Optimization of the Biomethane Production Process by Anaerobic Digestion of Wheat Straw Using Chemical Pretreatments Coupled with Ultrasonic Disintegration

Round 1

Reviewer 1 Report

Anaerobic digestion is one of the promising renewable energy sources; however, the process still needs the optimization to fulfill both, the economic and environmental aims. The manuscript entitled “Optimization of the process of methane production from wheat straw and bovine manure co-digestion by ultrasound disintegration and physicochemical treatments” deals with important issue which is increasing methane production per unit of added substrate. Pretreatment of straw is considered to be one of the options to enhance anaerobic digestion rate and though increase the biogas production. Therefore the study described in the manuscript may add some valuable information to the issue of straw pretreatment. However, the title, aim and further description of methods and results are unclear in nomenclature. I have also some reservations about the methods used in the study. The part of the study on comparison of bovine manure and sewage sludge seems to be not very relevant and could be omitted. The methods are very poorly described without many relevant technical data and assumptions of experiments. The main flaw is the short period of the biochemical methane potential test which was done in duplicate instead of triplicate. This did not allow using any statistical methods. Part of methods is described in results. The methodology does not describe the individual stages of the research. The results are also poorly described. The substrates are not characterized. The results are not compared with any other articles. The conclusions are partly derived from results, but part of them is more like an introduction or there are such conclusions which do not arise from research.

Below, there are several major comments:

1. I do not agree that anaerobic digestion is the best option of renewable source energy. There are some concerns about this solution such as demand of substrates, energy crops used as feedstock, digestate management.
2. The straw in first case should be used as bedding in animal housing. Then it can be considered as a waste. The bedding systems with straw or other material are considered the most proper for animals comfort and therefore should be used instead of other manure management systems.
3. The pretreatment systems are well described however I miss information on effect of these treatments on the biogas or methane production. I miss also more details about types of pretreatments, e.g. other chemical solutions used, the concentration of solutions and their effect, the biological pretreatment is not described at all. Also the combined methods are not described.
4. The description of organization of the paper (lines 77-83) is unnecessary and should be avoided in scientific articles which are not reviews.
5. Materials and Methods are written very poorly. It is difficult to follow and understand the principles and experiments assumptions. The title suggests that bovine manure and wheat straw are the co-digested substrates while in course of the manuscript it turns out that bovine manure is inoculum which is compared to the sewage sludge. This is not clearly written in the Methods and this should be unified in title, aim and methods.
6. Typical BMP test is performed on one substrate with inoculum which is digestate from biogas plant or sewage sludge or rarely animal slurry. In this case the reactors with inoculum only are control and the produced methane from inoculum is subtracted from methane produced in reactors with inoculum and substrate. If we would like to determine the methane production from co-digestion it is done mainly in CSTR but it is possible to do it as BMP test, however there has to be control in the experiment. In case of experiment in manuscript there is no information how it was performed and what was the control. Perhaps it was bovine manure with straw and the results were compared with BMP performed on bovine manure with variously pretreated straw but this is not written in Methods. Another possible criticism could be that, the BMP test with bovine manure was not performed.
7. The bovine manure should be described more precisely. Is it liquid or solid, what it consist of: feces, water and urine (liquid manure – slurry) or only water and urine (liquid manure – slurry) or feces and bedding material (solid manure)?
8. The part about comparing sewage sludge and bovine manure is not relevant in this study; especially that this could be taken from literature that sewage sludge contains heavy metals which impair the biogas production; the determination of inoculum performance is usually done by BMP of inoculum with cellulose, not by comparing the pH, volatile fatty acids and total alkalinity for only 10 days. The differences in results should be statistically analyzed to confirm the differences and the methane yield should be also taken into account.
9. The pretreatment aims in degradation of cellulose, lignin, and hemicelluloses. There should be characteristics of these parameters of wheat straw. I miss any chemical description (total solids, volatile solids, volatile fatty acid, nitrogen, organic carbon etc.) of straw and bovine manure.
10. Materials and Methods. 2.2 Analytical methods should be at the end of section 2. In Section 2.2 the description of methods used should be in one paragraph and the producer of the equipment, and the country where the equipment was produced should be given. COD, TS, VS and VFA, TA , pH were determined in substrates or inoculum or digestates? In how many replicates the analyses were performed? SEM analysis was performed on which material? Technical details of SEM should be given.
11. In Section 2.3. What do you mean by dry WS? The first two sentences repeat the content from the introduction.
12. The paragraph consisting of one sentence should be avoided. Use the paragraph only when you start discussing a new topic. If in the next one you continue the topic from the previous one, you just keep writing on the same line.
13. What device was used for grinding? Please give the type, model and producer.
14. Section 2.4 is not a description of method but the well-known theory. In this section the parameters of ultrasound pretreatment should be given e.g. frequency, ultrasonic power, time of treatment, amount of substrate, volume of vessel, solution. The device used in this pretreatment should be also named with producer. If the device is prototype (line 132), it should be described with all technical details.
15. Section 2.5 needs additions about reactor volume, inoculum volume, total solids of liquid in reactor, pH of liquid in reactor, ratio of VS_inoculum to VS_substrate, control, replicates, mixing, the time of experiment and the criterion of its termination. Since in Results it turns out that the time of termination the experiment was 10 days, it is too short. It should be at least 35 days or the termination should be done when daily methane or biogas production is less than 1% of cumulative production.
16. There is nothing in Methods about NaOH and H₂O₂ pretreatment.
17. In Methods the statistical analyses are missing, the calculation of specific methane yield is also missing. No replications is a very serious flaw of this study. Any comparison should be based on the statistical analysis and they can be performed only if the replications are done.
18. Table 1. Tables should be self-explanatory; therefore you should not use the abbreviations in title and in table. What are the values given as BM[3] etc. What means the values in italics? The mg/Kg is incorrect. You should use the SI units i.e. mg kg^-1.
19. Section 3.1 seems to be not in the article scope, especially that lines 161- 174 are more like an introduction and lines 175-177 and 188-192 are description of methods (the experiment assumptions, poorly described, without any important principles which should be given). The rest of the section deals with pH, VFA and TA in simple BMP tests; however, if these changes in parameters affected the methane or biogas production is not revealed.
20. Section 3.2 The lines 238-241 are the experiment assumption and should be in Methods. Was this biomass dry, grinding? It is not clearly written that the straw was treated with the solutions separately. There is no comparison between NaOH and H₂O₂ treatments. Why the concentrations of both reagents are different 0.5% NaOH and 8% H₂O₂ but there is no 0.5% H₂O₂ and 8%NaOH?
21. Figure 4 is difficult to read. The concentrations of Na OH and H₂O₂ should be given below the columns; the colors should represent the type of reagent which could be also given below the concentrations. What means DCO in title of vertical axis, the unit is written in incorrect way. This refers also to Figure 5 and 6.
22. Section 3.5 Here for the first time there is any information on H₂O treatment with US. there is nothing about it in Methods and in previous results.
23. Section 3.6 The lines 321-326 are very poor description of experiment without any important assumptions and should be added to Methods. Figure 9 suggests that methane production stopped after two days of BMP in samples with untreated straw and after 7 days in samples with pretreated straw while biogas production stops after 5 and 7 days, respectively. In most BMP the production is observed even after 60 days. This refers to straw co-digestion with animal manure. It seems strange that biogas production stops after few days. Perhaps longer BMP could bring more information; especially that sometimes there is long lag time or second period of high daily production. Ten days is too short to make the conclusions.
24. Section 3.7. Again very poorly described methods in lines 372-374 which should be in Methods. What do you mean by filtrate, how the experiment was conducted, what was used as inoculum, what was control?
25. Conclusions should be written in one or two paragraphs or listed. The style used in manuscript i.e. paragraphs consisting of one sentence should be avoided. The conclusion from lines 408-410 is not derived from the study and should not be presented in this form. The part of conclusions line 412-463 and Figure 11 is more like introduction and is not derived from results of this study.  The definition of methanization and anaerobic digestion should rather start the manuscript which is about this process.
26. In table titles, there should be full stop after number of table.
27. In brackets the subsequent references numbers should be after the decimal point e.g. [1,2] or if there is 1, 2 and 3 should be written as follows [1–3]. The references written as [1–15] (line 44) suggest 15 different references with numbers from 1 to 15. Please see the Instructions for Authors.
28. Figures 1 and 2 are very poor quality and have to be changed.
29. The SI units should be used tables, figures and text.

Below, there are several minor comments:

Line 28: should be “biomass”

Line 30-31:  Agricultural biomass is abundantly found in nature, such as wheat straw (WS). What do you mean? I do not think that agricultural wastes are very abundant in environment. Most of them are used, not abandoned.

Line 63: What do you mean by this: (●OH)

Line 86: Why bovine manure and wheat straw were taken from stable. Stable is rather used for a building for keeping horses.

Line 137: please remove double space

Line 139: there should be a space between “pH” and “2”

Line 197: please remove double space

Line 233: confirm sth or agree with sth

Line 274 and 275: check the spaces, please

Line 304: “The hydrolysis with the only H2O coupled with the US” Is it H2O or H2O2. If H2O was used, there is nothing in the results or methods.

Line 354: please check: …of the methane, The…

Line 406: what do you mean by American pretreatment?

Line 446: this is incorrect reference. There should be number in square brackets

Line 411-463: space between paragraphs should not exceed the space between lines

Author Response

Thank you for your review of our document.

Please find below our responses given point by point. The changes made to the manuscript are indicated in red. In this regard, it is useful to draw your attention to the fact that we have performed new experiments to ensure reproducibility of results and compared our results with those of recent methods. Statistical analyses have been performed for each experiment and represented in each figure by error bars.

In this new version of the manuscript, we have made important changes and have enriched the materials and methods section with the characterization of wheat straw, cattle manure and sewage sludge (total solids, volatile solids, pH...).

We believe that this new version has been significantly improved by your comments and those of other reviewers.

Thank you very much for your valuable suggestions

Prof. Dr. A. OUAHABI

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript aims at assessing a combination of methods for enhancing the methane potential of wheat straw. There are major weaknesses as mentioned below:

In the introduction, it is not obvious why the specific combination of methods was chosen. Is there a hint that this combination would be preferable? There are combinations in literature that prove to be superior to the single treatment methods. What are these? The authors also mention that "coupling the US to grinding and chemical pretreatment using NaOH and H2O2 solutions has been weakly studied for lignocellulosic degradation to improve biogas and biomethane production". What is the closest combination studied and what are the results.

I am not sure if the introduction should end in this way. Usually, the main concept of the paper is presented, but not every section is mentioned.

In section 2.4,  a description of the methodology of the tests sjould be presented. Instead of this, information fitting best to the results or the introduction are reported.

Figures 1 and 2 are not necessary

In section 2.5, more details should be given. VS loading ratio of WS to inoculum, source of inoculum, if blank tests were included etc

lines 200-213: this is common knowledge. It should be ommitted since it doesn't offer a new knowledge, nor explains something significant.

line 210: What is "value buffer of pH?" Please rephrase

What is mentioned in section 3.2, should be reported in the materials and methods with more details.

Characteristics of WS are not reported, such as VS, TS, TKN

line 244: "WS hydrolysis with water": these tests are not mentioned in the materials and methods and cannot be understood in this section.

Figure 4 and likewise figures which are used for comparison: Replicates were not made and statistical analysis  was not performed. This would show if there are statistically significant differences

Section 3.3: What was the power applied in the US experiments? This should be mentioned in the materials and methods

Section 3.6: After pretreatment, how the sample was transferred to the 
BMP vials, since it was solids in liquid? How was secured that samples were
homogeneous?

line 325: we cannot see if the results are reproducible.

In figure 9 and elsewhere, is gas calculated at STP conditions? The temperature and pressure should be mentioned.

lines 341-358: common knowledge as above.

section 3.7: what filtrate? This should be mentioned in the materials and methods. I cannot understand.

The results are not compared with similar findings from literature.

It is not clear why the specific combination of methods was chosen, and if this is superior to other methods

The cost entailed in these pretreatment methods is not referred if it is compensated by the increase of methane.

Some corrections were made directly in the mansucript. Please see the attachment

Comments for author File: Comments.pdf

Author Response

Thank you for your review of our document.

Please find below our responses given point by point. The changes made to the manuscript are indicated in red. In this regard, it is useful to draw your attention to the fact that we have performed new experiments to ensure reproducibility of results and compared our results with those of recent methods. Statistical analyses have been performed for each experiment and represented in each figure by error bars.

In this new version of the manuscript, we have made important changes and have enriched the materials and methods section with the characterization of wheat straw, cattle manure and sewage sludge (total solids, volatile solids, pH...).

We believe that this new version has been significantly improved by your comments and those of other reviewers.

Thank you very much for your valuable suggestions

Prof. Dr. A. OUAHABI

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments:

There are many articles about disintegration as stated. Poor discussion, some analysis results were given. No statistical analysis. No characterization of samples after disintegration before fermentation (dry organic matter, VOC, pH, etc.).

1. lines 77-81 are not needed
2. line 106 - maybe should to show more other studies of sewage sludge describe the validity of the choice of operating parameters of ultrasound
3. Why were heavy metals analyzed? The scope of objectives set for research should be supplemented and the title of the thesis extended. There is nothing in the discussion about heavy metals. The effect of e.g. pH/H2O2/heavy metals was not studied after disintegration.
4. Fig. 1 - COD not DCO
5. line 245 - ... improve the amount of dissolved organic matter .... explain how was dissolved organic matter analyzed.
6. methodology - where is it described how the samples were filtered?
7. methodology - it has not been written how many days the fermentation was carried out.
8.  the disintegration efficiency in comparison with the control sample should be given as a percentage in the discussion.
9. Fig. 9, 10 - add the disintegration parameters in the Fig.
10. explain why fermentation was carried out for only 10 days?
11. line 406 - American pretreatment?
12. line 406 -463 - the information does not relate to the research subject, it is general and should be in the introduction rather than in the summary of the research results. Fig. 11 in conclusion? - nothing new, what for?

Author Response

Thank you for your review of our document.

Please find below our responses given point by point. The changes made to the manuscript are indicated in red. In this regard, it is useful to draw your attention to the fact that we have performed new experiments to ensure reproducibility of results and compared our results with those of recent methods. Statistical analyses have been performed for each experiment and represented in each figure by error bars.

In this new version of the manuscript, we have made important changes and have enriched the materials and methods section with the characterization of wheat straw, cattle manure and sewage sludge (total solids, volatile solids, pH...).

We believe that this new version has been significantly improved by your comments and those of other reviewers.

Thank you very much for your valuable suggestions

Prof. A. OUAHABI

Author Response File: Author Response.pdf

Reviewer 4 Report

The paper deals with the pretreatment of wheat straw with ultrasonication and different chemical agents for improving biogas production. During the first part of the research, the authors did the optimization of the chemical pretreatments focusing on the solubilization of organic matter. In the next phase, they focused on the optimization of the ultrasonication, before the influence of the best pretreatment combination was analyzed on the biogas production.

The paper is interesting, although many papers in the past have investigated ultrasound and chemical pretreatments for improving biogas production from lignocellulosic material. However, in my opinion, there are many weaknesses in the paper. Therefore, it requires deep modifications before being published in a scientific journal. In the following comments, this reviewer will try to explain the points that require important changes for the authors' consideration.

#1 Title: the title can lead to misunderstandings. When checking the paper, one can see that bovine manure is used as a source of microorganisms. Therefore, the term co-digestion is confusing for referring to this paper.

#2 Abstract: authors stated that they applied statistical analyses to evaluate the results of the sonication, however, these statistical analyses do not appear anywhere in the paper.

Introduction:

#3 General comment: there is a lack of a proper literature review regarding the influence of pretreatments on lignocellulosic material. What have been the effects observed? Do they increase solubilization or biogas production?

#4 Lines 28-30: this sentence sounds somewhat exaggerated: Can the production of biogas from waste meet the world's energy demand? In my opinion, this should be rewritten.

#5 Lines 74-75: could you please include the main findings of the papers which studied the combination of these pretreatments before AD or other processes (e.g. bioethanol production)? It could be useful to evaluate the novelty of the paper. Do authors believe that there are any research needs?

#6 Lines 77-83: in my opinion, this part should be removed from the paper.

Materials and Methods:

In my opinion, this section lacks a proper construction and the needed minimum information for replicating the experiments. Therefore, it should be rewritten. Some specific comments can be found below.

Section 2.1.

#7 Line 87. Could authors please specify if manure was fresh when it was collected, how it was collected, from where…

#8 Line 88. Could authors explain something more from the sewage sludge which was collected to be used as inoculum? Was it taken from a biodigester treating activated sludge in a WWTP? At what temperature was this digester working? What type of digester (CSTR)?

#9. Both substrates should have been characterized for their physical and chemical properties, not only the heavy metals.

Section 2.2.

#10. How did you measure the COD in solid samples? Did you apply any pretreatment to bovine manure and wheat straw before its analysis?

#11. What kind of method did you use for VFA measurement? There are two different methods in Standard Methods.

Section 2.4.

#12. Lines 106-125 fit better as part of the introduction since authors are including scientific information which makes the background of this research.

#13. What experimental conditions (time, power, dilution of substrates…) did you apply during ultrasound disintegration? In my opinion, experimental conditions for the pretreatments applied to WS and BM should be indicated here, including the number of replicates. Moreover, the experimental conditions for NaOH and H2O2 applications are missing here. Authors should also indicate how are they going to evaluate the effectiveness of the pretreatments.

Section 2.5.

#14. In this section, authors should include replicates, number of experiments, duration, etc. Pretreatment conditions that the authors tested in BMP should be indicated.

Results and discussion

Section 3.1.

#15. Line 182. The minimum values required for AD are different from tolerance levels, which may indicate from which concentration you can find inhibition. Therefore, in my opinion, it would be better if authors compared HM concentration to the tolerance levels and not to the minimum concentration required for AD.

#16. Line 190. This part should be indicated in Materials and Methods. Also, that the authors did two batch assays to study the possible inhibitory effects of HM in AD.

#17. Line 199. Was BM used as inocula or it was used as a substrate to which pretreatment was applied?

#18. Lines 234-235. I do not believe, based on the results, that an inhibition of the anaerobic digestion can be concluded. Did you measure biogas production and composition during this tests? In my opinion, results only indicated a common anaerobic digestion process during which at the beginning a reduction of pH may occur which will be recovered later. If authors would like to do a study of the HM influence on the AD process a completely different evaluation should be performed, including different concentration levels of HM in the substrates, longer retention periods, etc.

Section 3.2.

#19. Lines 238-242. This part should be located in materials and methods. Did you do replicates for each pretreatment? Can you apply statistical analysis to be sure that you have significant differences between pretreatment conditions?

#20. Line 245. How did you measure dissolved organic matter?

#21. Figure 4. Error bars should be included and statistical analysis should be performed to conclude which chemicas and concentrations are better.

Section 3.3.

#22. Figure 5. Same comments as in previous section.

#23. Line 271. Regarding the mineralization of solubilized organic matter by hydroxyls radicals: has this been seen before? Can you include a reference.

Section 3.4.

#24. Same comments as in previous sections (replicates, statistical analysis, include error bars…)

Section 3.5

#25. This part of the research should also be indicated in Materials and Methods. There is no reference to this until this point in the paper.

#26. Line 301. What did Zheng et al. study? Why it is similar to what you obtained? This should be explained in the paper.

Section 3.6.

#27. Could you please include error bars in your graph? Could you conduct statistical analysis to confirm your results? It is also strange that biogas production stopped after 10 days when the process normally lasts for 30-40 days in BMP assays. Any ideas why this happened?

#28. Lines 342-358 do not add significant value to the paper since it is a plain description of the anaerobic digestion process.

Section 3.7.

#29. Can you explain what did you do in this section? Again all this information should be included before in materials and methods, as part of the explanation of the experimental condition. Moreover, if in section 3.6. you used only the WS without the filtrate it should be noted.

Section 4.

#30. Lines 392-396. In my opinion, this conclusion is not supported by the results.

#31. Lines 412-End should be separated from the conclusions. Authors are encouraged to include a section before conclusions to express their perspectives for biogas production based on their findings.

Author Response

Thank you for your review of our document.

Please find below our responses given point by point. The changes made to the manuscript are indicated in red. In this regard, it is useful to draw your attention to the fact that we have performed new experiments to ensure reproducibility of results and compared our results with those of recent methods. Statistical analyses have been performed for each experiment and represented in each figure by error bars.

In this new version of the manuscript, we have made important changes and have enriched the materials and methods section with the characterization of wheat straw, cattle manure and sewage sludge (total solids, volatile solids, pH...).

We believe that this new version has been significantly improved by your comments and those of other reviewers.

Thank you very much for your valuable suggestions

Prof. Dr. A. OUAHABI

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been corrected. There are some minor comments:

Line 38: there should be indent at a beginning of the paragraph

Line 46: please delete full stop after bracket and combine both sentences

Line 47: this sentence needs rearrangement. You cannot write “the % of biomass …. is 19%”

Line 68: the sentence “WS can be…” has no sense to me

Line 162 please check the size of letters in red

Line 308 please add space between full stop and “Based”

Line 333: please add the space between “H2O2” and “are” and between “illustrated” and “in”

Line 480: Conventional

Line 481: space between biogas and plant

Figures 4, 5, 6, 10 and 11 should be corrected because the letters are too small and the explanations in legend or under axis are illegible. All the figures should be prepared in the same way with the same size of fonts, the same size of the graph etc.

Author Response

Dear Colleague,

Thank you for your corrections and suggestions.

In this new version of the manuscript, we have made significant improvements, and we have responded favorably to the reviewers' comments.

Kind regards

Prof. Dr. A. OUAHABI

Reviewer 3 Report

I agree with the correction.

Author Response

Dear Colleague,

Thank you for your corrections and suggestions

In this new version of the manuscript, we have made significant improvements, and we have responded favorably to the reviewers' comments.

Kind regards

Prof. Dr. A. OUAHABI

University of Tours

France

Author Response File: Author Response.pdf