A Review of Basic Bioinformatic Techniques for Microbial Community Analysis in an Anaerobic Digester
, Elena A. Zhuravleva 3, Dmitriy A. Kovalev 2, Yuriy V. Litti 3, Shyam Kumar Masakapalli 4, Nidhi Pareek 5 and Vivekanand Vivekanand 1,*Round 1
Reviewer 1 Report
The review article by Upadhyay and colleagues provides an introduction/overview of bioinformatic techniques for analyzing microbial communities in anaerobic digesters. The manuscript attempts to provide context for a wide range of topics in the analysis pipeline, with a main focus on metagenomic/transcriptomic analysis and how they can be applied to improve efficiencies of anaerobic digesters. The manuscript has the opportunity to present a nice review of the application of bioinformatic pipelines to anaerobic digesters. However, by covering such a broad range of topics, there are sections where the information being presented is not clear and where there is not a nuanced or relevant review of the scientific literature in the field. For example, section 2 (starting on page 3 to page 5) reads more like a methods/catalog paper, and the topic is not adequately discussed in terms of the challenges presented when analyzing anaerobic digesters. There could be several ways to improve this section, such as simplifying the discussion and pointing to relevant methods, protocols and other reviews that discuss DNA isolation in the field or by expanding on with more thorough discussion of the problems related to isolating DNA from anaerobic digesters (efficiencies are briefly mentioned but not expanded on with nuanced discussion of the issues).
The review article in sections seems to fluctuate between trying to provide technical details (method) versus discussing how the techniques have been used in the context of the field (section 4 seems to focus on the later method while 2 and 3 lean towards the former).
Within the paper, section 3.6 “Statistical multivariate analysis” seems to follow this procedure by presenting a more nuanced discussion of the uses of PCoA vs PCA and their advantages/disadvantages.
Major Comments.
1. Related to the above comment: In this reviewer’s opinion, the manuscript can be significantly improved by focusing on how the problems are specific to the field of anaerobic digestion rather than trying to list all methods for DNA prep/extraction kits and pre-processing steps. Numerous publications exist that cover many of the bioinformatic pipelines that can be referenced and referred to rather than going into details. Key discussion on analysis can be referenced or included in a figure to more clearly present the information (i.e., expanding figure 1).
2. The list of DNA isolation kits does not add to the manuscript and there does not seem to be a comprehensive review of percentages presented in this section (with ~20 articles cited for metric generation
3. In section four, the authors’ state that they bioinformatic profile helps with developing understanding and that the findings can help improve efficiencies of the systems. Sections 4.2 and 4.3 ties into this overall theme of using the data to understand/improve systems. Section 4.1 does not fully tie into this analysis and couple sentences discussing how this information could be used to benefit operations should be added to the section. to understand and then potential optimize the systems. Section 4.1 and attempting to tie together that the DNA sequencing can help identify key species that respond to different stresses (temperature, feedstocks, etc). If that is the case, the information
4. (line 631) Section 5 – what do the authors mean by “Metagenomic sequencing of microbial community associated with anaerobic digestion have been undermined very frequently”? The readability of this section makes it a bit difficult to understand. Are the authors suggestion that there needs to be improved data storage techniques/standardization for microbial community tools or focusing on reproducibility issues?
The second half of this section seems to be focusing on the issues with extrapolating small scale laboratory systems to real world digestors, which seems decoupled and a different section than the first portion.
Other Comments.
-Define the abbreviation of AD before use. I.e., in the first sentence add (AD). “intricate microbial population in anaerobic digesters (AD)”.
The abbreviation (‘viz’) is used frequently throughout the manuscript, and can be replaced in some instances where it is used one after another.
Figure 1. The size of boxes, arrows and text are not consistent. Steps such as PCR seem redundant to the process and could be reorganized to be more informative of the steps.
Figure 2. The colors of the bubbles with the white text are very difficult to read. It is suggested to use all black text such as bubble 1.
A figure of the anaerobic digester steps would help readers have a clearer picture of the process that is being discussed.
Author Response
#Reviewer 1
- The review article by Upadhyay and colleagues provides an introduction/overview of bioinformatic techniques for analyzing microbial communities in anaerobic digesters. The manuscript attempts to provide context for a wide range of topics in the analysis pipeline, with a main focus on metagenomic/transcriptomic analysis and how they can be applied to improve efficiencies of anaerobic digesters. The manuscript has the opportunity to present a nice review of the application of bioinformatic pipelines to anaerobic digesters. However, by covering such a broad range of topics, there are sections where the information being presented is not clear and where there is not a nuanced or relevant review of the scientific literature in the field. For example, section 2 (starting on page 3 to page 5) reads more like a methods/catalogue paper, and the topic is not adequately discussed in terms of the challenges presented when analyzing anaerobic digesters. There could be several ways to improve this section, such as simplifying the discussion and pointing to relevant methods, protocols and other reviews that discuss DNA isolation in the field or by expanding on with more thorough discussion of the problems related to isolating DNA from anaerobic digesters (efficiencies are briefly mentioned but not expanded on with nuanced discussion of the issues).
Response: Authors would like to thank the reviewer for their useful suggestions. Authors have already included a separate part for the challenges faced while analysing the metagenomics of anaerobic digester. Request to kindly refer to the, ‘5. Restrictions and predictions of bioinformatics analysis on microbial metagenomics’ at Page no. 17-18. Also, pls refer at Page No. 5; Line No. 173-178.
- The review article in sections seems to fluctuate between trying to provide technical details (method) versus discussing how the techniques have been used in the context of the field (section 4 seems to focus on the later method while 2 and 3 lean towards the former). Within the paper, section 3.6 “Statistical multivariate analysis” seems to follow this procedure by presenting a more nuanced discussion of the uses of PCoA vs PCA and their advantages/disadvantages.
Response: Authors have tried to give a broader picture of the commonly used bioinformatic tools in the metagenomic analysis of anaerobic digester instead of discussing a particular type of tool or method. Also, in MVA the PCoA vs PCA are common tools to be discussed and focussed. Hence, presenting the aforesaid detail will make the review too lengthy. May be group will look in to the possibility of drafting a new review on the suggested topics.
- Related to the above comment: In this reviewer’s opinion, the manuscript can be significantly improved by focusing on how the problems are specific to the field of anaerobic digestion rather than trying to list all methods for DNA prep/extraction kits and pre-processing steps. Numerous publications exist that cover many of the bioinformatic pipelines that can be referenced and referred to rather than going into details. Key discussion on analysis can be referenced or included in a figure to more clearly present the information (i.e., expanding figure 1).
Response: Authors have expanded figure 1(now figure 2) as per the reviewer’s suggestion, which has improved the overall quality of the revised MS and the information it contains.
- The list of DNA isolation kits does not add to the manuscript and there does not seem to be a comprehensive review of percentages presented in this section (with ~20 articles cited for metric generation)
Response: Authors have listed the DNA kits which are mostly used in the metagenomic analysis of anaerobic digesters worldwide. The information will help ‘Fermentation’ readers to have a closer look on deciding which kit to be used as it will lead to save, the time, energy and resources of the reader for shortlisting the best-suited kits to be used.
- In section four, the authors’ state that they bioinformatic profile helps with developing understanding and that the findings can help improve efficiencies of the systems. Sections 4.2 and 4.3 ties into this overall theme of using the data to understand/improve systems. Section 4.1 does not fully tie into this analysis and couple sentences discussing how this information could be used to benefit operations should be added to the section. to understand and then potential optimize the systems. Section 4.1 and attempting to tie together that the DNA sequencing can help identify key species that respond to different stresses (temperature, feedstocks, etc). If that is the case, the information
Response: Authors have added few sentences on reviewer’s suggestion (P. No. 14, Lin No. 491-494 & 513-516).
- (line 631) Section 5 – what do the authors mean by “Metagenomic sequencing of microbial community associated with anaerobic digestion have been undermined very frequently”? The readability of this section makes it a bit difficult to understand. Are the authors suggestion that there needs to be improved data storage techniques/standardization for microbial community tools or focusing on reproducibility issues? The second half of this section seems to be focusing on the issues with extrapolating small scale laboratory systems to real world digestors, which seems decoupled and a different section than the first portion.
Response: Thanks for the observation and constructive suggestion. Authors have corrected the aforesaid line (line no.650, pg no.19). Authors have highlighted both, the data storage challenges and also the reproducibility.
- Define the abbreviation of AD before use. I.e., in the first sentence add (AD). “intricate microbial population in anaerobic digesters (AD)”.
Response: Authors have made the suggested correction.
- The abbreviation (‘viz’) is used frequently throughout the manuscript and can be replaced in some instances where it is used one after another.
Response: Authors have made the suggested correction.
- Figure 1. The size of boxes, arrows and text are not consistent. Steps such as PCR seem redundant to the process and could be reorganized to be more informative of the steps.
Response: As per the suggestion, authors have remade the figure 1 (now figure 2).
- Figure 2. The colors of the bubbles with the white text are very difficult to read. It is suggested to use all black text such as bubble 1.
Response: Authors have made the suggested correction, now in the revised MS, its Fig. 3 instead of Fig. 2.
- A figure of the anaerobic digester steps would help readers have a clearer picture of the process that is being discussed.
Response: As per the suggestion, a figure (now in the revised MS, its Fig. 1) depicting the anaerobic digestion process and common microbes involved has been added in the manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The review, dealing with the techniques for microbial analysis in anaerobic digesters, is interesting and rich in citations, although a bit difficult to follow in some points due to lack of explanations, figures and schemes.
This latter is a major point I would suggest the authors to revise: figure 3 is very poorly explained, Figure 4 and 5 do not cite the source of the data included and also other approaches are simply cited with no effort in providing the reader with schemes or summarizing tables. As a suggestion, similar review focusing on AD analysis of microbial populations from slightly different viewpoints might be of help (https://doi.org/10.1016/j.biortech.2021.126350 https://doi.org/10.3390/microorganisms9061162 )
A further point to be addressed is the cited analysis of separated step of the process (as rightfully pointed out at line 55 and 64 page 2 or 456 page 13 in more detail). Here some important papers describing the details on the metagenomic and or trascriptomic of single steps in the perspective of consortia stability and their effect on optimization of hydrogen and methane production might be also cited (as few examples: The ISME Journal volume13, pages2617–2632 (2019), Scientific Reports volume 11, Article number: 19777 (2021), Biotechnol Biofuels 2021 Sep 16;14(1):182)
MInor point:
OLR as organic loading rate is not explained (only cited as OLR, no glossary either)
Author Response
#Reviewer 2
- The review, dealing with the techniques for microbial analysis in anaerobic digesters, is interesting and rich in citations, although a bit difficult to follow in some points due to lack of explanations, figures and schemes. This latter is a major point I would suggest the authors to revise: figure 3 is very poorly explained, Figure 4 and 5 do not cite the source of the data included and also other approaches are simply cited with no effort in providing the reader with schemes or summarizing tables. As a suggestion, similar review focusing on AD analysis of microbial populations from slightly different viewpoints might be of help (https://doi.org/10.1016/j.biortech.2021.126350 https://doi.org/10.3390/microorganisms9061162 )
Response: Authors would like to appreciate all the valuable and useful suggestion. Figure 3,4,5 (now figure 4, 5, 6) are all made from the data obtained from the experiments done by authors. These figures are only used as an model graph to give readers an idea of the results obtained through the particular analysis. Hence, no paper has been cited. The afore-suggested literatures have been added by the authors in the manuscript.
- A further point to be addressed is the cited analysis of separated step of the process (as rightfully pointed out at line 55 and 64 page 2 or 456 page 13 in more detail). Here some important papers describing the details on the metagenomic and or trascriptomic of single steps in the perspective of consortia stability and their effect on optimization of hydrogen and methane production might be also cited (as few examples: The ISME Journal volume13, pages2617–2632 (2019), Scientific Reports volume 11, Article number: 19777 (2021), Biotechnol Biofuels 2021 Sep 16;14(1):182)
Response: Authors have elaborated on the afore-suggested lines and also inserted the work from the literature mentioned by the reviewers.
- OLR as organic loading rate is not explained (only cited as OLR, no glossary either)
Response: Authors have made the suggested corrections.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors have clarified portions of the manuscript and improved the parts of the figures.
Reviewer 2 Report
The authors applied the recommended changes and improvements
