The disk diameter in addition to depth should be reported.
- Line 146. Refer the DNA ladder used (size, brand, etc).
- Lines 230-231. Consider revising this sentence. It looks like a word is missing.
- Figure 4. Top image, last lane (left to right) correspond to the DNA ladder? If yes, correct the box over it.
- Line 352. Conclusion was a nice addition but in my opinion, it is too long. If the authors summarize the main findings, it would be more informative.
- Lines 379-380. Consider revising the last sentence since its meaning is not clear.
Author Response
Response to Reviewer 2 Comments
All correction are referred to line numbers of the revised manuscript.
Point 1: Line 126. Consider "..was prepared and immediately sterilized" instead of was prepared and was….
Response 1: Fixed
Point 2: Lines 129-130. For the trace elements, it would be more informative to give for each element (and not its source) the final concentration in the media.
Response 2: Fixed
Point 3: Line 139. The disk diameter in addition to depth should be reported.
Response 3: Fixed, we reported the electrode surface area.
Point 4: Line 146. Refer the DNA ladder used (size, brand, etc).
Response 4 Fixed – line 128.
Point 5: Figure 4. Top image, last lane (left to right) correspond to the DNA ladder? If yes, correct the box over it.
Response 5: Fixed.
Point 6: Line 352. Conclusion was a nice addition but in my opinion, it is too long. If the authors summarize the main findings, it would be more informative.
Response 6: The conclusions were shortened.
Point 7: Lines 379-380. Consider revising the last sentence since its meaning is not clear.
Response 7: The sentence was revised.
