Kinetics of Lignin Removal from Rice Husk Using Hydrogen Peroxide and Combined Hydrogen Peroxide–Aqueous Ammonia Pretreatments

Round 1

Reviewer 1 Report

The manuscript presents research on Kinetic Modeling of Hydrogen Peroxide and Combined Hydrogen Peroxide-Aqueous Ammonia Pretreatments of Rice Husk. Overall, the manuscript collected system experiments and got some reliable data. The manuscript still needs improvement in the abstract, introductin, result analysis and conclusion. The manuscript cannot be recommended in its current form. Major revise is needed.

1. Title,Kinetic Modeling?  You're just done the kinetics of lignin removal. I suggest that state this clearly in the title.
2. Abstract:to maintain independence of abstract, it should include purpose, method, result and conclusion. do you have any information about lignin removal Kinetic Modeling in your abstract since it is your title? List so many data is meanless.
3. Introduction, Line83-91, I think this is the content of materials and methods, your logic is not clear.
4. You shouldfocus on the pretreatment of combined alkaline peroxide pretreatment in the introduction section. Authors should read at least the following literature: Modified alkaline peroxide pretreatment: Alkaline hydrogen peroxide pretreatment of bamboo culm for improved enzymatic release of reducing sugars using recombinant cellulases. Cellulose, 2020. 27(2): p. 769-779. Application of hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment of energy crops. Fuel, 2017. 205(5): p. 184-191.
5. Forequation 7-9, the multiplication sign looks a lot like a letter X, please revise
6. So manytypos, please revise it carefully. For example (not limited):  Line 203 “……5 g/L (NH4)2SO4, 3.5 g/L K2HPO4, 0.75 g/L MgSO4•7H2O, and 1 g/L CaCl2•2H2O w……”;  Line 132“……the solid phase and the H2O2……”
7. FIGURE 1 and 2, The units of the legend are oC rather than C.
8. Line 395 “The impact of cellulose hydrolysis is highly correlated with the effect of pretreatment,where the less lignin in biomass, the simpler to enzymatic hydrolysis.”  Authors haven’t discussed the relationship between lignin removal and enzymatic hydrolysis. In fact, enzymatic hydrolysis is also related to the structure of lignin, not just the amount of content. You give us the result directly. In addition, for the degradation mechanism of lignin in AHP pretreatment, authors should carefully read at least the following literature: Synergistic effect of hydrogen peroxide and ammonia on lignin. Industrial Crops and Products, 2020. 146(1): p. 112177.
9. Conclusion, Too much, please concise it, most of the second paragraph belongs to the introduction Line 412-419, add the information of Kinetic Modeling in the conlusion.

Author Response

Point 1: Title, Kinetic Modeling? You're just done the kinetics of lignin removal. I suggest that state this clearly in the title.

Response 1: Title was changed to: “Kinetics of lignin removal of Hydrogen Peroxide and Combined Hydrogen Peroxide-Aqueous Ammonia Pretreatments of Rice Husk”. (in red)

Point 2: Abstract: to maintain independence of abstract, it should include purpose, method, result and conclusion. do you have any information about lignin removal Kinetic Modeling in your abstract since it is your title? List so many data is meanless.

Response 2: The information about lignin removal kinetic modeling already be inserted in abstract. The activation energy of both HP and CHPA also be included (in red-line 22-25). Lignin removal percentage of HP and CHPA was changed (in red-line 21-22).

Point 3: Introduction, Line83-91, I think this is the content of materials and methods, your logic is not clear.

Response 3: Line 83-88 was already removed. Line 89-91 was joined to previous paragraph (in red-line 79-82).

Point 4: You should focus on the pretreatment of combined alkaline peroxide pretreatment in the introduction section. Authors should read at least the following literature: Modified alkaline peroxide pretreatment: Alkaline hydrogen peroxide pretreatment of bamboo culm for improved enzymatic release of reducing sugars using recombinant cellulases. Cellulose, 2020. 27(2): p. 769-779. Application of hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment of energy crops. Fuel, 2017. 205(5): p. 184-191.

Response 4: “The pretreatment of combined alkaline peroxide pretreatment” was already inserted. (in red-line 79-82). “AHP treatment” was already put (in red-line 76-78).

Point 5: For equation 7-9, the multiplication sign looks a lot like a letter X, please revise.

Response 5: It was already revised (in red).

Point 6: So many typos, please revise it carefully. For example (not limited):  Line 203 “……5 g/L (NH4)2SO4, 3.5 g/L K2HPO4, 0.75 g/L MgSO4•7H2O, and 1 g/L CaCl2•2H2O w……”;  Line 132“……the solid phase and the H2O2……”

Response 6: It was already revised to: 5 g/L yeast, 7.5 g/L (NH₄)₂SO₄, 3.5 g/L K₂HPO₄, 0.75 g/L MgSO₄•7H₂O, and 1 g/L CaCl₂•2H₂O (in red-line 204-205); …the solid phase and the H₂O₂ solution  (in red-line 133)

Point 7: FIGURE 1 and 2, The units of the legend are oC rather than C.

Response 7: It was already revised as reviewer suggestion.

Point 8: Line 395 “The impact of cellulose hydrolysis is highly correlated with the effect of pretreatment, where the less lignin in biomass, the simpler to enzymatic hydrolysis.”  Authors haven’t discussed the relationship between lignin removal and enzymatic hydrolysis. In fact, enzymatic hydrolysis is also related to the structure of lignin, not just the amount of content. You give us the result directly. In addition, for the degradation mechanism of lignin in AHP pretreatment, authors should carefully read at least the following literature: Synergistic effect of hydrogen peroxide and ammonia on lignin. Industrial Crops and Products, 2020. 146(1): p. 112177.

Response 8: It was already revised to: (in red-line 397-406)

Previous author [56] found that the combination impact of hydrogen peroxide and ammonia not only breaks lignin successfully, but also significantly reduces its polydis-persity index (PI), resulting in homogeneous lignin fragments. Their results show that ammonia damaged the ester link preferentially, whereas hydrogen peroxide destroyed the resinol structure selectively. Both ammonia and hydrogen peroxide have the potential to act on the lignin side chain's alkyl ether and aryl ether linkages. Both, how-ever, had a negligible effect on the p-coumaran structure. The synergy of hydrogen peroxide and ammonia on lignin indicated the fundamental mechanism of the pre-treatment and provided insight into lignin structure and biomass resistance, with the goal of increasing enzymatic hydrolysis.

Point 9: Conclusion, Too much, please concise it, most of the second paragraph belongs to the introduction Line 412-419, add the information of Kinetic Modeling in the conlusion.

Response 9: Line 413-420 was removed in the article. Line 423-427 was removed to previous paragraph. The information of Kinetic Modeling was already inserted. (in red-line 434-438).

Reviewer 2 Report

This original article deals with the study hydrogen peroxide (mixed or not with aqueous ammonia) pretreatments to fractionate rice husk, and the subsequent enzymatic hydrolysis. The article is in accordance with the journal’s scope, however, some points must be improved before its publication:

• There are major English mistakes that must be improved in the manuscript. Also, there are incomplete sentences.
• Line 19. Subscript in hydrogen peroxide (H2O2).
• Line 20 and following. I reckon that you rather use a range for the lignin removal, so it is easier for the readers, i.e., HP ranging 39.96-62.24, whereas CHPA ranged 64.74-81.97 of lignin removal.
• Lines 23-24. Join both sentences about higher temperatures.
• Line 24. CHPA pretreatment did not produce glucose via enzymatic hydrolysis. Arrange that phrase to something like: ‘CHPA pretreatment was subjected to enzymatic hydrolysis (6% enzyme loading), enabling the production of 6.58 g glucose/L at 25 h’.
• Line 31. Renewable should not be in capital letters.
• Line 43. Erase the extra ‘s’.
• Line 49. ‘…in the production of second generation bioethanol’.
• Line 49. Exciting? Change for something more objective.
• Line 55. Why rice husk is a ‘more basic material’?
• Line 57. Bioethanol is not obtained via chemical, but biological or biotechnological pathway. Change that, and improve the concordance in the phrase.
• Lines 64-65. The sentence seems incomplete.
• Line 69. Extraordinary?
• Lines 85-87. The sentence is incomplete.
• Line 88. ‘5 h’.
• Line 90. Why Si?
• Line 120. What do you mean with ‘HP refers to this process 120 throughout the rest’?
• Line 128. Correct the degrees Celsius (°C).
• Line 134. Subscript the numbers in H2O2.
• Line 144. Eq. 1. The lignin content is measured in g/100 g raw material?
• Line 179. Correct the degrees Celsius (°C) in that line and throughout the manuscript.
• Line 190. ‘Aspergillus niger’ in italics.
• Lines 203-204. Subscript the numbers.
• Line 240. Table 1. % regarding what? 100 g of pretreated biomass? In addition, ‘hemicelluloses’ is written incorrectly in both table 1 and 2.
• Table 1 and 2. As I understand, the results from tables 1 and 2 are expressed in % regarding pretreated biomass. In this sense, you cannot subtract the amount of lignin obtained after the pretreatment to that obtained in untreated biomass since the unit is not the same (% regarding pretreated biomass and % regarding untreated biomass). In this sense, all the following calculus is erroneous. To perform the calculation correctly you should take into account the solid yield within the pretreatment.

Author Response

Point 1: There are major English mistakes that must be improved in the manuscript. Also, there are incomplete sentences.

Response 1: The final version of revised paper is send to MDPI proofreading.

Point 2: Line 19. Subscript in hydrogen peroxide (H2O2).

Response 2: It was revised to: H₂O₂ (in red: line 19).

Point 3: Line 20 and following. I reckon that you rather use a range for the lignin removal, so it is easier for the readers, i.e., HP ranging 39.96-62.24, whereas CHPA ranged 64.74-81.97 of lignin removal.

Response 3: It was changed to: “Lignin removal percentage of HP varied from 48.25 to 66.50, whereas CHPA removed lignin at a rate of 72.22 to 85.73.”. (in red: line 20-21)

Point 4: Lines 23-24. Join both sentences about higher temperatures.

Response 4: One of both sentences was removed.

Point 5: Line 24. CHPA pretreatment did not produce glucose via enzymatic hydrolysis. Arrange that phrase to something like: ‘CHPA pretreatment was subjected to enzymatic hydrolysis (6% enzyme loading), enabling the production of 6.58 g glucose/L at 25 h’.

Response 5: It was already revised (in red: line 23-24).

Point 6: Line 31. Renewable should not be in capital letters.

Response 6: It was already revised (in red: line 29).

Point 7: Line 43. Erase the extra ‘s’.

Response 7: It was already revised (in red: line 41)

Point 8: Line 49. ‘…in the production of second generation bioethanol’.

Response 8: It was already revised (in red: line 47)

Point 9: Line 49. Exciting? Change for something more objective.

Response 9: It was already revised to: “complex” (in red: line 47)

Point 10: Line 55. Why rice husk is a ‘more basic material’?

Response 10: It was already revised to ‘by-product of rice milling industry’ (in red: line 53-54)

Point 11: Line 57. Bioethanol is not obtained via chemical, but biological or biotechnological pathway. Change that, and improve the concordance in the phrase.

Response 11: It was already revised to: “biotechnological pathway” (in red: line 55)

Point 12: Lines 64-65. The sentence seems incomplete.

Response 12: It was already added to: “have been shown to be successful at removing lignin in previous research” (in red: line 65)

Point 13: Line 69. Extraordinary?

Response 13: It was already removed (in red: line 70).

Point 14: Lines 85-87. The sentence is incomplete.

Response 14: It was already removed because this is the content of materials and methods.

Point 15: Line 88. ‘5 h’.

Response 15: : It was already removed because this is the content of materials and methods.

Point 16: Line 90. Why Si?

Response 16: The primary goal of the pretreatment is to remove lignin and silica, which provide mechanical and chemical robustness to the biomass structures. The amount of silica in the samples prepared with HP was 6.02 %, according to SEM-EDS analysis. On the other hand, Si was not discovered in the samples prepared with CHPA. This result indicates that lignin and silica were removed in large quantities from the rice husk during processing. (in red: line 328-330 and line 334-338).

Point 17: Line 120. What do you mean with ‘HP refers to this process 120 throughout the rest’?

Response 17: It was already removed to prevent missunderstanding.

Point 18: Line 128. Correct the degrees Celsius (°C).

Response 18: It was already revised (in red: line 122).

Point 19: Line 134. Subscript the numbers in H2O2.

Response 19: It was already revised (in red: line 126).

Point 20: Line 144. Eq. 1. The lignin content is measured in g/100 g raw material?

Response 20: It was already revised: Where CL0 is the initial lignin in untreated rice husk, CL(t) is the lignin in pretreated rice husk at any time t, L(t) is the percentage of lignin removal at any time (in red: line 139-140).

Point 21: Line 179. Correct the degrees Celsius (°C) in that line and throughout the manuscript.

Response 21: It was already revised (in red: line 122; 199; 227; 321)

Point 22: Line 190. ‘Aspergillus niger’ in italics.

Response 22: It was already revised (in red: line 184).

Point 23: Lines 203-204. Subscript the numbers.

Response 23: It was already revised (in red: line 197-198).

Point 24: Line 240. Table 1. % regarding what? 100 g of pretreated biomass? In addition, ‘hemicelluloses’ is written incorrectly in both table 1 and 2.

Response 24: It was already revised. (wt %) means:

Weight of lignin (g) / Weight of the test speciment (g).

'Hemicellulose’ was written correctly in both table 1 and 2.

Point 25: Table 1 and 2. As I understand, the results from tables 1 and 2 are expressed in % regarding pretreated biomass. In this sense, you cannot subtract the amount of lignin obtained after the pretreatment to that obtained in untreated biomass since the unit is not the same (% regarding pretreated biomass and % regarding untreated biomass). In this sense, all the following calculus is erroneous. To perform the calculation correctly you should take into account the solid yield within the pretreatment.

Response 25:

The calculation was taken into account again, with the addition of the solid yield ((in red: equation 1; table 1 and table 2; Figure 1 and Figure 2). All of the kinetic value was already changed (in red: table 3 and table 4; Figure 4; table 6).

Reviewer 3 Report

During the hydrogen peroxide pretreatment, the hemicellulose is also degraded. The kinetics should be also investigated. In addition, the main focus is the Kinetic Modeling. The results of enzymatic hydrolysis is not necessary

Author Response

Point 1: During the hydrogen peroxide pretreatment, the hemicellulose is also degraded. The kinetics should be also investigated.

Response 1: In this reasearch, we just focus on kinetic modeling of lignin removal. Generally, hemicellulose is desired to produce bioethanol. To accomode this suggestion, we change the title to: “Kinetics of lignin removal of Hydrogen Peroxide and Combined Hydrogen Peroxide-Aqueous Ammonia Pretreatments of Rice Husk”. (in red)

Point 2: In addition, the main focus is the Kinetic Modeling. The results of enzymatic hydrolysis is not necessary.

Response 2: The results of enzymatic hydrolysis is still needed to know the glucose content after pretreatment.

Point 3: Lines 203-204. Subscript the numbers.

Response 3: It was already revised (in red: line 204-205).

Round 2

Reviewer 1 Report

The authors stated my concerns and carefully revised the article, and I suggested publish as it is.

Author Response

Authors would like to thank the Reviewers and Editors due their appropriate and constructive suggestions as well as their proposed corrections, which have been utilized in improving the quality of this manuscript.

Reviewer 2 Report

Although the authors have improved the manuscript, there are still some modifications that have to be accomplished.

- Solid yield has ben added to tables 1 and 2. However, if we calculate the cellulose content regarding the raw material (multiplying solid yield and cellulose), the amount of cellulose after the pretreatment is impressively higher than that before pretreatment. For example, last experiment from table 1: 63.84 x 0.7915 = 50.53, which would mean that the cellulose is created, since the raw material only has 22.34 g/100 g...

- Besides that, the enzymatic hydrolysis (figure 5) is modelled, but that is not reflected in the material and methods section.

Author Response

Although the authors have improved the manuscript, there are still some modifications that have to be accomplished.

Point 1: Solid yield has ben added to tables 1 and 2. However, if we calculate the cellulose content regarding the raw material (multiplying solid yield and cellulose), the amount of cellulose after the pretreatment is impressively higher than that before pretreatment. For example, last experiment from table 1: 63.84 x 0.7915 = 50.53, which would mean that the cellulose is created, since the raw material only has 22.34 g/100 g...

Response 1: As expected, the percentages of cellulose and hemicellulose in rice husks processed with CHPA were impressively higher than those in untreated husks. This occurred because some of the lignin in the husks was removed during the processing.

We put these sentences in manuscript (in red-line 246-248).

Point 2: - Besides that, the enzymatic hydrolysis (figure 5) is modelled, but that is not reflected in the material and methods section.

Response 2: We agreed with the reviewer. We removed the model, since it is not appropriate with the tittle. We will use this model for next publication. (in red-line 392-393).

Author Response File: Author Response.pdf

Reviewer 3 Report

accept

Author Response

Authors also would like to thank the Reviewers and Editors due their appropriate and constructive suggestions as well as their proposed corrections, which have been utilized in improving the quality of this manuscript.