Geobacter grbiciae—A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, after carefully studying the article, I have decided that it requires major revision. Below I list my suggestions. 

1. Descriptions of recent studies must be excluded from the Abstract. In addition, the research methodology must be added to the Abstract.

2. In the Introduction section, it is necessary to add subsections Problem Statement, purpose and objectives of the study.

3. In the Materials and Methods section, you need to add a flowchart that would describe the implementation of further research.

4. Figure 2 needs improvement. The vertical scale should be reduced from -0.25 to 0.25. In Fig. 5 no markings where Fig. 5a, and where is Fig. 5b.

5. The Discussion section must be separated into a separate section and the number of articles in it increased for 2022-2023.

6. The Conclusions section is very small and does not reflect all the results of the study. In this section it is necessary to add the results of the experiment in numerical form, as well as the possible application of the results obtained.

7. It is necessary to add more articles from 2022-2023 to the article, and also exclude articles before 2000.

Author Response

Responses to reviewer

Please also see the attachment for responses to reviewers.

Comments and Suggestions for Authors

Dear authors, after carefully studying the article, I have decided that it requires major revision. Below I list my suggestions. 

1. Descriptions of recent studies must be excluded from the Abstract. In addition, the research methodology must be added to the Abstract.

Descriptions of recent studies have been excluded from the Abstract. In addition, the research methodology has been added to the abstract. Please see line 18-24.

2. In the Introduction section, it is necessary to add subsections Problem Statement, purpose and objectives of the study.

The problem Statement, purpose and objectives of the study have been added in the end of introduction section. Please see line 75-89.

3. In the Materials and Methods section, you need to add a flowchart that would describe the implementation of further research.

The flowchart that would describe the implementation of further research has been added in the Materials and Methods section (Please see line 100 to 108 and Figure 1).

4. Figure 3 needs improvement. The vertical scale should be reduced from -0.25 to 0.25. In Fig. 6 no markings where Fig. 6a, and where is Fig. 6b.

The vertical scale of Figure 3 has been reduced from 0.0 to 0.25 (Please see line 245 and Figure 3). The markings have been added in Fig. 6. (Please see line 289 and Figure 6)

5. The Discussion section must be separated into a separate section and the number of articles in it increased for 2022-2023.

The Discussion section has been separated into a separate section and the number of articles in it increased for 2022-2023. Please see the discussion section( line 294-374).

6. The Conclusions section is very small and does not reflect all the results of the study. In this section it is necessary to add the results of the experiment in numerical form, as well as the possible application of the results obtained.

The results of the experiment in numerical form were added to the Conclusions section( line 294-374). The possible application of the results obtained was added in the conclusion(Please see line 399-402 of the conclusion section).

7. It is necessary to add more articles from 2022-2023 to the article, and also exclude articles before 2000.

More references from 2022 to 2023 have been added. All references before 2000 have been deleted except reference 65 and 73. Because the metabolic substrate characteristics of G. sulfurreducens and G. metallireducens GS-15 were reported in these two references, respectively. The substrate characteristics of these two strains were needed in the discussion(Please see the discussion section).

Thank reviewers for their valuable comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

IThe authors of the study: Geobacter grbiciae — A new Electron Donor in the Formation of
Co-cultures via Direct Interspecies Electron Transfer present a new strain able to perform DIET in co-cultures. For this, Geobacter grbiciae was tested for its ability to grow in co-culture with M. barkeri 800, G. sulfurreducens Δhyb and Methanospirillum hungatei. Study results indicate that Geobacter grbiciae is able to form co-cultures based on DIET with M. barkeri 800 in presence granular activated carbon or magnetite, whereas it was able to perform DIET with G.sulfurreducens also in absence of such mediators. The lack of the formation of co-cultures with Methanospirillum hungatei, requiring H2 or formate as electron shuttles further supports that the electron interspecies transfer via Geobacter grbiciae is based on DIET and not H2 or formate production.

General comments:

The title leaves the reader wondering whether an alternative electron donor instead of ethanol will be described in this study or if a new strain passing on the electrons from the substance to a different strain is presented. Possibly a title which does not raise wrong assumptions can be selected.

Revise literature and pay attention to italic writing of genus and species names

Most concentrations are given only in mmol, occasionally in mmol/Liter or mg/L, a harmonization of the units if possible would stronglw improve clarity and comparability

Figure 1: check polyester spelling

Figure 3: check spelling of polyester, check your concentration information mmol or mM? What means Fumarate/mmol, do you mean Fumarate (mmol)? (check also in other figures)

Figure 5: it is confusing the the Y-Axis starts at -0.25, isn’t there just no CH4 detected? In the figure captions mmol per Liter is indicated in the graphs only mmol, is this intentional?

 

Specific comments:

Line 44: find terms to differentiate between the molecule as an electron donor and the prokaryotic organism as an electron donor. Here you describe ethanol as the electron donor and at a later stage or in the title the strain. For clarity it is recommended to differentiate between organisms that pass on electrons and molecules such as EtOH

Line 80: what was in the headspace? And where the flasks only sealed with rubber septa or also crimped?

Line 84: give the reference for the trace mineral solution as well.

Line 90: Explain briefly abbreviation of NBF medium and content.

Line 93: Explain abbreviation of DSM 120 medium, explain: modified DSM 120

Line 97: How were the media prepared for the Geobacter strains?

Line 99: To a final concentration of 1 mM?

Line 105: Explain abbreviation of NBM medium

Line 105: How old were the precultures, in which growth phase and were similar cell numbers used to guarantee viability of both cell lines?

Line 113: Again to a final concentration of 1mM or the stock was 1 mM?

Line 115: Describe briefly how they were sterilized.

Line 117: Describe briefly why polyester felt was used as a control.

Line 119: The analytical method used should be briefly described

Line 139: Why the references are given in two sections?

Line 139: Very long sentence, shorten to improve readability.

Line 147 and 148: Stay exact, the serum bottles do not generate the methane but the cultures. The previous line was more exact (methane generated in the anaerobic serum bottle)

Line 150: Sentence structure could be improved

Line 154: check word separation

Line 155: could you harmonize for your conductive material the units provided for better comparability of the quantities used? (GAC: 20 g/L; magnetite 10 mmol/L, 5 pieces of 20×20×3 mm)

Line 217: indicate figure with right and left or add A and B to the figures, check the size of the figures which seems to be different

Line 227: check italic writing

Line 235: Check sentence structure

Line 240: Again not clear what is referred to as electron acceptor, strain or substance (compare line 243 here you refer to ethanol as electron donor, make difference clear, later you use for instance “electron-donating partners in DIET”)

Line 249: Explain better. G. metallireducens and Methanosaeta harundinacea metabolize propanol or butanol as the sole electron donor, but not propionate or butyrate, which does not hinder other strains to be able to use butyrate or propionate.

Comments on the Quality of English Language

Line 127: can form...

Sometimes the sentence structure seems not clear or sentences are too long.

Author Response

Responses to reviewers

Please also see the attachment for responses to reviewers.

Comments and Suggestions for Authors

IThe authors of the study: Geobacter grbiciae — A new Electron Donor in the Formation of
Co-cultures via Direct Interspecies Electron Transfer present a new strain able to perform DIET in co-cultures. For this, Geobacter grbiciae was tested for its ability to grow in co-culture with M. barkeri 800, G. sulfurreducens Δhyb and Methanospirillum hungatei. Study results indicate that Geobacter grbiciae is able to form co-cultures based on DIET with M. barkeri 800 in presence granular activated carbon or magnetite, whereas it was able to perform DIET with G.sulfurreducens also in absence of such mediators. The lack of the formation of co-cultures with Methanospirillum hungatei, requiring H2 or formate as electron shuttles further supports that the electron interspecies transfer via Geobacter grbiciae is based on DIET and not H2 or formate production.

General comments:

1. The title leaves the reader wondering whether an alternative electron donor instead of ethanol will be described in this study or if a new strain passing on the electrons from the substance to a different strain is presented. Possibly a title which does not raise wrong assumptions can be selected.

If the electron donor is ethanol, it has been changed to electron donor substance. If the electron donor is a microbe, it has been changed to electron donor microbe. “Electron Donor” in the title has been changed to “ Electron Donor Microbe”.

2. Revise literature and pay attention to italic writing of genus and species names

We will pay more attention to italic writing of genus and species names in this manuscript.

3. Most concentrations are given only in mmol, occasionally in mmol/Liter or mg/L, a harmonization of the units if possible would stronglw improve clarity and comparability

Concentrations given in mmol or mM have been changed to mmol/L.

4. Figure 2: check polyester spelling

“Polyeaster” in Figure 2 and other Figures has been changed to “polyester”(Please see line 226 and Figure 2).

5. Figure 4: check spelling of polyester, check your concentration information mmol or mM? What means Fumarate/mmol, do you mean Fumarate (mmol)? (check also in other figures)

“Polyeaster” in Figure 4 has been changed to “polyester” (Please see line 271 and Figure 4). All mM have been changed to mmol/L. Fumarate/mmol means Fumarate (mmol). All “Fumarate/mmol” has been changed to Fumarate (mmol). All “CH₄/mmol” has been changed to “CH₄ (mmol)”.

6. Figure 6: it is confusing the the Y-Axis starts at -0.25, isn’t there just no CH4 detected? In the figure captions mmol per Liter is indicated in the graphs only mmol, is this intentional?

There is just no CH₄ detected at 0 day. If the Y-Axis starts at 0, the amount of methane at 0 day cannot be displayed in Figure 6. Now the Y-Axis starts at 0 (please see line 289 and Figure 6). In the figure captions mmol means the amount of methane, not the concentration of methane. Now the CH₄/mmol has been changed to CH₄ (mmol)(Please see line 226 and Figure 2, line 245 and Figure 3, line 289 and Figure 6) .

Specific comments:

1. Line 44: find terms to differentiate between the molecule as an electron donor and the prokaryotic organism as an electron donor. Here you describe ethanol as the electron donor and at a later stage or in the title the strain. For clarity it is recommended to differentiate between organisms that pass on electrons and molecules such as EtOH

If the electron donor is ethanol, it has been changed to electron donor substance. If the electron donor is a microbe, it has been changed to electron donor microbe.

2. Line 80: what was in the headspace? And where the flasks only sealed with rubber septa or also crimped?

The mixture of CO₂ and N₂ (20:80, V/V) was in the headspace. Cultures were grown under strict anaerobic conditions in anaerobic pressure tubes (27 mL) or serum bottles (156 mL) sealed with thick butyl rubber stoppers and crimped with aluminum covers. We have described these in the revised manuscript now(see line 96-99.).

3. Line 84: give the reference for the trace mineral solution as well.

We have given the reference for the trace mineral solution as well (see line 112-113).

4. Line 90: Explain briefly abbreviation of NBF medium and content.

NBF medium means NBAF medium that was first mentioned in reference “M. V. Coppi, C. Leang, S. J. Sandler, D. R. Lovley, Appl Environ Microbiol 67, 3180 (2001).” It doesn’t explain the abbreviation of NBAF medium. We have changed NBF to NBAF(see line 138). In addition, we have added the content of NBAF medium to the revised manuscript (see line 140-144).

5. Line 93: Explain abbreviation of DSM 120 medium, explain: modified DSM 120

DSM 120 medium is DSMZ_Medium120 (https://www.dsmz.de) that is used for methanogen culture. The website doesn’t explain the abbreviation of DSMZ_Medium120. We have changed DSM 120 medium to DSMZ_Medium120 (see line 158 ). We have added the origin of DSMZ_Medium120 (see line 158 ). Modified DSM 120 means Modified DSMZ_Medium120. Modified DSMZ_Medium120 is modified from DSMZ_Medium120 by removing yeast extract, casitone, resazurin, Na-acetate, and methanol. We have added the explanation of the DSMZ_Medium120 (see line 240 ) and the Modified DSMZ_Medium120(see line 240 ) in the revised manuscript.

6. Line 97: How were the media prepared for the Geobacter strains?

The preparation of media for the Geobacter grbiciae has been added to the revised manuscript (see line 115-136 ).

The preparation of media for the G. sulfurreducens Δhyb has been added to the revised manuscript (see line 140-156 ).

7. Line 99: To a final concentration of 1 mM?

1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O means a final concentration of 1 mmol/L and 0.5 mmol/L. We have changed the description for better understanding. “a premix of 1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O” has been changed to “0.5 mL premix of cysteine (100 mmol/L) and Na₂S·9H₂O (50 mmol/L)” (see line 182-183 ). The volume of the culture medium is 50 mL. A final concentration of 1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O will be obtained by Adding 0.5 mL premix of cysteine (100 mmol/L) and Na₂S·9H₂O (50 mmol/L) to the medium.

8. Line 105: Explain abbreviation of NBM medium

NBM medium is modified from NBAF medium by removing fumaric acid from NBAF medium. We have added the explanation of abbreviation of NBM medium to the revised manuscript (see line 167-168 ).

9. Line 105: How old were the precultures, in which growth phase and were similar cell numbers used to guarantee viability of both cell lines?

The coculture inocula were obtained from cultures that were in late exponential phase, as determined by monitoring the optical density at 600 nm. The growth phase of inoculum has been added to this manuscript (see line 166-167, line 171-172 and line 175-176 ).

10. Line 113: Again to a final concentration of 1mM or the stock was 1 mM?

1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O means a final concentration of 1 mmol/L and 0.5 mmol/L. We have changed the description for better understanding. “a premix of 1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O” has been changed to “ 0.5 mL premix of cysteine (100 mmol/L) and Na₂S·9H₂O (50 mmol/L)” (see line 163-164, line 182-183 ). The volume of the culture medium is 50 mL. A final concentration of 1 mmol/L cysteine and 0.5 mmol/L Na₂S·9H₂O will be obtained by Adding 0.5 mL premix of cysteine (100 mmol/L) and Na₂S·9H₂O (50 mmol/L) to the medium,

11. Line 115: Describe briefly how they were sterilized.

“All electron donors were added from anaerobic sterilized stocks.” has been changed to “All electron donors were added from anaerobic heat-sterilized stocks”.(see line 183-184 )

 “Noted GAC or magnetite nanoparticles were added to the suitable medium.” has been changed to “GAC (8–20 mesh, 80.7 Ω^–1·m^–1, Sigma-Aldrich, St Louis, MO, USA) (20 g/L) or magnetite nanoparticles (34.0 Ω^–1·m^–1, a final concentration 10 mmol/L) were added to a suitable medium before heat-sterilization of the medium.” (see line 184-186)

12. Line 117: Describe briefly why polyester felt was used as a control.

The increase in the methane production rate after adding activated carbon and magnetite to the co-culture system may be attributed to two reasons: (i) activated carbon and magnetite act as attachment carriers for microbial growth, which increases the chances of contact between the two microbes; (ii) activated carbon and magnetite promote electron transfer between two microbes because of their conductivity. To eliminate the influence of (i) as much as possible, five pieces of polyester felt (Heavy Duty Fabric) (20 × 20 × 3 mm) were added to the control anaerobic serum bottle. A brief description that why polyester felt was used as a control has been added to the revised manuscript.(see line 187-193. )

13. Line 119: The analytical method used should be briefly described

Headspace methane (0.5 mL) was sampled and injected onto a GC-2014 equipped with an Rtx^®-1(30 m) column heated at 110 °C. The injector port and flame ionization detector (FID) were set at 200 °C. Samples for fumarate/succinate analysis were filtered with a syringe needle filter (0.22 μm pore diameter) and then separated by HPLC (Aminex HPX-87H column, 300 × 7.8 mm) with a mobile phase of 10.0 mmol/L H₂SO₄ flowing at 0.6 mL/min and detection at 215 nm. We have added this analytical method to the analytical method used in this manuscript (see line 199-204) .

14. Line 139: Why the references are given in two sections?

We made a mistake. Now we have incorporated the references(see line 306).

15. Line 139: Very long sentence, shorten to improve readability.

“Also, the anaerobic serum bottle supplemented with GAC exhibited faster and higher methane production than those with magnetite in the early phase, elucidating that GAC not only gives better interspecies electrical connections than magnetite due to the high specific surface area of GAC, it also gives a better environmental niche for the growth of microbes and provides better access to the substrate since GAC is a great adsorbent.” has been changed to “ Methane was generated at a faster rate and higher levels in co-cultures supplemented with GAC than in co-cultures supplemented with magnetite during the early growth phase. This observation indicates that GAC may afford better interspecies electrical connections than magnetite because of the high specific surface area of GAC. Moreover, this result also indicates that GAC may provide a better environmental niche for the growth of microbes and provide better access to the substrate because GAC is a strong absorbent.” (see line 306-312)

16. Line 147 and 148: Stay exact, the serum bottles do not generate the methane but the cultures. The previous line was more exact (methane generated in the anaerobic serum bottle)

Line 147 and 148 have been changed as follows:

“However, with more days of incubation, the anaerobic serum bottle supplemented with magnetite generated almost the same methane as those supplemented with GAC.” has changed to “However, similar levels of methane production were observed for co-cultures in anaerobic serum bottles supplemented with either magnetite or GAC after 90 days of culturing.”(see line 310-312 )

 

17. Line 150: Sentence structure could be improved

“To ensure that G. grbiciae and M. barkeri 800 interact through DIET, we confirmed this, further forming a co-culture with G. grbiciae and the strict H₂/formate-utilizing methanogen M. hungatei.” has been changed to “Co-culturing of G. grbiciae with the strict H₂/formate-utilizing methanogen M. hungatei was carried out to confirm that G. grbiciae and M. barkeri 800 interact through DIET rather than H₂/formate.” (see line 223-225)

18. Line 154: check word separation

“metaboliz-ing” has been changed to “metabolizing” in the revised manuscript.

19. Line 155: could you harmonize for your conductive material the units provided for better comparability of the quantities used? (GAC: 20 g/L; magnetite 10 mmol/L, 5 pieces of 20×20×3 mm)

It is difficult to unify the units of these materials’ concentrations. For example, we don't know the molecular weight of activated carbon. As far as I know, there is not unified concentration of activated carbon and magnetite used in the relevant research literatures.

20. Line 217: indicate figure with right and left or add A and B to the figures, check the size of the figures which seems to be different

We have added A and B to the figures. We have made the two figures the same.(see line 289 and Figure 6)

21. Line 227: check italic writing

“M. harundinacea 8Ac” has been changed to “M. harundinacea 8Ac”. (see line 360)

22. Line 235: Check sentence structure

“Even though some enrichments that might interact through DIET with propionate, butyrate or acetate as the sole electron donor have been described, neither of the enrichments were isolated with the purpose of DIET. Also, none of the pure co-cultures formed through DIET with propionate or butyrate or acetate as the sole electron donor has been acquired till date.” has been changed to “ Although some enrichments that might interact through DIET with propionate or butyrate as the sole electron donor substance have been described, none of the enrichments was isolated with the purpose of DIET. Additionally, no pure co-culture formed through DIET with propionate or butyrate as the sole electron donor substance has been acquired.”(see line 365-369)

23. Line 240: Again not clear what is referred to as electron acceptor, strain or substance (compare line 243 here you refer to ethanol as electron donor, make difference clear, later you use for instance “electron-donating partners in DIET”)

We have changed these. “ethanol as electron donor” has changed to “ ethanol as electron donor substance”. When microbe acts an electron donor, we describe it as “an electron donor microbe”.

24. Line 249: Explain better. G. metallireducensand Methanosaeta harundinaceametabolize propanol or butanol as the sole electron donor, but not propionate or butyrate, which does not hinder other strains to be able to use butyrate or propionate.

Line 249, we just say that claims of propionate and butyrate metabolism through DIET in mixed microbial communities with methane production are subject to further confirmation. However, we don’t deny the possible existence of co-cultures capable of using butyrate or propionate to produce methane through DIET. The pure co-cultures that can use butyrate or propionate to produce methane through DIET hasn’t been reported up to now. Perhaps it's just that it hasn't been discovered yet.

 

We have changed it to “Although the co-cultures of G. grbiciae and M. barkeri 800 syntrophically converted ethanol to methane through DIET, they did not convert propionate or butyrate to methane. However, this does preclude the possibility that other strains can use butyrate or propionate to produce methane through DIET. For example, propionate and butyrate metabolism through DIET in mixed microbial communities with methane production have been reported, and further efforts with pure co-cultures are needed to confirm these previous observations.”(see line 402-407 )

 

Comments on the Quality of English Language

25. Line 127: can form...

We have changed “ can format” to “ can form”. In addition, we have revised the English Language to make it easier to understand.

Sometimes the sentence structure seems not clear or sentences are too long.

Thank reviewers for their valuable comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, this version of the article is much better.