Enhancement of Biological Pretreatment on Rice Straw by an Ionic Liquid or Surfactant

Round 1

Reviewer 1 Report

The major weakness of the manuscript are the main focus on the production of sugars for bioethanol. The novelty of a study should not be the increase of glucose concentration during hydrolysis, but the catalytic performance of laccase in IL or with surfactant. More attention should be paid to these processes, including in the title.

Comment 1.

Enzymatic saccharification was not carried out at a high level from a technological point of view.

Low substrate concentration, high dosage of cellulases, long hydrolysis time and at the same time low efficiency.

Comment 2.

The description of the catalysts used is not complete. There is no data on the main and side activities, protein content, optimal pH and temperature. When describing laccase, there is no information about mediators, whether they are necessary, and what compounds can play this role in the catalytic processes.

Comment 3.

Despite the applied modern instrumental methods, the authors do not always fully manage to use them. For example, Figure 3 ambiguously indicates the effect of the biocatalysts on the fiber, since there can be different plant anatomical elements in the camera lens, and this can be much more clearly represented at higher magnification. In paragraph 3.6 there is no reference to the methodology used. Currently, new, alternative approaches are emerging (for example https://0-link-springer-com.brum.beds.ac.uk/article/10.1134%2FS0036024420060059).

Other comments.

Line 50- 51 The authors should decide whether microbes or enzymes separate from microbes carry out deconstruction.

Table 1 It is desirable to provide data in terms of dry weight.

Figure 2 improve the quality or reduce based on previous comments.

Author Response

We appreciate the detailed review and constructive suggestions for improving the manuscript. We have responded in detail to all suggestions for revisions or requests for clarification below and have made revisions to the manuscript as noted (blue words). We would love to thank you for allowing us to resubmit a revised copy of the manuscript and we highly appreciate your time and consideration.

Reviewer #1:

The major weakness of the manuscript are the main focus on the production of sugars for bioethanol. The novelty of a study should not be the increase of glucose concentration during hydrolysis, but the catalytic performance of laccase in IL or with surfactant. More attention should be paid to these processes, including in the title.

Thank you for your comments. We have revised the title to “Enhancement of biological pretreatment on biomass by an ionic liquid or surfactant”.

Comment 1.

Enzymatic saccharification was not carried out at a high level from a technological point of view.

Low substrate concentration, high dosage of cellulases, long hydrolysis time and at the same time low efficiency.

We sincerely appreciate your comment. In this study, we focus on improving biodelignification pretreatment of rice straw. Therefore, substrate concentration, cellulose dosage and hydrolysis time were not optimal in this study. We also added that the reaction conditions still need to be further optimized to approach industrial conditions in the conclusion section.

Comment 2.

The description of the catalysts used is not complete. There is no data on the main and side activities, protein content, optimal pH and temperature. When describing laccase, there is no information about mediators, whether they are necessary, and what compounds can play this role in the catalytic processes.

Thank you for your comments. The laccase used in this study was a commercial product purchased from Sigma (St. Louis, Mo, USA). In the pretreatment process, rice straw (0.5 g) was pretreated with laccase only (2U/g substrate) or with laccase in the presence of [AMIM]Cl  or TritonX-100  in 5 mL buffer with 0.1 M citrate, pH 4.5 (Substrate concentration: 10%). The pretreatment was carried out in a 50 ml conical flask incubated at 50°C using a shaking incubator operating at 150 rpm for 24 h. The optimal pH and temperature were referred from the literature.

Comment 3.

Despite the applied modern instrumental methods, the authors do not always fully manage to use them. For example, Figure 3 ambiguously indicates the effect of the biocatalysts on the fiber, since there can be different plant anatomical elements in the camera lens, and this can be much more clearly represented at higher magnification. In paragraph 3.6 there is no reference to the methodology used. Currently, new, alternative approaches are emerging (for example https://0-link-springer-com.brum.beds.ac.uk/article/10.1134%2FS0036024420060059).

We sincerely appreciate your suggestion. We have taken higher magnification photos. However, the images were not clear and different to see the effect. So our choice is 500 magnifications.

We have added the reference to the methodology used in 3.6.

Other comments.

Line 50- 51 The authors should decide whether microbes or enzymes separate from microbes carry out deconstruction.

We are grateful for the suggestion. We have revised the statement to “Rice straw contains cellulose, hemicellulose, and lignin, which naturally block its deconstruction by microbes.” Please refer to line 51.

Table 1 It is desirable to provide data in terms of dry weight.

We sincerely appreciate your comment. The data in Table 1 is in dry weight and we added an explanation in the caption.

Figure 2 improve the quality or reduce based on previous comments.

Thank you for your correction. We have adjusted and improved the quality of Figure 2.

Reviewer 2 Report

This article contains some significant data and scientific effort. I recommend this paper for publication in the catalysis journal after careful consideration of the following comments:

• The introduction section should include
• The English language needs revision for grammar and form. There are some minor and errors such as:

- line 120.  The cellulose contents are, not ‘The cellulose content is’

- line 126. In line with our results, several recent studies have …, not  ‘In addition, recent studies have’

• Lines 131 – 134 should move to materials and methods
• Increase the quality of Figure 2, perhaps move the color description icons from the left-hand side to the top of the graphs. Too much ‘materials and methods’ information in the figure caption should be deleted
• The manuscript needs more discussion for example comparing the results of the IL pretreatment with microwave pretreatment and other commercial enzymes, refer to the following paper:

https://0-www-tandfonline-com.brum.beds.ac.uk/doi/abs/10.1080/17597269.2021.1955585

The manuscript should discuss the importance of second-generation biomass in water-saving and reducing the Waterfoot of biofuels and biobased chemicals, refer to the papers:

https://0-www-mdpi-com.brum.beds.ac.uk/2227-9717/9/8/1399

https://0-www-sciencedirect-com.brum.beds.ac.uk/science/article/abs/pii/S0959652617302342

https://0-link-springer-com.brum.beds.ac.uk/article/10.1007/s40710-018-0311-x

• The experimental section should be moved up (before the Results and Discussion) and should be re-named to Materials and Methods
• The authors need to explain, how many replicates have been used in these experiments? and what statistical analysis was used.
• The conclusion should include a statement on the work novelty, the challenges and limitations faced in the current research, and the perspective for future research.

Author Response

We appreciate the detailed review and constructive suggestions for improving the manuscript. We have responded in detail to all suggestions for revisions or requests for clarification below and have made revisions to the manuscript as noted (blue words). We would love to thank you for allowing us to resubmit a revised copy of the manuscript and we highly appreciate your time and consideration.

Reviewer #3: This article contains some significant data and scientific effort. I recommend this paper for publication in the catalysis journal after careful consideration of the following comments:

• The English language needs revision for grammar and form. There are some minor and errors such as:

- line 120.  The cellulose contents are, not ‘The cellulose content is’

Thank you for your correction. We have revised to “The cellulose contents are 38.50%, 39.57%, and 40.83% after pretreating the rice straw with laccase only…..”. Please refer to line 121.

- line 126. In line with our results, several recent studies have …, not  ‘In addition, recent studies have’

Thank you for your correction. We have revised to “In line with our results, several recent studies have indicated that IL is effective in removing lignin [20,21]”. Please refer to lines 126-127.

• Lines 131 – 134 should move to materials and methods

We are grateful for the suggestion. We have removed the statement cause seminar describe already in the materials and methods.

• Increase the quality of Figure 2, perhaps move the color description icons from the left-hand side to the top of the graphs. Too much ‘materials and methods’ information in the figure caption should be deleted

We sincerely appreciate your suggestion. We have improved the quality of Figure 2 and moved the information to materials and methods.

• The manuscript needs more discussion for example comparing the results of the IL pretreatment with microwave pretreatment and other commercial enzymes, refer to the following paper:

We are grateful for the suggestion. We have added the reference and discussed our results.

The manuscript should discuss the importance of second-generation biomass in water-saving and reducing the Waterfoot of biofuels and biobased chemicals, refer to the papers:

https://0-www-mdpi-com.brum.beds.ac.uk/2227-9717/9/8/1399

https://0-www-sciencedirect-com.brum.beds.ac.uk/science/article/abs/pii/S0959652617302342

https://0-link-springer-com.brum.beds.ac.uk/article/10.1007/s40710-018-0311-x

We sincerely appreciate your suggestions. We have added the reference and statement in the text.

• The experimental section should be moved up (before the Results and Discussion) and should be re-named to Materials and Methods.

We are grateful for the suggestion. We have re-named the Experimental section to Materials and Methods. But experimental section follows by Results and Discussion is the format for Catalysts. We can’t change this order.

• The authors need to explain, how many replicates have been used in these experiments? And what statistical analysis was used.

Thank you for your comments. We have added the statement “All experiments were performed in triplicate under the same conditions and the average results were reported.” Please refer to lines 255-256.

• The conclusion should include a statement on the work novelty, the challenges and limitations faced in the current research, and the perspective for future research.

Thank you for your comments. We have added the statement of novelty, challenges, and perspective for future research in the conclusion.

Reviewer 3 Report

The manuscript from Dr Yang and co-workers reported on the enzymatic production of sugars from rice straw biomass assisting by ion-liquid or surfactant. I should note that this manuscript clearly described and summarized to support the authors’ assertions. Therefore, I recommend acceptance of the manuscript after minor revision based on the following comments:

1. Line 92: for reader interests, suggest including other screening results of ILs and surfactant.
2. Line 189: typo mistake ‘–C–6CH2–.
3. Line 189: typo mistake of reference [25.26]
4. Line 212: suggest giving full words for ‘CrI’. Although probably understood by some readers in the field, the abbreviation should still be explained when first used.
5. Line 265: ‘et al.’ should be in italic with dot
6. Are there any synergistic effect of [AMIM]Cl (ionic liquid) and TritonX-100 (surfactant) for hydrolysis of lignocellulose in your case? There were several reports on this such as improve the stability of Paenibacillus LLZ1 cellulase.

Author Response

We appreciate the detailed review and constructive suggestions for improving the manuscript. We have responded in detail to all suggestions for revisions or requests for clarification below and have made revisions to the manuscript as noted (blue words). We would love to thank you for allowing us to resubmit a revised copy of the manuscript and we highly appreciate your time and consideration.

Reviewer #5:

Dear Authors,

The manuscript from Dr Yang and co-workers reported on the enzymatic production of sugars from rice straw biomass assisting by ion-liquid or surfactant. I should note that this manuscript clearly described and summarized to support the authors’ assertions. Therefore, I recommend acceptance of the manuscript after minor revision based on the following comments:

1. Line 92: for reader interests, suggest including other screening results of ILs and surfactant.

We sincerely appreciate your suggestion. We added the tested ILs and surfactants in the text.

2. Line 189: typo mistake ‘–C–6CH2–.

Thank you for your correction. We have revised to –C–6CH2–.

3. Line 189: typo mistake of reference [25.26]

Thank you for your correction. We have revised it to [25,26].

4. Line 212: suggest giving full words for ‘CrI’. Although probably understood by some readers in the field, the abbreviation should still be explained when first used.

We sincerely appreciate your suggestion. We have added full words for ‘CrI’. Please refer to line 211.

5. Line 265: ‘et al.’ should be in italic with dot

Thank you for your correction. We have revised to et al..

6. Are there any synergistic effect of [AMIM]Cl (ionic liquid) and TritonX-100 (surfactant) for hydrolysis of lignocellulose in your case? There were several reports on this such as improve the stability of PaenibacillusLLZ1 cellulase.

Thank you for your comment. We have added reference 22. In the study, ILs have been found to be capable of supporting and improving activity and stability of enzymes including laccases.

Round 2

Reviewer 1 Report

Author Response

We appreciate the detailed review and constructive suggestions for improving the manuscript.

Reviewer 2 Report

Dear Authors, 

You have shown good effort to address my comments but the English still needs improvement. I have given you one or two examples in the original review but the paper has many more English errors. Even the new statement you added in the conclusion was not linguistically correct, to help you, I have rephrased it for you, see below. I would say, even with the presence of the language errors, the paper still can be accepted but how readers will look at you? remember this will stay therefore ever and perhaps so many people will read it over time, so please spend some effort to improve it. 

'Although promising results have been obtained in the present work, the reaction conditions need further optimization, mainly by increasing the solid loading and decreasing the laccase doses, in order to reach industrial feasibility.'

Author Response

We sincerely appreciate your comment and correction.