Two-Stage Anaerobic Digestion for Green Energy Production: A Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

There are many flaws in the article. Some related to language that really downgrade the overall quality of the article. Some terms are not well define and explained. Too lengthy and can be summarized for better and clearer guide to reader.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The article require professional proofreading service prior submission.

Author Response

Response to the Comments of Reviewer 1

 

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Most of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Reviewer 1comments:

1. The manuscript has many flaws in language that downgrade the overall quality. It require a professional proofreading service prior submission.

 

Authors' response:

We accept the remark. A professional proofreading service has been provided to the entire text.

 

2. The author `s way for citing article by writing ``In (Nabaterega et al.,2021)” and other same example is not a proper way for citing others work. These example can be seen throughout the manuscript, please make sure to change it all using proper sentences for citation.

 

Authors' response:

We accept the remark. Changes have been made to the entire text of the article.

 

3. The arrangement of the article content and section can be improved further.

Authors' response:

We accept the remark. Changes have been made to the entire text of the article.

 

4. Introduction part is too lengthy and out of direction. The introduction is too long .

 

Authors' response:

We accept the remark. The introduction has been shortened considerably.

 

5. The TSAD part should be written separately.

 

Authors' response:

We accept the remark. A separate section „2. Тwo-stage anaerobic digestion description“ has been created.

 

6. Most of the section part writing is lengthy – can be shorten and summarize.

 

Authors' response:

We accept the remark. Abbreviations have been made in almost all sections and figures have been removed. But adding two new tables kept the length of the article the same size.

 

7. Title - Can be improved – using better words.

 

Authors' response:

We accept the remark. Another shorter title „Two-stage anaerobic digestion for green energy production: a review“ has been created..

 

8. Please divide part of the introduction part into new section ( 1 or 2 section)- Possibily name as TSAD or any suitable name to better guide the reader.

 

Authors' response:

We accept the remark. A separate section „2. Тwo-stage anaerobic digestion description“ has been created, in which part of the Introduction has been included

 

9. Line 123-126. This sentence is confusing. Please rewrite it and maybe put in in two sentence to give a clearer explanation

 

Authors' response:

We accept the remark. This passage (lines 123-126) has been removed.

 

10. Line 127. Please extend the sentence and provide a complete one.

 

Authors' response:

We accept the remark. This passage (line 127) has been removed.

 

11. Line 156-157, the sentence is not complete.

 

Authors' response:

We accept the remark. This passage (lines 156-157) has been removed.

 

12. In section 2.0 Feedstock. Please provide a table of data to simplify the explanation regarding type of substrate used. Please add also some explanation and comparison of the substrates in the same table of data.

 

Authors' response:

Different substrates have different compositions (even if we limit ourselves to carbohydrates, proteins and fats) and therefore the result is different, especially in the first phases, where the product is hydrogen. In addition, the different cited articles indicate different results for the same substrate. If this remark is reflected, then a separate review can be made for almost every substrate, as was done in some of the cited reviews. AD of waste mixtures (AcoD) seems to be the most promising, but with TSAD new difficulties and limitations appear precisely in these first phases. However, the articles in this direction are relatively fewer in number and it seems to be the most promising for future research. We accept the remark as an idea for a new article.

 

13. Similarly in section 3.0. The section title- please rewrite in to better title – Process and configuration in biohythane production can be summarized in table of Data.

 

Authors' response:

We accept the remark. Тhe new title is: “4. Process Parameters and configurations in biohythane production”. In this section of the article (as subsections) has been incorporated sections 3, 4 and 5 of the old version of the article. Two new tables are included:

Table 1. Some results for existing combinations of bioreactors in TSAD

Table 2. Some results obtained with a combination of different temperatures in both BRs.

Tables 1 and 2 complement each other.

 

14. Section 4.0. Please rewrite the title to Process Parameter or any suitable title. Provide a table of data for comparison of previous research.

 

 

Authors' response:

We accept the remark. Section 4.0 is included in the new Section “4. Process Parameters and configurations in biohythane production” as a subsection “4.1. Reactors configuration for biohythane production”. Two new tables are included:

Table 1. Some results for existing combinations of bioreactors in TSAD

Table 2. Some results obtained with a combination of different temperatures in both BRs.

Tables 1 and 2 complement each other.

 

15. Section 5.0, is removed and combine in to section 4.0

 

Authors' response:

We accept the remark. Section 5.0 is included in Section “4. Process Parameters and configurations in biohythane production”.

 

16. Section 6.0 should be rename as microorganism in TSAD. Provide a summary table of comparison the microbes used in the different process.

 

Authors' response:

We accept the remark. Section 6.0 (new number 5) was renamed as: 6. Microbiology – participating microorganisms in TSAD.

The microbial communities participating in the TSAD include various microorganisms, depending on the process conditions like temperature, pH, hydraulic retention time, type of substrate, co-substrates etc. Every single process involves a specific content of microbes in the community structure. In the case of TSAD the most important are hydrogen-consuming bacteria and hydrogen-producing bacteria. A comparison could not be made because of the specificity of each process. Table 1 (new number 3) reveals the prevailing microorganisms in the first stage - H₂ production, and the second stage CH₄ production again at different conditions. Different methods had been applied to investigate the microbial diversity. Knowing the exact constituent groups of microorganisms in the two bioreactors could help in providing suitable conditions for acidogenic and methanogenic microorganisms growth, development and action for a stable and efficient process implementation for clean energy production.

 

17. Section 7.0. Please provide table of data summarize the writing; by comparing important models used in the TSAD and AD

 

Authors' response:

Mathematical models in TSAD and AD are developed on the same basis, differing mainly in their complexity, depending on how many dominant reactions are involved. The difference with TSAD is that there are two bioreactors, and for this it must be reflected which part of the reactions take place in BR₁ and which in BR₂ and the exchange of flows from one to the other. The available articles on modeling of TSAD are still too few, therefore we believe that creating a table will only increase the volume of the article without improving its quality.

 

18. Section 8.0. Can be shorten and summarize with table of data

 

Authors' response:

We accept the remark. Abbreviations have been made in this paragraph by removing figures 4, 5 and 6 and the text associated with them. The available articles on control of TSAD are still too few, therefore we believe that creating a table will only increase the volume of the article without improving its quality.

 

19. Section 9.0. Title to rewrite to Energy consideration

Authors' response:

We accept the remark. The title has been changed.

 

20. TFAD ? What is the meaning of TFAD?

 

Authors' response:

We accept the remark. These are equivalent abbreviations. All are uniform to TSAD

 

21. Section 10. Figure 7- TPAD ? What is the meaning of TPAD?

 

Authors' response:

We accept the remark, and the figure has been removed as not representative of this article.

 

22. Please provide a table of Data to compare the advantages and disadvantages of the technologies.

 

Authors' response:

We believe that the introduction of a table will only lengthen the article without contributing to its improvement.

 

23. Section 11. Please rewrite the title to Future works

 

Authors' response:

We accept the remark. The title has been changed to “9. Future works”.

 

24. Conclusion. Please remove the citation for this part. Provide original opinion of the authors.

 

Authors' response:

We accept the remark. Citations removed. Added some own comments.

 

25. Clarity and context. Require major amendments based on comments

 

Authors' response:

We accept the remark. Made some changes based on comments.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

he authors of this paper deal with the processing of organic waste in a two-stage anaerobic system. Anaerobic processes of organic waste processing are important in terms of reducing emissions into the environment as well as alternative sources of clean energy.

The two-stage reactor technique allows, in comparison with the single-reactor arrangement (AD), where the optimization of the anaerobic process is subordinated to the priority creation of conditions for the growth of slowly growing methanogenic bacteria, the two-stage system (TSAD) allows to take into account the specifics of both slowly and rapidly growing microorganisms and independently optimize the conditions for their growth.

In this review paper, the authors present the results of a review of the state of the art, advantages and disadvantages, as well as some perspectives, including individual aspects of TSAD (the influence of parameters, types of bioreactors, microbiology, mathematical modeling, automatic control and energy considerations of processes) based on more than 210 references from the period 2002 to 2024 as well as their own studies.

The published results show that, compared to the traditional single-reactor process for CH4 production, the separation of the processes into a cascade of two bioreactors (BR) represents a significantly higher energy yield (H2 + CH4).

In addition to the decontamination of highly polluted organic wastewater and municipal waste, this anaerobic biotechnology allows, using various wastes and residues as substrates and mixed cultures, to produce hydrogen and volatile fatty acids (VFAs) including acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acids and other carboxylic acids such as succinic and lactic acids, which are important precursors for various industries as mixed or purified chemicals with high market value.

However, some organic wastes cannot be easily biotransformed due to their low nutrient levels, insufficient for anaerobic digestion, therefore co-digestion (AcoD) (digestion of mixtures of organic wastes) is a viable option. Numerous studies have shown that the use of co-substrates in anaerobic digestion systems improves methane yields, as positive synergisms are created in the digestion medium and the co-substrates introduce the supply of missing nutrients. The synergistic effects between the different chemical components within the AcoD process played an important role in improving its performance. In the TSAD system, relatively fast-growing acidogens and H2-producing microorganisms are cultured in the first hydrogen bioreactor and participate in the formation of volatile fatty acids (VFA) and H2. Slow-growing acetogens and methanogens are cultured in the second, i.e. methanogenic bioreactor (), in which the previously produced VFA are further transformed into CH4 and CO2.

The main aspects of TSAD have been confirmed by experimental tests (in batch or continuous mode) using two feeds as organic wastes. The results show that the energy produced in the form of hydrogen and methane gas is 1.5–2.7 times higher compared to the energy obtained in a single-stage AD system. By combining the production of biohydrogen and biomethane from organic wastes in TSAD, biomethane gas with high potential for use as fuels could be obtained. TSAD processes can ensure the biological production of hydrogen and methane using organic waste and wastewater as feedstock. The potential of TSAD to reduce greenhouse gas emissions has been demonstrated.

The implementation of TSAD can be simplified by converting existing biogas plants by adding an additional bioreactor. This would avoid the construction of new plants, a factor that significantly increases the implementation costs. The identification of the correlation between the type of feedstock and the abundance of existing microbial consortia species can be used to optimize AcoD systems through the correct selection of feedstocks and their combination ratio.

From a technical point of view, it is essential to focus future research on the isolation and immobilization of stable and adapted bacteria for the digestion process, together with studies on the combination of specific biodigesters, pretreatments and substrate mixtures. The development of this sector is fundamental to achieving a high level of technological maturity that allows the expansion of two-stage digestion, which in most cases occurs only at a test or pilot scale. In addition to the economic interest, the payback of the TSAD process was evaluated over approximately 2–6 years, depending on the costs of organic waste/residue disposal.

Author Response

Response of Reviewer 2.

This reviewer does not explicitly state any comments or recommendations.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

I had the chance to review your manuscript entitled “Two-stage anaerobic digestion of organic wastes: state of the art, advantages, disadvantages and some perspectives”. In general, I find it a very complete review, but I have several issues with the way some information is presented to the readers.

I believe chapter 2 in general could be easily converted into a table with the format “Feedstock | Simplified description | Productivity | References”, because it is difficult to read in the current format. The yields/main results of each of the references should be presented in this table format in a succinct form, with numbers but not too much text, which is largely missing in the current situation. All extraneous data can be discussed after the table by referring to a specific entry on the table. Several of the details present in “5. Molasses wastewater” could be discussed in chapter 4 instead, or maybe show that the order of the chapters should be redesigned, so that operational parameters come before feedstock. Similar logic could easily be applied to chapter 3.

Bio-gas production tends to be self-contained, in the sense that the produced gas is immediate combusted for the production of heat (and sometimes power generation). However, there is a push for the injection of produced methane in the natural gas grid, which requires removal of the produced CO2. Further valorizations of the bio-gas that do not involve injection also require the purification of different potential gas streams. I believe the paper would benefit from a discussion on methods for downstream treatment of both TSAD effluents, when the use is not direct combustion.

I find the chapters about mathematical modeling (7) and advantages and disadvantages (10), as well as the future work one (11), very complete and full of guidance. Both abstract and conclusions are very solid and will surely guide future readers to the topic, and I appreciate that they complement each other, instead of being very similar, as is frequently seen.

Make sure to properly format scientific names of microorganisms. This doesn’t seem to be done consistently. Attention to detail is important and positive for increased engagement.

There are several issues with the formatting of references throughout the text, stemming mostly from the lack of checking the references in the reference manager software used. It seems to happen with large frequency with Asian names, where it may unclear which is the first name and which is/are family name(s). The formatting of the references is not consistent either, with different paragraphs often using different logic (contrast the references in the paragraph in ll. 293-299 with those in the paragraph in ll. 304-313). Please be consistent.

There are minor English and formatting errors throughout the text, which, while not affecting greatly the inferred meaning of the text, could improve the overall perception and quality of the final product.

Some minor comments:

l. 328-337: It would be very interesting to present these options in a figure/scheme format;

l. 405-418: It would be very interesting to present these options in a figure/scheme format;

section 5.1: I feel like this could easily be merged into chapter 6, and should be further developed. It is an interesting area of discussion that has practical applications for both academia and industry;

Figure 4: the figure seems to be compressed vertically, consider providing a better version with a higher DPI. Also, you don’t include copyright information;

Table 2 and 3: while you are referring to your own work reported in another publication, you should still add the proper references to the captions.

 Thank you.

Author Response

Response to the Comments of Reviewer 3.

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Most of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Comments and Suggestions for Authors

1. I believe chapter 2 in general could be easily converted into a table with the format “Feedstock | Simplified description |Productivity References”, because it is difficult to read in the current format. The yields/main results of each of the references should be presented in this table format in a succinct form, with numbers but not too much text, which is largely missing in the current situation.

Authors' response:

The reviewer's remark seems quite logical, but according to the authors, its implementation is not only extremely difficult, but will also lead to a very voluminous table and, therefore, to an increase in the volume of the already long article. The arguments for this are as follows:

- 20 groups of waste are listed, but in each group there are several types;

- For some type of waste, different results are given in the literature depending on the type of bioreactors, the temperature regime, etc.;

- Most of the cited reviews are precisely about one type or group of waste and each of these rerviews is sufficiently detailed and voluminous;

- The two new tables made (Table 1 and Table 2) confirm that this would lead to a large increase in the volume of the article.

 

2. Several of the details present in “5. Molasses wastewater” could be discussed in chapter 4 instead.

Authors' response:

We agree that “Molasses wastewater” could be discussed in 4. “Industrial wastes” and we have put it there.

 

3. Similar logic could easily be applied to chapter 3

Authors' response:

We agree that Organic fraction of “municipal solid waste” could be discussed in 2’’Municipal Wastewater” and we have put it there.

 

4. “or maybe show that the order of the chapters should be redesigned, so that operational parameters come before feedstock”.

Authors' response:

It is the opinion of the authors that „feedstock“ should come before the „operational parameters“ paragraph, which at the suggestion of other reviewers has been revised in a much more general way.

 

5. Biogas production tends to be self-contained, in the sense that the produced gas is immediate combusted for the production of heat (and sometimes power generation). However, there is a push for the injection of produced methane in the natural gas grid, which requires removal of the produced CO₂. Further valorizations of the biogas that do not involve injection also require the purification of different potential gas streams.

Authors' response:

We know that one of the most promising methods of hydrogen transfer is its injection into existing natural gas pipelines. At the same time, most of the authors in the field of TSAD indicate that one of the best (in a practical sense) qualities of TSAD is the possibility of easily mixing the two types of gas obtained (hydrogen and methane) in a certain ratio, which is close to that obtained in most real cases. The resulting mixture, for which a new term (hythane) has even been introduced, has improved characteristics for use in internal combustion engines, so its use in co-generator groups (most widespread now) with the simultaneous production of electrical and thermal energies seems the most prospective.

 

6. I believe the paper would benefit from a discussion on methods for downstream treatment of both TSAD effluents, when the use is not direct combustion.

Authors' response:

The authors agree with this recommendation, but the article has become rather long anyway, so it will be covered in another paper.

 

7. Make sure to properly format scientific names of microorganisms. This doesn’t seem to be done consistently. Attention to detail is important and positive for increased engagement.

Authors' response:

Scientific names of microorganisms were checked and corrected.

 

8. There are several issues with the formatting of references throughout the text, stemming mostly from the lack of checking the references in the reference manager software used. It seems to happen with large frequency with Asian names, where it may unclear which is the first name and which is/are family name(s). The formatting of the references is not consistent either, with different paragraphs often using different logic (contrast the references in the paragraph in ll. 293-299 with those in the paragraph in ll. 304-313). Please be consistent.

Authors' response:

We fully accept the remark and, as far as we could, have taken it into account in the corrected version of the article.

9. There are minor English and formatting errors throughout the text, which, while not affecting greatly the inferred meaning of the text, could improve the overall perception and quality of the final product.

 

Authors' response:

 

Some English and formatting errors were corrected throughout the text.

Some minor comments:

- 328-337: It would be very interesting to present these options in a figure/scheme format;

Authors' response:

We agree with the reviewer's recommendation, but it would add to the already large volume of the article. Various figures and diagrams are given in the cited articles.

 

- 405-418: It would be very interesting to present these options in a figure/scheme format;

Authors' response:

We agree with the reviewer's recommendation, but it would add to the already large volume of the article. Various figures and diagrams are given in the cited articles.

 

• section 5.1 Inocula: I feel like this could easily be merged into chapter 6, and should be further developed. It is an interesting area of discussion that has practical applications for both academia and industry;

Authors' response:

Section 5.1 was merged into chapter 4, according to the suggestion. Additional text with references was included to the already mentioned.

 

• Figure 4: the figure seems to be compressed vertically, consider providing a better version with a higher DPI. Also, you don’t include copyright information;

Authors' response:

The authors decided that this Figure 4 was not defining for the paper and removed it.

 

Table 2 and 3: while you are referring to your own work reported in another publication, you should still add the proper references to the captions.

Authors' response:

The relevant references were added to the captions.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors set the stage to present the state-of-art of the two-stage anaerobic digestion (TSAD) of organic wastes. The TSAD is a two-stage process, in which in the fist stage relatively fast growing acidogens and H₂ producing microorganisms are grown (hydrogenic bioreactor) and in the second stage slow growing acetogens and methanogens are grown. As a result in the first bioreactor volatile fatty acids (VFAs) and H₂ is produced, whereas in the second bioreactor the VFAs are converted to CH₄ and CO₂. In this review, the authors included successfully all the aspects of the TSAD process, the influence of different parameters, types of bioreactors, microbiology, mathematical modelling, automatic control and energetical considerations on TSAD. This study is interesting but there are some issues to be addressed.   

 

1) The authors should include some figures of the different type of the bioreactors mentioned in the text (section 3. Reactors configuration of biohythane production)  

 

2) The authors should reconstruct the section that is referred the temperature influence on AD (and TSAD) process (4.1. Temperature) in order to be more comprehensive,  e.g.  first the dark fermentation, second the AD classification according to temperature (lines 375 - 380), then the analysis (thermophilic, mesophilic etc).  

 

3) The authors should reconstruct the section that is referred to mathematic modeling (section 7) in order to be more comprehensive. eg. first, the 1D or 2D steady state models, secondly CFD models and last machine learning model. The control (section 8 Control) and Artificial Intelligence could be a subsection in the section 7  

 

4) In Integrating meta-omics approaches (section 11.1) some figures with omic (e.g. metabolomic) heat map or pathway should be included.

Author Response

Response to the Comments of Reviewer 4.

 

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Some of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Comments Form Reviewer 4 :

• The authors should include some figures of the different type of the bioreactors mentioned in the text (section 3. Reactors configuration of biohythane production)

 

Authors' response:

Section 3 has been reconfigured as subsection „4.1. Reactors configuration for biohythane production“ of the consolidated section „4. Process Parameters and configurations in biohythane production“. А large number of figures of different types of bioreactors in both phases are are shown in cited references. In our opinion, adding such figures would increase the volume of the already quite long article.

 

2) The authors should reconstruct the section that is referred the temperature influence on AD (and TSAD) process (4.1. Temperature) in order to be more comprehensive, e.g. first the dark fermentation, second the AD classification according to temperature (lines 375 - 380), then the analysis (thermophilic, mesophilic etc).

 

Authors' response:

In the new version of the article, subsection “4.1. Temperature” has been reconfigured as subsection “4.2. Temperature” of the consolidated section “4. Process Parameters and configurations in biohythane production” and two new tables have been added (Table 1 and Table 2), which we hope has responded to the reviewer's recommendation.

 

3) The authors should reconstruct the section that is referred to mathematic modeling (section 7) in order to be more comprehensive. eg. first, the 1D or 2D steady state models, secondly CFD models and last machine learning model. The control (section 8 Control) and Artificial Intelligence could be a subsection in the section 7

 

Authors' response:

The available articles on modeling of TSAD are still too few, so in our opinion it is still too early to make strict classifications. Steady state models are obtained from dynamic models, so their separation does not seem appropriate to us. The inclusion of “Control” as a subsection of “Modeling” seems to us inappropriate, at least due to the fact that there are also model-free control algorithms (i.e., they are not synthesized based on process models).

 

4) In Integrating meta-omics approaches (section 11.1) some figures with omic (e.g. metabolomic) heat map or pathway should be included.

 

Authors' response:

To our knowledge, there are very few articles in this area about the two-step process. Moreover, the article has already become quite long, so adding more figures seems inappropriate to us.

 

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript offers a detailed discussion of the two-stage anaerobic digestion (TSAD) process, focusing on various aspects such as microbiology, process parameters, and the potential for biohydrogen and biomethane production. However The innovation of the manuscript has not been well reflected.

The topic is highly relevant to current research in the field of sustainable energy, waste management, and biofuels.

But I think there 's something else in this manuscript that needs to be revised:

1.     The introduction provides an adequate background but could benefit from more details about the motivations for researching TSAD. Specifically, the authors should explain in more depth why TSAD is an important area of study and what gaps in existing research this review aims to address.

2.     In Chapter 2, the comparison of the characteristics of various feedstocks can be discussed to better explain the influence of different feedstocks on digestion process.

3.     In 11.3, it would be helpful to further discuss the performance of AI models in TSAD, particularly the optimization of different algorithm combinations and the potential technical bottlenecks.

Author Response

Response to the Comments of Reviewer 5.

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Some of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Comments and Suggestions for Authors

1. However the innovation of the manuscript has not been well reflected.

 

Authors' response:

Our review provides new information in all aspects of TSAD, including mathematical modeling, automatic control, and the use of AI, which, to our knowledge, has not been included in any other review, and also presents some new perspectives for development in this field.

 

2. The introduction provides an adequate background but could benefit from more details about the motivations for researching TSAD. Specifically, the authors should explain in more depth why TSAD is an important area of study and what gaps in existing research this review aims to address.

 

Authors' response:

The article states (Chapter „Advantages and disadvantages”) what the main advantages of this approach are and why it has been the subject of intensive research in recent years, as well as what its disadvantages are. Therefore, it seems to us that it is not appropriate to insert additional explanations into the already very long Introduction.

 

3. In Chapter 2, the comparison of the characteristics of various feedstocks can be discussed to better explain the influence of different feedstocks on digestion process.

 

Authors' response:

Different substrates have different compositions (even if we limit ourselves to carbohydrates, proteins and fats) and therefore the result is different, especially in the first phases, where the product is hydrogen. In addition, the different cited articles indicate different results for the same substrate. If this remark is reflected, then a separate review can be made for almost every substrate, as was done in some of the cited reviews. AD of waste mixtures (AcoD) seems to be the most promising, but with TSAD new difficulties and limitations appear precisely in these first phases. However, the articles in this direction are relatively fewer in number and it seems to be the most promising for future research.

 

4. In 11.3, it would be helpful to further discuss the performance of AI models in TSAD, particularly the optimization of different algorithm combinations and the potential technical bottlenecks.

Authors' response:

Publications on the application of AI in TSAD are extremely few and therefore it would be difficult to have such a discussion at this stage. The potential difficulties are the same as in the single-phase process – the small amount of available experimental data, the possible ongoing changes in the individual phases of the process (especially in the first BR) in the absence of opportunities to obtain experimental data in real time, as well as the “black box” nature of these models and their difficult acceptance by the biological community. Such kind of reviews are known to the authors, but for AD and AcoD, and not for TSAD.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

some minor error in line 991, exemples- should be written examples

Author Response

Response to the Comments of Reviewer 1

 

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Most of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Reviewer 1comments:

1. The manuscript has many flaws in language that downgrade the overall quality. It require a professional proofreading service prior submission.

 

Authors' response:

We accept the remark. A professional proofreading service has been provided to the entire text.

 

2. The author `s way for citing article by writing ``In (Nabaterega et al.,2021)” and other same example is not a proper way for citing others work. These example can be seen throughout the manuscript, please make sure to change it all using proper sentences for citation.

 

Authors' response:

We accept the remark. Changes have been made to the entire text of the article.

 

3. The arrangement of the article content and section can be improved further.

Authors' response:

We accept the remark. Changes have been made to the entire text of the article.

 

4. Introduction part is too lengthy and out of direction. The introduction is too long .

 

Authors' response:

We accept the remark. The introduction has been shortened considerably.

 

5. The TSAD part should be written separately.

 

Authors' response:

We accept the remark. A separate section „2. Тwo-stage anaerobic digestion description“ has been created.

 

6. Most of the section part writing is lengthy – can be shorten and summarize.

 

Authors' response:

We accept the remark. Abbreviations have been made in almost all sections and figures have been removed. But adding two new tables kept the length of the article the same size.

 

7. Title - Can be improved – using better words.

 

Authors' response:

We accept the remark. Another shorter title „Two-stage anaerobic digestion for green energy production: a review“ has been created..

 

8. Please divide part of the introduction part into new section ( 1 or 2 section)- Possibily name as TSAD or any suitable name to better guide the reader.

 

Authors' response:

We accept the remark. A separate section „2. Тwo-stage anaerobic digestion description“ has been created, in which part of the Introduction has been included

 

9. Line 123-126. This sentence is confusing. Please rewrite it and maybe put in in two sentence to give a clearer explanation

 

Authors' response:

We accept the remark. This passage (lines 123-126) has been removed.

 

10. Line 127. Please extend the sentence and provide a complete one.

 

Authors' response:

We accept the remark. This passage (line 127) has been removed.

 

11. Line 156-157, the sentence is not complete.

 

Authors' response:

We accept the remark. This passage (lines 156-157) has been removed.

 

12. In section 2.0 Feedstock. Please provide a table of data to simplify the explanation regarding type of substrate used. Please add also some explanation and comparison of the substrates in the same table of data.

 

Authors' response:

Different substrates have different compositions (even if we limit ourselves to carbohydrates, proteins and fats) and therefore the result is different, especially in the first phases, where the product is hydrogen. In addition, the different cited articles indicate different results for the same substrate. If this remark is reflected, then a separate review can be made for almost every substrate, as was done in some of the cited reviews. AD of waste mixtures (AcoD) seems to be the most promising, but with TSAD new difficulties and limitations appear precisely in these first phases. However, the articles in this direction are relatively fewer in number and it seems to be the most promising for future research. We accept the remark as an idea for a new article.

 

13. Similarly in section 3.0. The section title- please rewrite in to better title – Process and configuration in biohythane production can be summarized in table of Data.

 

Authors' response:

We accept the remark. Тhe new title is: “4. Process Parameters and configurations in biohythane production”. In this section of the article (as subsections) has been incorporated sections 3, 4 and 5 of the old version of the article. Two new tables are included:

Table 1. Some results for existing combinations of bioreactors in TSAD

Table 2. Some results obtained with a combination of different temperatures in both BRs.

Tables 1 and 2 complement each other.

 

14. Section 4.0. Please rewrite the title to Process Parameter or any suitable title. Provide a table of data for comparison of previous research.

 

 

Authors' response:

We accept the remark. Section 4.0 is included in the new Section “4. Process Parameters and configurations in biohythane production” as a subsection “4.1. Reactors configuration for biohythane production”. Two new tables are included:

Table 1. Some results for existing combinations of bioreactors in TSAD

Table 2. Some results obtained with a combination of different temperatures in both BRs.

Tables 1 and 2 complement each other.

 

15. Section 5.0, is removed and combine in to section 4.0

 

Authors' response:

We accept the remark. Section 5.0 is included in Section “4. Process Parameters and configurations in biohythane production”.

 

16. Section 6.0 should be rename as microorganism in TSAD. Provide a summary table of comparison the microbes used in the different process.

 

Authors' response:

We accept the remark. Section 6.0 (new number 5) was renamed as: 6. Microbiology – participating microorganisms in TSAD.

The microbial communities participating in the TSAD include various microorganisms, depending on the process conditions like temperature, pH, hydraulic retention time, type of substrate, co-substrates etc. Every single process involves a specific content of microbes in the community structure. In the case of TSAD the most important are hydrogen-consuming bacteria and hydrogen-producing bacteria. A comparison could not be made because of the specificity of each process. Table 1 (new number 3) reveals the prevailing microorganisms in the first stage - H₂ production, and the second stage CH₄ production again at different conditions. Different methods had been applied to investigate the microbial diversity. Knowing the exact constituent groups of microorganisms in the two bioreactors could help in providing suitable conditions for acidogenic and methanogenic microorganisms growth, development and action for a stable and efficient process implementation for clean energy production.

 

17. Section 7.0. Please provide table of data summarize the writing; by comparing important models used in the TSAD and AD

 

Authors' response:

Mathematical models in TSAD and AD are developed on the same basis, differing mainly in their complexity, depending on how many dominant reactions are involved. The difference with TSAD is that there are two bioreactors, and for this it must be reflected which part of the reactions take place in BR₁ and which in BR₂ and the exchange of flows from one to the other. The available articles on modeling of TSAD are still too few, therefore we believe that creating a table will only increase the volume of the article without improving its quality.

 

18. Section 8.0. Can be shorten and summarize with table of data

 

Authors' response:

We accept the remark. Abbreviations have been made in this paragraph by removing figures 4, 5 and 6 and the text associated with them. The available articles on control of TSAD are still too few, therefore we believe that creating a table will only increase the volume of the article without improving its quality.

 

19. Section 9.0. Title to rewrite to Energy consideration

Authors' response:

We accept the remark. The title has been changed.

 

20. TFAD ? What is the meaning of TFAD?

 

Authors' response:

We accept the remark. These are equivalent abbreviations. All are uniform to TSAD

 

21. Section 10. Figure 7- TPAD ? What is the meaning of TPAD?

 

Authors' response:

We accept the remark, and the figure has been removed as not representative of this article.

 

22. Please provide a table of Data to compare the advantages and disadvantages of the technologies.

 

Authors' response:

We believe that the introduction of a table will only lengthen the article without contributing to its improvement.

 

23. Section 11. Please rewrite the title to Future works

 

Authors' response:

We accept the remark. The title has been changed to “9. Future works”.

 

24. Conclusion. Please remove the citation for this part. Provide original opinion of the authors.

 

Authors' response:

We accept the remark. Citations removed. Added some own comments.

 

25. Clarity and context. Require major amendments based on comments

 

Authors' response:

We accept the remark. Made some changes based on comments.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

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Author Response

Response to the Comments of Reviewer 4.

 

We thank the reviewer for his serious and thorough review of the content of our review article and the creative remarks made. Some of these comments have been reflected in the new version of the article and it is hoped that it has improved its quality. Below we give our comments in red, with amendments and additions also in red in the text of the new version.

 

Comments Form Reviewer 4 :

• The authors should include some figures of the different type of the bioreactors mentioned in the text (section 3. Reactors configuration of biohythane production)

 

Authors' response:

Section 3 has been reconfigured as subsection „4.1. Reactors configuration for biohythane production“ of the consolidated section „4. Process Parameters and configurations in biohythane production“. А large number of figures of different types of bioreactors in both phases are are shown in cited references. In our opinion, adding such figures would increase the volume of the already quite long article.

 

2) The authors should reconstruct the section that is referred the temperature influence on AD (and TSAD) process (4.1. Temperature) in order to be more comprehensive, e.g. first the dark fermentation, second the AD classification according to temperature (lines 375 - 380), then the analysis (thermophilic, mesophilic etc).

 

Authors' response:

In the new version of the article, subsection “4.1. Temperature” has been reconfigured as subsection “4.2. Temperature” of the consolidated section “4. Process Parameters and configurations in biohythane production” and two new tables have been added (Table 1 and Table 2), which we hope has responded to the reviewer's recommendation.

 

3) The authors should reconstruct the section that is referred to mathematic modeling (section 7) in order to be more comprehensive. eg. first, the 1D or 2D steady state models, secondly CFD models and last machine learning model. The control (section 8 Control) and Artificial Intelligence could be a subsection in the section 7

 

Authors' response:

The available articles on modeling of TSAD are still too few, so in our opinion it is still too early to make strict classifications. Steady state models are obtained from dynamic models, so their separation does not seem appropriate to us. The inclusion of “Control” as a subsection of “Modeling” seems to us inappropriate, at least due to the fact that there are also model-free control algorithms (i.e., they are not synthesized based on process models).

 

4) In Integrating meta-omics approaches (section 11.1) some figures with omic (e.g. metabolomic) heat map or pathway should be included.

 

Authors' response:

To our knowledge, there are very few articles in this area about the two-step process. Moreover, the article has already become quite long, so adding more figures seems inappropriate to us.

Author Response File: Author Response.pdf