Emerging Technologies in Pretreatment and Hydrolysis for High-Solid-Loading Bioethanol Production from Lignocellulosic Biomass

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review "Emerging Technologies in Pretreatment and Hydrolysis for High-Sol-Loading Bioethanol Production" addresses the topic of bioethanol production from a specific type of lignocellulose feedstock under conditions of high initial substrate concentrations. The 28-page manuscript, with a 117-item bibliography, most of which are fairly recent, is well-written and organized. The authors are knowledgeable in this topic and focus their review on new types of lignocellulose pretreatment. They also thoroughly discuss the challenges arising during the enzymatic hydrolysis of lignocellulosic substrates, outline several enzymatic hydrolysis options, and demonstrate the advantages of combined processes. A list of deficiencies is provided below. These should be addressed.

Disadvantages:

1. It is recommended to indicate the object of study in the article title; in this case, it is "lignocellulosic biomass (LCB)".
2. If comment 1 is accepted by the authors, it is recommended to exclude from the review other types of research objects, such as "sugarcane molasses, agave juice, potato processing waste," which are unexpectedly discussed in Section 6, line 698.
3. Figure 3. It is recommended to amend the fourth stage, which includes "crystallization." This stage describes the production of pure ethanol, so it is unclear what crystallization is being discussed.
4. It is recommended to check the correct formatting of references, especially when referencing book chapters (items 35, 36, 38).

Author Response

Response to Reviewer 1:

The review "Emerging Technologies in Pretreatment and Hydrolysis for High-Solid-Loading Bioethanol Production" addresses the topic of bioethanol production from a specific type of lignocellulose feedstock under conditions of high initial substrate concentrations. The 28-page manuscript, with a 117-item bibliography, most of which are fairly recent, is well-written and organized. The authors are knowledgeable in this topic and focus their review on new types of lignocellulose pretreatment. They also thoroughly discuss the challenges arising during the enzymatic hydrolysis of lignocellulosic substrates, outline several enzymatic hydrolysis options, and demonstrate the advantages of combined processes. A list of deficiencies is provided below. These should be addressed.

Response: We sincerely thank the esteemed reviewer for their invaluable time, insightful comments, and detailed evaluation of our manuscript. Your feedback has greatly helped us improve the clarity, quality, and depth of our research. All corresponding changes have been highlighted in yellow in the revised manuscript.

Comment 1:  It is recommended to indicate the object of study in the article title; in this case, it is "lignocellulosic biomass (LCB)".

Author’s Response: Thank you for your valuable comment. We have revised the title of the study and changed it to “Emerging Technologies in Pretreatment and Hydrolysis for High Solid Loading Bioethanol Production from Lignocellulosic Biomass”.

Comment 2:  If comment 1 is accepted by the authors, it is recommended to exclude from the review other types of research objects, such as "sugarcane molasses, agave juice, potato processing waste," which are unexpectedly discussed in Section 6, line 698.

Author’s Response: Thank you for your valuable comment. Line 698 has been revised based on the changes as per comment 1. See the highlight part of table 5.

Comment 3:  Figure 3. It is recommended to amend the fourth stage, which includes "crystallization." This stage describes the production of pure ethanol, so it is unclear what crystallization is being discussed.

Author’s Response: Thank you for your valuable comment. Figure 3 has been revised by removing the crystallization.

Comment 4:  It is recommended to check the correct formatting of references, especially when referencing book chapters (items 35, 36, 38).

 

Author’s Response: Thank you for your valuable comment. All the references have been formatted as per the author guidelines of the journal.

Reviewer 2 Report

Comments and Suggestions for Authors

Please confirm the attachment.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

There are many typos, especially in the references. Thus, this manuscript requires major revision for publication. 

Author Response

Response to Reviewer 2:

We sincerely thank the esteemed reviewer for their invaluable time, insightful comments, and detailed evaluation of our manuscript. Your feedback has greatly helped us improve the clarity, quality, and depth of our research. All corresponding changes have been highlighted in green in the revised manuscript.

Comment 1: The first paragraph of the introduction section is too abstract. Please provide specific examples to explain why demand for biofuels is increasing.

Author’s Response: Thank you for your valuable comment. The first paragraph of the introduction has been revised to make it more realistic.

Comment 2: To give a better understanding to readers, add the units of documents in Figure 1.

Author’s Response: Thank you for your valuable comment. Upon re-examining the data, we found that the number of publications per year in this research area is relatively limited, which makes it difficult to show a statistically meaningful trend. Therefore, we have decided to remove the figure to avoid any possible misinterpretation of the data.

Comment 3: Line 162, it is described as “remove lignin”, but surely it should be “decompose”? If “remove” is intended, does this involve membrane separation or similar processing? If so, please state this accurately.

Author’s Response: Thank you for your valuable comment. Line 162 has been revised to provide a more accurate statement. Please see the highlighted revision in lines 158–161.

 Comment 4: In sections 2.1.1–2.14, the description is abstract. Please explain more specifically, such as the type of lignocellulosic biomass in the target and the name of the microorganism used.

Author’s Response: Thank you for your valuable comment. Sections 2.1.1 to 2.1.4 provide a general overview of the four key steps in the biorefinery process rather than focusing on specific studies, types of lignocellulosic biomass, or microorganisms. Therefore, detailed examples were not included in these sections to maintain a broader conceptual description.

Comment 5: Line 179, it is described as “selecting efficient microorganisms”, but does this assume the utilization of enzymes secreted outside the microbial cells? If so, a detailed explanation is required as this could lead to misunderstanding regarding enzymatic saccharification versus microbial utilization.

Author’s Response: Thank you for your valuable comment. Line 179 has been revised to ensure a more accurate statement. Please see the highlighted revision at line 175.

Comment 6: Lines 257–259, pretreatment methods vary depending on the type of lignocellulosic biomass. Please specify for which types of lignocellulosic biomass the pretreatment methods described in Sections 3.1 to 3.4 are effective.

Author’s Response: Thank you for your valuable comment. The type of lignocellulosic biomass has been added for all the pretreatments mentioned in sections 3.1 to 3.4.

Comment 7: Lines 317–324, when describing previous study, please describe the type of biomass, not the biomass.

Author’s Response: Thank you for your valuable comment. The type of biomass has been incorporated in the details of lines 310-317.

Comment 8: In the 4. Hydrolysis Methods for High Solid Processing section, when describing biofuel production concentrations, please provide details of previous studies, such as the name of the microorganism used and the experimental conditions.

Author’s Response: Thank you for your valuable comment. The name of the strain and experimental conditions have been added in section 4.

Comment 9: The description of the "5. 1.One-pot System" section should be deleted because it overlaps with the explanation in the SSF mentioned above. If the description is not deleted, please explain the differences, such as by providing examples of practical applications.

Author’s Response: Thank you very much for your insightful comment. We would like to clarify that the one-pot system differs from SSF in terms of process integration. In the one-pot system, pretreatment, enzymatic hydrolysis, and fermentation occur sequentially in a single reactor without the washing step after pretreatment, allowing for a more streamlined and cost-effective process. In contrast, SSF generally involves a washing step following pretreatment before proceeding to enzymatic hydrolysis and fermentation.

Comment 10: In the 5.2. Consolidated bioprocessing (CBP) section, if possible, add the discussion with the yield relative to the raw material.

Author’s Response: Thank you for your valuable comment. The yield of ethanol has been incorporated in lines 595-596.

Comment 11: In the 6. Future Directions and Recommendations section, to give a better understanding to readers unders, please summarise the future direction and recommendations in a table.

Author’s Response: Thank you for your valuable comment. Section 6, "Future Directions and Recommendations," has been summarized in Table 5.

Comment 12: Please revise the description of the abstract according to the above corrections.

Author’s Response:  Thank you for your valuable comment.  The description of the abstract aligns with the changes made in the revision.

Comment 13: Lines 158, 174, 186, 198, the title should be in italics.

Author’s Response:  Thank you for your valuable comment. According to the journal’s formatting guidelines, subsubsection titles are not required to be in italics. Therefore, the headings of Sections 2.1.1 to 2.1.4 have been kept in their current format to maintain consistency with the journal template.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The following corrections are required for your manuscript to be accepted:

Line 459: "FPU" is an enzyme unit. Please confirm that the enzyme name is "pretreated biomass" and that the description is correct.

Lines 469 and 541: "WXY74" should not be written in italics.

Line 521: Please correct "β glucosidase" to "β-glucosidase.

Comments on the Quality of English Language

OK.

Author Response

Response to Reviewer:

Comment 1:  Line 459: "FPU" is an enzyme unit. Please confirm that the enzyme name is "pretreated biomass" and that the description is correct.

Author’s Response: Thank you for your valuable comment. We confirm that "FPU" refers to the enzyme activity unit (Filter Paper Unit). The enzyme used is SacchariSEBC6, and the enzyme loading was 10 FPU per gram of pretreated biomass. We confirm that the description is correct.

Lines 469 and 541: "WXY74" should not be written in italics.

Author’s Response: Thank you for your valuable comment. WXY74 has been corrected in line 469 and 541.

Line 521: Please correct "β glucosidase" to "β-glucosidase.

Author’s Response: Thank you for your valuable comment. "β glucosidase" has been corrected to "β-glucosidase” in line 521.