Development of Digested Sludge-Assimilating and Biohydrogen-Yielding Microflorae
Round 1
Reviewer 1 Report
The manuscript deals with an interesting topic, namely how to enrich cultures for hydrogen production out of biogenic residues. This operation mode is widely called dark fermentation, the phrase should be used here more consistently, not only in the discussion.
Some concerns arise, however, when reading the manuscript.
In general, it is not surprising that a pH control and less hydrogen in the headspace would both have been better, as the authors conclude in their discussion. This is, however, a trivial finding.
The question remains why closed bottles and little headspace have sometimes been used as this has a tremendeous effect on the hydrogen synthesis. The hydrogen is, however, the parameter on which decisions were made. This part of the manuscript is thus doubtful concerning its relevance. More interestingly is the survival/abundance and performance of microorganisms in general. This is kept rather short, especially on the strain level. It needs to be discussed how trustful the method is, whether more is known about hydrogen synthesis of the individual strains and if, based on this, a certain productivity can be assumed, also under consideration of known hydrolytic enzymes that are produced by these strains.
It is strange that there are no proteases in. This needs to be dsicussed and confirmed by similar findings of others or a thorough discussion about potential errors needs to be made.
Minor comments
The different conditions should be put in one table rather than discussed in the main text. It is hard to understand/follow.
The conditions need to be put in figure legends when shown, not the abbreviations.
The reason for green/yellow in fig.3 needs to be explained in the legend.
What was done prior to the exposure to low pH? Not the low pH as such, but a sharp sudden drop of the pH can be harmful for many bacteria.
line 273: needs to be "thermophilic"
Author Response:
1
Response to Reviewer 1 Comments
Point 1: The manuscript deals with an interesting topic, namely how to enrich cultures for hydrogen production out of biogenic residues. This operation mode is widely called dark fermentation, the phrase should be used here more consistently, not only in the discussion.
Response 1: According to the suggestion, words “cultivation” and “gas production” were replaced with the word “dark fermentation” or “fermentation” throughout the revised manuscript.
Point 2: The question remains why closed bottles and little headspace have sometimes been used as this has a tremendeous effect on the hydrogen synthesis. The hydrogen is, however, the parameter on which decisions were made. This part of the manuscript is thus doubtful concerning its relevance. More interestingly is the survival/abundance and performance of microorganisms in general. This is kept rather short, especially on the strain level. It needs to be discussed how trustful the method is, whether more is known about hydrogen synthesis of the individual strains and if, based on this, a certain productivity can be assumed, also under consideration of known hydrolytic enzymes that are produced by these strains.
Response 2: Closed vials and bottles were used because of a limitation of our laboratory space. Since data for hydrogen yield and enzyme activity (Figs. 3, 5, 6, and 7) in the present study are shown as an avarage ± standard deviation with statistical analysis (Student’s t-test), we think they are reliable. We also find impotance of analysis in DS hydrolysis and hydrogen production for the individual strains in the microflorae as the reviewer’s comment, but it is difficult for us to fulfill it, because we does not possess an anaerobic chamber which is necessary for research of anaerobes. Please understand our difficult situation. Instead, we commented above issues in the the revised manuscript (page 10 lines 336–338).
Point 3: It is strange that there are no proteases in. This needs to be dsicussed and confirmed by similar findings of others or a thorough discussion about potential errors needs to be made.
Response 3: Casein is widely employed as a substrate for non-specific protease assay, by which the protease activity for other bacterial flora was detected. Therefore we think our experiment works properly, and the heat-treated DABYS-A mainly hydrolyze polysaccharides in DS by their cellulase and chitinase.
Point 4: The different conditions should be put in one table rather than discussed in the main text. It is hard to understand/follow.
Response 4: A new table was constructed to summarize the different conditions for the heat treatment and gas production by the treated bacterial florae in the revised manuscript (Table 1).
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Point 5: The conditions need to be put in figure legends when shown, not the abbreviations.
Response 5: The abbreviated word for an experiment (the word DGGE in Fig. 4a legend) was changed to a compete expression (Denaturing gradient gel electrophoresis) in the revised manuscript.
Point 6: The reason for green/yellow in fig.3 needs to be explained in the legend.
Response 6: There is no special reason for green/yellow color, which indicates enzyme activity for parent (CH4-producing) flora and H2 gas-producing flora, respectively. As the reviewer commented, however, it is confsing for journal readers. Therefore, color of the columns in fig. 3 was improved to simple one in the revised manuscirpt.
Point 7: What was done prior to the exposure to low pH? Not the low pH as such, but a sharp sudden drop of the pH can be harmful for many bacteria.
Response 7: We did not perform any pretreatment prior to acidification of culture medium. Because hydrogen yield by the DABYS-A and -B significantly reduced at pH 4.1 and 4.8, respectively, they seem to receive some harmful effects by a sharp drop of pH, as the reviewer suggested . We thus commented this point in the revised manuscript (page 10 lines 306–308).
Point 8: line 273: needs to be "thermophilic"
Response 8: Thank you for finding a typo, which was corrected in the revised manuscirpt (page 9 line 298).
Thank you for your useful suggestions.
Reviewer 2 Report
The Manuscript ID: fermentation-2160695 “Development of Digested Sludge-Assimilating and Biohydrogen-Yielding Microflorae” requires revision before accepted for publication. The specific comments are given below.
1. The "Introduction" section should follow the state of the art of this field and review what has been done, for supporting the research gap and the significance of this study. Please improve the state of the art overview, to clearly show the progress beyond the state of the art.
2. In the last paragraph of the introduction, clearly indicate the research hypothesis.
3. Ln 64 – In chapter 2.2. Preparation of DS provide operating parameters (such as OLR, HRT, sewage flow) municipal sewage treatment plant in Yokohama City, Japan.
4. Statistical research is very important in experiments. How were the significances of the differences between the variables determined? Complete the methodology.
5. Research requires repetition. How many repetitions did you test? In chapter 3.1. Heat treatment of the DABYS microflora, add the standard deviations.
6. The results of the obtained studies are very poorly described. Describe and interpret them and compare them with the results of other authors.
7. I am asking you to perform an energetic analysis in order to assess the legitimacy.
8. It is also recommended to discuss and explain what should be the appropriate policies based on the findings of this study.
9. To add chapter Conclusions. Indicate the most important results of your research, problems and directions for the future.
10. Review the latest research from 2019 - 2023. Refresh the literature.
Author Response
1
Response to Reviewer 2 Comments
Point 1: The "Introduction" section should follow the state of the art of this field and review what has been done, for supporting the research gap and the significance of this study. Please improve the state of the art overview, to clearly show the progress beyond the state of the art.
Response 1: Description of the research in sludge hydrolysis technologies (eg., thermal, ultrasonic, oxidizing agents, electrolysis, pyrolysis, and enzymatic treatments) with recent review literatures as references was added in the revised manuscirpt (page 1 lines 37-40 and the refernce list).
Point 2: In the last paragraph of the introduction, clearly indicate the research hypothesis.
Response 2: Our research hypothesis “If the methanogenic DABYS microflorae are transformed to hydrogenic ones, renewable biohydrogen can be produced from DS, one of major industrial wastes for many developed nations, and contribute to the sustainable society” was described in the revised manuscript (page 2 lines 53–55).
Point 3: Ln 64 – In chapter 2.2. Preparation of DS provide operating parameters (such as OLR, HRT, sewage flow) municipal sewage treatment plant in Yokohama City, Japan.
Response 3: Information for the municipal sewage treatment plant with HRT and calorific value for DS used in the present study was added in the revised manuscript (page 2 lines 69–71, page 2 lines 80–82).
Point 4: Statistical research is very important in experiments. How were the significances of the differences between the variables determined? Complete the methodology.
Response 4: Statistical analyses were performed using Student’s t-test (P<0.05), which was added to the Method section (2.8.) of the revised namuscript. This information is also included in figure legends.
Point 5: Research requires repetition. How many repetitions did you test? In chapter 3.1. Heat treatment of the DABYS microflora, add the standard deviations.
Response 5: The data for gas production of bacterial flora after heat treatment (Fig. 1) and that for 10-times repetitive subculture (Fig. 2) were obtained as means for two-time GC quantification, from which it is unsuitable to calculate standard deviation (We think that correct SD value can be obtained by at least three-time experiment). We therefore added sentence “data are means for two-time GC analysis” in the legends for Figs. 1 and 2 of the revised manuscript.
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Point 6: The results of the obtained studies are very poorly described. Describe and interpret them and compare them with the results of other authors.
Response 6: The results were described in more detail the revised manuscript than the old version (page 4 lines 164–180, page 6, lines 204–218). Additionally, Interpretation of the results were made with comparing to the published relevant data (page 9 lines 279–285, page 10 lines 324–338).
Point 7: I am asking you to perform an energetic analysis in order to assess the legitimacy.
Response 7: Because we have a data for calorific value of the digested sludge, energetic balance for hydrogen production was discussed (page 10 lines 324–333).
Point 8: It is also recommended to discuss and explain what should be the appropriate policies based on the findings of this study.
Response 8: We think that additional improvement in fermentation condition of the microflorae and empoyment of physico-chemical treatment to make fine DS particles are indispensable for the industrial application. Our opinion was described in the revised manuscript (page 10 lines 333–335).
Point 9: To add chapter Conclusions. Indicate the most important results of your research, problems and directions for the future.
Response 9: The chapter Conclusion was added in the revised manuscript (page 10 lines 340–349).
Point 10: Review the latest research from 2019 - 2023. Refresh the literature.
Response 10: We replaced old literatures in the reference list to new ones in the revised manuscript as well as possible, and 15 and 8 literarures out of 29 in the reference list were replaced to the latest research (from 2019-2023 and 2015-2018, respectively). Some literatrures however could not be replaced because of their rarity or respect for their originality (eg., experimental protocols).
Thank you for your useful suggestions.
Round 2
Reviewer 1 Report
Many thanks to the authors for the detailled answers. Please consider point 3: "Results about the protease activity need to be dsicussed and confirmed by similar findings of others or a thorough discussion about potential errors needs to be made." I think this is mandatory to show that you have taken the rather unusual result under a deeper investigation.
Author Response:
Response to Reviewer 1 Comments
Point 1: Many thanks to the authors for the detailled answers. Please consider point 3: "Results about the protease activity need to be dsicussed and confirmed by similar findings of others or a thorough discussion about potential errors needs to be made." I think this is mandatory to show that you have taken the rather unusual result under a deeper investigation.
Response 1: Thank you so much for the suggestion. By heat treatment of the DABYS parent microflorae, the DABYS-A80 and -A100 lost protease activity, as shown in Fig. 3. DGGE analysis showed that an Enterobacteriaceae family strain which generates the amplicon-n was found extinct in those microflorae (Fig. 4). Because some strains of Enterobacter asbunae and Escherichia coli, the closest known relative to the amplicon-n, are reported to show proteolytic activity by secreting extracellular protease (Gheibipour et al (2022) Bioresource Technol. Rep. and Oh et al (2018) J. Microbiol. Biotechnol., for instance), we think the strain is a major contributor to protease activity of the DABYS parent microflorae, and its extinction in the DABYS-A80 and -A100 brought a loss of their protease activity. We added above discussion with new references in the revised manuscript (page 9 line 300 – page 10 line 307 and page 12 lines 421–424).
Reviewer 2 Report
Please complete the manuscript with OLR and HRT values for dark fermentation in all experimental variants - it is very important.
Author Response:
Response to Reviewer 2 Comments
Point 1: Please complete the manuscript with OLR and HRT values for dark fermentation in all experimental variants - it is very important.
Response 1: Thank you for your suggestion. We, however, apologize that we cannot determine OLR and HRT values for a serices of our fermentation experiments, because all experiments were carried out in a glass vial under a batch mode (NOT continuous mode). We appreciate you for understanding our experimental situation.
