The Impact of Antimicrobial Substances on the Methanogenic Community during Methane Fermentation of Sewage Sludge and Cattle Slurry

Round 1

Reviewer 1 Report

This manuscript reports on the effects of selected antibiotics on anaerobic digestion of various wastes. The manuscript is impossible to review as some essential  information is missing. “Table 2” absent, and the description of the how the experiments were set up is lacking as well. With respect to the general approach: the authors only evaluate methane production and methanogens.  Anaerobic digestion is however accomplished by a community/food web; the non-methanogens play an essential role in the process and should be included in the manuscript.

 

Below are some comments pencilled in when I was going through the manuscript. Hope this helps in rewriting and in providing a more complete publishable picture.

 

Specific comments:  

 

1. 12: “the effect of” is missing
2. 17: explain MSc and MST
3. 25: after having read the abstract the reader wonders what antibiotic concentrations you used, and to what extent these concentrations would be expected to enter AD systems.

 

More in general, the reader wonders to what extent the biomass (inoculum) in these experiments had been previously exposed to these antibiotics. This too needs to be mentioned in the abstract.

 

1. 83: provide the references for these “relevant scientific references”.
2. 93: you are talking about the 16S rRNA genes here I presume?
3. 104: explain D^-1
4. 113: explain your line of reasoning here.

You also need to specify the amounts of antibiotics fed in the materials and methods section.

L120: briefly summarise the experimental set up. Size of the reactors, how much inoculum, how much substrate, size of the headspace, how many replicates, etc.

L.166: show the two graphs with time courses for methane production for SS and CS (with and without antibiotics).

Table 2. is missing.

1. 168: when were the VFA samples taken?
2. 175: this statement reinforces my previous statements that you need to be more explicit about the concentrations you used, and the concentrations found in influent and sludge of “real” reactors.

Section 3.1 It is not possible for me to review this section with Table 2 missing; but even if this Table would have been provided, my impression is that you need to be more quantitative and explicit about loads and concentrations when you compare your results to those in the literature and in actual waste streams.

1. 223 and throughout: gene rather than gen.
2. 231: well argued; nice!

Figure 1: show the SD or the SE, and indicate how significant these differences were. When I eyeball the data I would say that in panel A only MET was significantly lower. In B OXY is higher than the control. If this is really significant ( I doubt it; depends on the chose P) you should use letters to indicate what was different from what.

1. 246: the reader wants/needs to see the methane data!

Figure 2: give the data per individual treatment, not all (for example) methane data lumped together in one point in the graph.

Figure 3: I do not understand this figure. What am I looking at here? Make a more illustrative and intuitive figure instead.

Figure 4: see my comments to Figure 1.

Section 3.3 hard to evaluate without Table 2; also it seems a rather wordy for what is has to offer. Condense!

 

What I am missing in this manuscript is a brief mention that antibiotics will also affect the bacteria that provide the methanogens with their substrates. Thus it may well be that the effects on methanogens you are observing here are actually indirect effects.    

 

Author Response

Answers to Reviewer 1’ comments:

1. Line 12: “the effect of” is missing.

Thank you for this comment. The word „effect” was added in the text.

2. Line 17: explain MSC and MST.

Thank you for your comment. MSC and MST abbreviations are explained. All changes are marked in red. Changes can be found in the “Abstract” in line 18.

3. Line 25: after having read the abstract the reader wonders what antibiotic concentrations you used, and to what extent these concentrations would be expected to enter AD systems. More in general, the reader wonders to what extent the biomass (inoculum) in these experiments had been previously exposed to these antibiotics. This too needs to be mentioned in the abstract.

Thank you for this comment. The abstract was additionally supplemented with data on antibiotic doses and the effect of antibiotics on microorganisms. Information on selection pressure has been added. For this purpose, the abstract has been rebuilt, all changes are marked in red. The changes can be found in the lines number 12- 14; 18; 22; 24; 25; 26. All changes have been marked in red.

4. Line 83: provide the references for these “relevant scientific references”.

Thank you for the comment. Two new references have been added to the manuscript. Below, there is a list of publications attached to the References section. The changes can be found in the lines number 99 and 546- 550. Changes in the text and the list of references are marked in red.

[30] Spielmeyer, A. Occurrence and fate of antibiotics in manure during manure treatments: A short review. Sustain. Chem. Pharm. 2018, 9, 76- 86. https://doi.org/10.1016/j.scp.2018.06.004.

[31] Zhang, X. and Zhang, K. Effects of antibiotics on anaerobic digestion resistance genes and archaeal communities in cattle manure. Journal of Physics: Conference Series. 2020, 1549, 032076. doi:10.1088/1742-6596/1549/3/032076..

5. Line 93: you are talking about the 16S rRNA genes here I presume?

Thank you for the valuable comment. Yes, we thought about the 16S rRNA fragment which is characteristic for the methanogenic Archaea families, i.e. Methanosarcinaceae (MSC) and Methanosaetaceae (MST). The changes can be found in the lines number 109-110.

6. Line 104: explain _D^-1.

Thank you for the comment. An explanation of the abbreviation has been added in the text and Table 1. All changes are marked in red. The changes can be found in the lines number 120- 121.

7. Line 113: explain your line of reasoning here.

Thank you for the comment. The information that the effect of antibiotics was measured using the Automatic Methane Potential Test System II (Bioprocess Control), testing the production of biogas and its CH₄ content has been added to the paper. The changes can be found in the lines number 130- 131. All changes have been marked in red.

8. You also need to specify the amounts of antibiotics fed in the materials and methods section.

Thank you for the comment. In section 2.2. the information about the amounts of antibiotic fed in the materials has been added. The changes can be found in the lines number 129- 134. All changes have been marked in red.

9. L120: briefly summarise the experimental set up. Size of the reactors, how much inoculum, how much substrate, size of the headspace, how many replicates, etc.

Thank you for the comment. Information about the experiment has been added, i.e. size of the reactors, how much inoculum, how much substrate, size of the headspace, how many replicates. The changes can be found in the lines number 137- 140. All changes are marked in red.

10. What I am missing in this manuscript is a brief mention that antibiotics will also affect the bacteria that provide the methanogens with their substrates. Thus it may well be that the effects on methanogens you are observing here are actually indirect effects.

Thank you for the comment. In the “Introduction” section, we added information on the topic about the effect of antibiotics on bacteria and Archaea, and about the relationships between these groups of microorganisms. The changes can be found in the lines number 76- 86.

11. 166: show the two graphs with time courses for methane production for SS and CS (with and without antibiotics).

As recommended by the reviewer, the relevant graphs have been attached to the Supplementary Materials as Fig. S1.

12. Table 2. is missing.

We sincerely apologize for this mistake. As a result of our error, not all tables were included in PDF format available to the reviewers. All tables have been included only in DOCX format. In revised version of the manuscript, all graphics and tables were properly included.

13. 168: when were the VFA samples taken?

Measurements of VFA production were carried out systematically throughout the experiment. The mean values of the VFA produced together with the standard deviation are presented in Table 2.

14. 175: this statement reinforces my previous statements that you need to be more explicit about the concentrations you used, and the concentrations found in influent and sludge of “real” reactors.

In our previous research (quoted in section 3.1., https://doi.org/10.3390/proceedings2201274), the addition of low doses of antibiotics to bioreactors did not cause significant differences in the efficiency of the process. The high concentrations of the antibiotics used in the research were aimed at obtaining a pronounced response of the microorganisms involved in the process. We believe that this allowed us to observe visible trends, and thus determine the differences in the effects of individual antibiotics on the microbiota characteristic of sewage sludge and cattle slurry. Some new information and references about the concentration of antibiotics in the manure and sludge samples have been also added to the manuscript (lines 68-73).

15. Section 3.1 It is not possible for me to review this section with Table 2 missing; but even if this Table would have been provided, my impression is that you need to be more quantitative and explicit about loads and concentrations when you compare your results to those in the literature and in actual waste streams.

Table 2 has been attached to the manuscript. Once again, we apologize for not having this table in the version received by the reviewer. We hope this table contains all the necessary information. As suggested by the reviewer, more detailed information on the concentrations of antimicrobial substances was included in Abstract and in the section No 2.2.

16. 223 and throughout: gene rather than gen.

Thank you for the comment, the typo has been corrected (line 263).

17. 231: well argued; nice!

Thank you!.

18. Figure 1: show the SD or the SE, and indicate how significant these differences were. When I eyeball the data I would say that in panel A only MET was significantly lower. In B OXY is higher than the control. If this is really significant ( I doubt it; depends on the chose P) you should use letters to indicate what was different from what.

Fig. 1 has been replaced in revised version of the manuscript by Fig. 2. Figure 2 shows the average mcrA gene concentrations in 1 gram of digestate from individual bioreactors. Corrected graphs were made taking into account SD. The statistical significance of the differences with the proper control (p<0.05) was marked with *. The previous version of the bar graphs included a logarithmic scale on the y-axis. This could give the impression that there was no significance between the concentrations. We believe that the current form of presenting Figure 2 better illustrates the results of work.

19. 246: the reader wants/needs to see the methane data!

Thank you for the comment. Appropriate data on methane production have been presented in Table 2.

20. Figure 2: give the data per individual treatment, not all (for example) methane data lumped together in one point in the graph.

We thank the reviewer for this comment. After analysing it, however, we found that we would like to retain it unchanged in the manuscript. The PCA analysis shows the dispersion of methane fermentation parameters as well as process and control bioreactors in space. Their mutual distribution and distances show the scale of the influence of individual antimicrobial substances on the change of individual process parameters.

21. Figure 3: I do not understand this figure. What am I looking at here? Make a more illustrative and intuitive figure instead.

We thank the reviewer for this comment. This figure was indeed not intuitive or easy to interpret. Therefore, we decided to remove it from the manuscript. At the same time, we enriched the manuscript with two heatmaps based on the Ward method of clustering (Fig. 1).

22. Figure 4: see my comments to Figure 1.

Fig. 4 has been replaced in revised version of the manuscript by Fig. 2. Figure 2 (C-F) shows the average MST and MSC genes concentrations in 1 gram of digestate from individual bioreactors. Corrected graphs were made taking into account SD. The statistical significance of the differences with the proper control (p<0.05) was marked with *. We believe that the current form of  Figure 2 better illustrates the results of work.

23. Section 3.3 hard to evaluate without Table 2; also it seems a rather wordy for what is has to offer. Condense!

Thank you for this comment. According to the note, the section 3.3 has been shortened to make it more legible.

24. What I am missing in this manuscript is a brief mention that antibiotics will also affect the bacteria that provide the methanogens with their substrates. Thus it may well be that the effects on methanogens you are observing here are actually indirect effects.

Thank you for this comment. An excerpt on the effect of antibiotics on the microorganisms involved in the first three stages of methane fermentation has been added. Moreover, it was also clarified in more detail that this work focuses on the effects of antimicrobial substances on methanogenic consortia. The changes can be found in the lines number 76- 86. All changes are marked in red.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript investigates impact of antimicrobials on anaerobic digestion of sewage sludge and cattle slurry sludge. The text requires revision before its publication in this journal. The specific comments are given below:

• Antimicrobial concentration range observed in sewage sludge and cattle slurry should be given in introduction section.
• Line 112: The selected antimicrobial concentrations have to appear in the text by briefly explaining the reason for selection.
• Table 1: What is the reason for selecting given TS concentrations in sewage and cattle slurry sludge? The reason for not adjusting the initial conditions should be explained to be able to compare experimental results.
• Lines 139-141: This claim should be explained clearly.
• Lines 155-159: Too long sentences should be avoided.
• Lines 169-175, 194-196: Applied concentrations should be given numerically in the text to be able to comment on the concentration effect.
• Table 2: Antibiotic concentrations should appear in the table. The authors should comment on the slightly increased/stable methane content on contrary to reduced methane production for OXYSS, OXYCS, SMXSS, SMXCS, and METCS.
• Lines 239-242: Comments should be included regarding this sentence.
• Figure 3: Explanation is not enough in the text.

Author Response

Answers to Reviewer 2’ comments:

1. Antimicrobial concentration range observed in sewage sludge and cattle slurry should be given in introduction section.

Thank you for the comment. Information on antimicrobials concentrations observed in cattle manure and sewage sludge was added in the introduction. The changes can be found in the lines number 68- 73. All changes have been marked in red.

2. Line 112: The selected antimicrobial concentrations have to appear in the text by briefly explaining the reason for selection.

Thank you for the comment. In the experiment, high doses of antibiotics were intentionally injected into bioreactors in order to induce a reaction of microorganisms. We added this information in the M&M section, subchaper 2.2 Methane fermentation, lines 132- 134.

3. Table 1: What is the reason for selecting given TS concentrations in sewage and cattle slurry sludge? The reason for not adjusting the initial conditions should be explained to be able to compare experimental results.

Thank you for the comment. Our aim was to adjust an organic load to 5 g VS L-1 in all bioreactors with a volume of 250 mL. Therefore, the level of Total Solids was different for cattle slurry and sewage sludge. The information was included in the text of the manuscript (lines 137-139)

4. Lines 139-141: This claim should be explained clearly.

Thank you for the comment. Our preliminary investigations by sequencing of the 16S rRNA (data not presented) enabled us to exclude any significant share in the samples of other genera from the domain Archaea, such as Methanococcales, Methanobacteriales, or Methanomicrobiales (the relevant information is contained in the text -  lines 163-165).

5. Lines 155-159: Too long sentences should be avoided.

Thank you for the comment. We divided the long sentence to two new sentences. The changes can be found in the lines number 180- 183. All changes have been marked in red.

6. Lines 169-175, 194-196: Applied concentrations should be given numerically in the text to be able to comment on the concentration effect.

We would like to thank the Reviewer for that comment. The concentrations of antimicrobial substances applied in individual research study have been given numerically in the text (lines 195, 222-223).

7. Table 2: Antibiotic concentrations should appear in the table. The authors should comment on the slightly increased/stable methane content on contrary to reduced methane production for OXYSS, OXYCS, SMXSS, SMXCS, and METCS.

According to the suggestion of the reviewer, the antibiotic concentrations have been listed in Table 2. We also thank to the reviewer for the valuable comment. The relevant information has been added to the manuscript (lines 242-249).

8. Lines 239-242: Comments should be included regarding this sentence.

According to the reviewer's suggestion, a comment has been included regarding this sentence (lines 282-284). The topic is further developed at the end of subchapter 3.2.

9. Figure 3: Explanation is not enough in the text.

This figure was indeed not intuitive or easy to interpret. Considering the low weight of the data that we presented graphically on it, we decided to completely remove it from the manuscript. We also decided to enrich the manuscript with two heatmaps based on the Ward method of clustering (Fig. 1 and Fig. 4).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has improved, but further changes are necessary before the manuscript can be published.

 

Line 76: the phrasing suggests that antibiotics only have an indirect on methanogens. I doubt that is true. However, it is known that archaea can have a different response to antibiotics than bacteria. All this should be mentioned/discussed and proper references should be used.

 

Fig. S1. Averages? Indicate how many replicates were used, and what the (dis)agreement was between replicates.

 

1. 140: later? When?

 

1. 177: indicate in this section what Wards method is and give a reference.

 

Fig. 1 and Fig. 4. Are good additions to the manuscript. However, they should be discussed more comprehensively. What can we learn from these data if we look at it collectively? Do they show similar or opposite trends, and what can we learn from that? Were specific groups of methanogens affected to the same extent by the antibiotics (relative to the control)? And was this reflected in the methane production data? If yes, what does this mean? If no, what does this mean? Discuss!  

Author Response

Dear Reviewer,

Thank you very much for the comments to our manuscript titled “The impact of antimicrobial substances on the methanogenic community during methane fermentation of sewage sludge and cattle slurry”. Below we provide our responses and hope that the paper is now acceptable. Introduced changes in the text have been highlighted in green.

Best regards,

Corresponding author

Answers to Reviewer 1’ comments:

1. Line 76: the phrasing suggest that antibiotics only have an indirect on methanogens. I doubt that is true. However, it is known that Archaea can have a different response to antibiotics than bacteria. All this should be mentioned/ discussed and proper references should be used.

Thank you for this comment. Information on the effects of antibiotics on methanogens has been expanded to include the suggested information. A new literature item has been added to fill the missing gap. The changes can be found in the lines number 86-91; 573-574.

2. Fig. S1. Averages? Indicate how many replicates were used, and what the (dis)agreement was between replicates.

Thank you for your comment. In the Materials and Methods section, subsection 2.2 Methane fermentation in line 144, the authors described that the experiment was carried out in replicates, and also the measurements of biogas production. We added the standard deviation and changes can be found in the Figure S1 in Supplementary Materials.

3. 140: later? When?

Thank you for this comment. The information about experiment has been added. The changes can be found in the lines number 145.

4. 177: indicate in this section what Wards method is and give a reference.

Thank you for the comment. New reference related to description of Ward’s method has been added to the manuscript. Below, there is a list of publications attached to the References section. The changes can be found in the lines number 189-191; 602-603.

[37] Joe H. Ward Jr. (1963) Hierarchical Grouping to Optimize an Objective Function, Journal of the American Statistical Association, 58:301, 236-244, DOI: 10.1080/01621459.1963.10500845

5. Fig.1 and Fig. 4. Are good additions to the manuscript. However, they should be discussed more comprehensively. What can we learn from these data if we look at it collectively? Do they show similar or opposite trends, and what can we learn from that? Were specific groups of methanogens affected to the same extent by the antibiotics (relative to the control)? And was this reflected in the methane production data? If yes, what does this mean? If no, what does this mean? Discuss!

Thank you for the comment. These figures have been discussed in more details. The changes can be found in the lines number 253-260; 416-432.

Author Response File: Author Response.docx