Microbial Lignocellulolytic Enzymes for the Effective Valorization of Lignocellulosic Biomass: A Review

Round 1

Reviewer 1 Report

some language corrections are highlighted in the attached manuscript

Comments for author File: Comments.pdf

Author Response

Comment: some language corrections are highlighted in the attached manuscript.

Response: Thank you so much for your valuable comments. We have made the changes in the revised manuscript as suggested.

Reviewer 2 Report

The manuscript is interesting and brings a lot of valuable information about lignocellulosic hydrolytic enzymes, which is also well organized. Before it can be published, there are some points that must be improved:

·       Line 42: please update this information once world’s population reached 8 billion people already.

·       Considering the new trends about microorganisms producing lignocellulolytic enzymes, it is important to include one or two paragraphs about the recombinant microorganisms that secret these enzymes once they were developed aiming consolidated bioprocessing of biomass. This technology should also be included in the future prospects topic.

·       Line 627: please update this information too once we already are in the end of 2022.

·       Authors have explored the importance of enzyme immobilization to allow its application at industrial scale due to its high cost. In this sense, a table or a paragraph exploring the significance of enzyme cost on process and final product will bring much more robustness to this topic. These information about enzyme costs and impact on process feasibility can be found on https://doi.org/10.1016/j.esd.2022.03.007

·       Line 708: “surface chemistry that establishes the binding chemistry for enzyme attachment”. Enzymes immobilization can be performed by adsorption, in this case the interaction between enzyme and support is physical, not chemical. Please correct this sentence.

·       Please add in the last paragraph of topic 5 an example of beta-xylosidase immobilization work, once this enzyme is well known to be very sensitive and its immobilization is of great importance.

·       When performing immobilization of enzymes, its nature becomes heterogeneous. Please discuss how is it possible that immobilized enzyme could be used to hydrolyze biomass once substrate and biocatalysts are solids.

Author Response

The manuscript is interesting and brings a lot of valuable information about lignocellulosic hydrolytic enzymes, which is also well organized. Before it can be published, there are some points that must be improved:

Response: Thank you for your comments and suggestions for improving the contents of the present Manuscript (MS). We have taken care of each comment and suggestion to make MS more informative.

 

Comment:  Line 42: please update this information once world’s population reached 8 billion people already.

Response: As per your suggestion, we have now updated the world’s population to 8 billion in the said line of the revised MS.

 

Comment: Considering the new trends about microorganisms producing lignocellulolytic enzymes, it is important to include one or two paragraphs about the recombinant microorganisms that secret these enzymes once they were developed aiming consolidated bioprocessing of biomass. This technology should also be included in the future prospects topic.

Response: Thank you for highlighting the importance of using recombinant microorganisms for enhanced production of commercial lignocellulolytic enzymes. In the revised MS, we have made the desired content in a separate new section 3.3. (Lignocellulolytic enzyme production from recombinant microorganisms). The text related to the microbial genetic engineering has also been added in the Future prospects section.

 

Comment: Line 627: please update this information too once we already are in the end of 2022.

Response: The suggested change has been made and updated in the revised MS.

Comment: Authors have explored the importance of enzyme immobilization to allow its application at industrial scale due to its high cost. In this sense, a table or a paragraph exploring the significance of enzyme cost on process and final product will bring much more robustness to this topic. These information about enzyme costs and impact on process feasibility can be found on https://doi.org/10.1016/j.esd.2022.03.007

Response: Enzyme cost is an imperative factor in the overall success of the industrial bioprocesses. A paragraph related to the significance of the enzyme cost on the final product/process has now been added in the revised MS under section 5 (Enzyme immobilization). 

 

Comment: Line 708: “surface chemistry that establishes the binding chemistry for enzyme attachment”. Enzymes immobilization can be performed by adsorption, in this case the interaction between enzyme and support is physical, not chemical. Please correct this sentence.

Response: We have now corrected the statement in the revised MS.

 

Comment: Please add in the last paragraph of topic 5 an example of beta-xylosidase immobilization work, once this enzyme is well known to be very sensitive and its immobilization is of great importance.

Response: As per you advise, we have now added a brief discussion on immobilization of β-xylosidases for potential use in the bioethanol production along with the reference from literature in the revised MS.

 

Comment: When performing immobilization of enzymes, its nature becomes heterogeneous. Please discuss how is it possible that immobilized enzyme could be used to hydrolyze biomass once substrate and biocatalysts are solids.

Response: The immobilization of enzyme as a solid heterogeneous catalyst which is insoluble in water is of great importance in the hydrolyzing the solid pretreated lignocellulosic biomass. The enzyme-substrate matrix so formed assists in the effective saccharification of complex sugars into simpler forms, thereby, also recycling the enzyme for repeated cycles. As per your kind suggestion, we have added a text on this aspect in the revised MS. The reference 155 has been cited, there is many examples about using immobilized cellulase hydrolyzed cellulose in the table 1 of this reference.

Reviewer 3 Report

Many abbreviations are included in the research. Abbreviations may not be made in parts that are not very repetitive. For example 2.1.1, 2.1.2, 2.1.3 .

Figure 1 should be edited.

Line 148 degradation

Line 664

table 2 check

References 57

References 75

Author Response

Comment: Many abbreviations are included in the research. Abbreviations may not be made in parts that are not very repetitive. For example 2.1.1, 2.1.2, 2.1.3 . Response: Acronyms which are used multiple times in the MS have been elaborated in the first instance when they appear, whereas, those which are not repetitive have been removed from the revised MS.

Comment: Figure 1 should be edited.

Response: Figure 1 has been edited, with the addition of few more types of important lignocellulolytic enzymes in the revised MS.

 

Comment: Line 148 degradation

Response: The suggested change has been made in the revised manuscript.

 

Comment: Line 664

Response: The error has now been corrected in the revised MS.

Comment: Table 2 check

Response: The error in Table 2 has been corrected in the revised MS.

 

Comment: References 57

Response: The error in reference number 57 which is now reference no. 68 in the revised MS has been corrected.

 

Comment: References 75

Response: The error in reference 75 which is now reference no. 86 in the revised MS has been corrected in the revised MS.

Reviewer 4 Report

The literature review submitted by Parushi Nargotra et al. aims to give a comprehensive overview of lignocellulose degradation and discusses different microbial lignocellulolytic enzymes with further focus on their production by solid state fermentation and immobilization techniques for the valorization of plant biomass. The topic is of importance to establish a circular economy based on renewable resources and therefore I believe it is of interest for the readers of “Catalysts”. However, a large number of review articles have been published during the last few years. In my personal opinion it would be better to focus more on one defined topic (e.g. SSF or enzyme immobilization or microbial lifestyles) than trying to cover all these topics in a rather shallow manner.

Furthermore, the article in its current form suffers from multiple weaknesses. Several points need to be improved before the manuscript meets the quality criteria for publication. I hope that the following critiques will aid the authors in refining and revising their manuscript:

 

Major:

·         The authors missed discussing er even mentioning the CAZy database system. The CAZy database represents a comprehensive and commonly accepted classification system for carbohydrate-active enzymes including all major lignocellulolytic enzymes. https://doi.org/10.1093/nar/gkab1045 or https://doi.org/10.1093/nar/gkt1178.

·         Line 258: here the authors are actually referring to the CAZy system. However, the correct designation is “auxiliary activities” and not “ancillary activities (AA)”. Furthermore, the information here seems to be not up-to-date. The authors are just discussing AA9, AA10 and AA11. However, besides the three mentioned families, there are additional families of LPMOs known (AA13 (2014), PMID 25201969; AA14 (2018), PMID=29377002; AA15 PMID=29472725; AA16 (2019); PMID=30923563; AA17, 10.1126/science.abj1342). This literature and information provided by the authors need to be updated and corrected

·         Figure 1: Important lignocellulolytic enzymes are missing (e.g. versatile peroxidases, H2O2-producing enzyme (CAZy family 3 of the auxiliary activities (AA3), e.g. glucose oxidase, aryl alcohol oxidase, etc.; glyoxal oxidase (AA5); glucooligosaccharide oxidase (AA7)) and others. In my opinion, the authors should definitely reconsider their selection of the enzymes presented. Furthermore, there is a typo in the Figure legend. “degradadtion”

·         The authors tried to focus on the newest literature and the majority of the references has been published within the last 3-5 years. However, some important findings are not mentioned and especially the description of enzyme classes seems in some passages rather superficial.

o   Cellulosomes are not mentioned in the sections about bacterial cellulolytic systems

o   For LPMO the authors only mention the reactivity towards oxygen. However, there are convincing evidences reported in the last ~5 years that H2O2 is the preferred cosubstrate. A discussion of this reports is definitely relevant within a review describing LPMO as cellulolytic enzyme. See e.g. https://doi.org/10.1038/nchembio.2470 or https://doi.org/10.1038/s41467-020-19561-8

·         The manuscript suffers from a variety of spelling/grammar errors (not limited to my further comments) and therefore I would strongly recommend proof reading and language editing to improve the quality of the manuscript

·         Literature references: In my opinion, the authors too often cite other review articles and they do not refer to the original research articles. However, most details the authors are discussing using such reference originally arise from other articles.

E.g. Chapter 4 “Solid State Fermentation for Enzyme Production”: The authors use the reference to the review article [119] by Leite et al. 2021 five times distributed over the entire length of the chapter. As an example, the authors write “It is expected that the global enzyme market may reach $7 billion by the end of 2022….” referring to reference 119. In this referenced article by Leite et al. stated “The world market of enzymes could reach $7.0 billion in 2023 [9]” which is referring to https://doi.org/10.1016/j.ijbiomac.2020.06.047. Please also note that the year (2022 vs. 2023) is different in the referenced article and the submitted manuscript. I would therefore recommend a thorough revision of the references

Minors:

Some of the selected keywords are too general in my opinion.

Line 72: wrong wording. I think the authors mean “resins”, not “raisins”

Line 142: “…can be used for the production of various chemicals like biofuels, hydrogels [4].” Are there other examples missing at the end of the sentence?

Line 250: The authors write “The reduction of a catalytic cupric ion, which 250 has a methylated terminal histidine, and a tyrosine as ligands, is necessary for LPMO action.” This is only true for fungal LPMOs. Even for fungal LPMO, not all LPMOs are methylated. Bacterial LPMOs show no methylation and a PHE residue is present instead of a TYR.

Line 275: “Hemicellulose is a heteropolymer and second most prevalent polysaccharide in plant cell wall.”

Line 306: Please correct “back bone” to “backbone”

Line 348: I think the authors mean acetylxylan esterase instead of “acetoxylan esterase”

Line 409: “Laccases (EC 1.10.3.2), one of the common oxidases produced by bacteria, fungi and plants are largest blue multicopper oxidases”. What do the authors mean by “largest multicopper oxidases”? Size? Phylogenetic group?

Line 412: ”They use oxygen as an electron 412 acceptor and copper’s redox capacities to oxidize phenolic compounds in lignin 413 because of low redox potential [19,72,75]” What do the authors mean? Laccase has a very high redox potential!

Line 433: “alpha hidroxiacids“ Please correct

Line 444 “Lignin peroxidases (EC.1.11.1.14) are glycosylated enzyme which contain heme protein in active centre”. I guess the authors mean “…contain a heme cofactor in the active centre”

Line 720: Word is missing

Line 738 – 764: Here the authors present some selected examples of immobilized enzymes and some performance criteria. There is a variety of examples available in literature. Why have the authors focused on these examples? A more critical assessment of the different processes would be more helpful for the reader than just presenting randomly selected examples.

Line 784 – 787: The authors should better differentiate between “enzyme engineering” and “metabolic engineering”

Comments for author File: Comments.pdf

Author Response

The literature review submitted by Parushi Nargotra et al. aims to give a comprehensive overview of lignocellulose degradation and discusses different microbial lignocellulolytic enzymes with further focus on their production by solid state fermentation and immobilization techniques for the valorization of plant biomass. The topic is of importance to establish a circular economy based on renewable resources and therefore I believe it is of interest for the readers of “Catalysts”. However, a large number of review articles have been published during the last few years. In my personal opinion it would be better to focus more on one defined topic (e.g. SSF or enzyme immobilization or microbial lifestyles) than trying to cover all these topics in a rather shallow manner.

Furthermore, the article in its current form suffers from multiple weaknesses. Several points need to be improved before the manuscript meets the quality criteria for publication. I hope that the following critiques will aid the authors in refining and revising their manuscript:

Response: Thank you so much for your in-depth investigation of the manuscript and suggesting the changes to make it into a much better form. We have thoroughly addressed your comments and updated the MS for its potential acceptance in the Catalysts journal.

 

Major:

Comment: The authors missed discussing er even mentioning the CAZy database system. The CAZy database represents a comprehensive and commonly accepted classification system for carbohydrate-active enzymes including all major lignocellulolytic enzymes. https://doi.org/10.1093/nar/gkab1045 or https://doi.org/10.1093/nar/gkt1178

Response: As per your suggestion, we have now added a paragraph on carbohydrate-active enzymes (CAZymes) in section 2 of the revised MS.

Comment: Line 258: here the authors are actually referring to the CAZy system. However, the correct designation is “auxiliary activities” and not “ancillary activities (AA)”. Furthermore, the information here seems to be not up-to-date. The authors are just discussing AA9, AA10 and AA11. However, besides the three mentioned families, there are additional families of LPMOs known (AA13 (2014), PMID 25201969; AA14 (2018), PMID=29377002; AA15 PMID=29472725; AA16 (2019); PMID=30923563; AA17, 10.1126/science.abj1342). This literature and information provided by the authors need to be updated and corrected

Response: The corrections have been made in the paragraph and information on the additional families of LPMOs has now been added along with the references in the revised MS.

 

Comment: Figure 1: Important lignocellulolytic enzymes are missing (e.g. versatile peroxidases, H2O2-producing enzyme (CAZy family 3 of the auxiliary activities (AA3), e.g. glucose oxidase, aryl alcohol oxidase, etc.; glyoxal oxidase (AA5); glucooligosaccharide oxidase (AA7)) and others. In my opinion, the authors should definitely reconsider their selection of the enzymes presented. Furthermore, there is a typo in the Figure legend. “degradadtion”

Response: Figure 1 has now been edited and updated with the addition of some other important lignocellulolytic enzymes. Furthermore, the typo error has been corrected in the revised MS.

 

Comment: The authors tried to focus on the newest literature and the majority of the references has been published within the last 3-5 years. However, some important findings are not mentioned and especially the description of enzyme classes seems in some passages rather superficial.

Response: We have now gone through the description of enzyme classes, and have added some text with respect to the classes of enzymes to add more value to the MS.

 

Comment: Cellulosomes are not mentioned in the sections about bacterial cellulolytic systems

Response: As per your inputs, a new paragraph has now been added on cellulosomes from bacterial systems in the revised MS.

 

Comment:  For LPMO the authors only mention the reactivity towards oxygen. However, there are convincing evidences reported in the last ~5 years that H2O2 is the preferred cosubstrate. A discussion of this reports is definitely relevant within a review describing LPMO as cellulolytic enzyme. See e.g. https://doi.org/10.1038/nchembio.2470 or https://doi.org/10.1038/s41467-020-19561-8

Response: According to your suggestion, we have now added the importance of H₂O₂ as an enhancer of LPMO activity and discussed the same in the revised MS.

 

Comment: The manuscript suffers from a variety of spelling/grammar errors (not limited to my further comments) and therefore I would strongly recommend proof reading and language editing to improve the quality of the manuscript

Response: We have thoroughly gone through the MS, made changes, corrected the grammatical errors and updated the suggested changes to improve its quality and make the MS in a more readable form.

 

Comment:  Literature references: In my opinion, the authors too often cite other review articles and they do not refer to the original research articles. However, most details the authors are discussing using such reference originally arise from other articles.

Response: In the revised MS, we have cited some more original articles with proper discussion and explanation, and tried to inculcate our own analysis of the previously published research and review papers so make it unique and interesting.

 

Comment: E.g. Chapter 4 “Solid State Fermentation for Enzyme Production”: The authors use the reference to the review article [119] by Leite et al. 2021 five times distributed over the entire length of the chapter. As an example, the authors write “It is expected that the global enzyme market may reach $7 billion by the end of 2022….” referring to reference 119. In this referenced article by Leite et al. stated “The world market of enzymes could reach $7.0 billion in 2023 [9]” which is referring to https://doi.org/10.1016/j.ijbiomac.2020.06.047. Please also note that the year (2022 vs. 2023) is different in the referenced article and the submitted manuscript. I would therefore recommend a thorough revision of the references

Response: We have now revised the statement in chapter 4 as “The global enzyme market in different industrial sectors is expected to reach $7 billion in 2023”. Regarding the reference of Leite et al. 2021, we have added more references along with the reference of Leite et al. 2021 to justify the text in the said Chapter 4 on Solid state fermentation.

 

Minors:

Comment: Some of the selected keywords are too general in my opinion.

Response: As suggested, some of the keywords have now been updated.

 

Comment: Line 72: wrong wording. I think the authors mean “resins”, not “raisins”

Response: The word has now been corrected in the revised manuscript.

 

Comment: Line 142: “…can be used for the production of various chemicals like biofuels, hydrogels [4].” Are there other examples missing at the end of the sentence?

Response: The missing examples such as biofilms, drug delivery coatings, organic acids have now been added in the revised MS.

 

Comment: Line 250: The authors write “The reduction of a catalytic cupric ion, which 250 has a methylated terminal histidine, and a tyrosine as ligands, is necessary for LPMO action.” This is only true for fungal LPMOs. Even for fungal LPMO, not all LPMOs are methylated. Bacterial LPMOs show no methylation and a PHE residue is present instead of a TYR.

Response: The statement has now been revised and updated in the MS.

 

Comment: Line 275: “Hemicellulose is a heteropolymer and second most prevalent polysaccharide in plant cell wall.”

Response: The grammatical error has now been corrected in the statement.

 

Comment: Line 306: Please correct “back bone” to “backbone”

Response: The correction has now been made.

Comment: Line 348: I think the authors mean acetylxylan esterase instead of “acetoxylan esterase”

Response: The correction has now been made in the revised MS.

 

Comment: Line 409: “Laccases (EC 1.10.3.2), one of the common oxidases produced by bacteria, fungi and plants are largest blue multicopper oxidases”. What do the authors mean by “largest multicopper oxidases”? Size? Phylogenetic group?

Response: Laccases are multicopper oxidases that oxidize the substrate by transferring electrons from a mononuclear copper center to a trinuclear copper center. Dioxygen binds to the trinuclear center and is reduced to two molecules of water after receiving four electron transfers from the mononuclear copper center. To avoid any confusion, the statement has been corrected in the revised statement.

 

Comment: Line 412: ”They use oxygen as an electron 412 acceptor and copper’s redox capacities to oxidize phenolic compounds in lignin 413 because of low redox potential [19,72,75]” What do the authors mean? Laccase has a very high redox potential!

Response: The redox potential of laccase enzyme varies from low to high (0.4 to 0.8 V) which however, is less than other lignolytic enzymes, such as lignin peroxidases (greater than 1 V). The statement has been revised and the updated in the revised MS.

 

Comment: Line 433: “alpha hidroxiacids“ Please correct

Response: The content has been corrected in the revised manuscript.

 

Comment: Line 444 “Lignin peroxidases (EC.1.11.1.14) are glycosylated enzyme which contain heme protein in active centre”. I guess the authors mean “…contain a heme cofactor in the active centre”

Response: The correction has been made now.

 

Comment: Line 720: Word is missing

Response: The statement has been corrected in the revised manuscript.

 

Comment: Line 738 – 764: Here the authors present some selected examples of immobilized enzymes and some performance criteria. There is a variety of examples available in literature. Why have the authors focused on these examples? A more critical assessment of the different processes would be more helpful for the reader than just presenting randomly selected examples.

Response: We have discussed the previously reported studies in the said portion with respect to the immobilization of different lignocellulolytic enzymes with a range of immobilization supports/materials with high reusability to enhance the process productivity and cost effectiveness. The initial statement of the paragraph has now been revised in the MS to let the reader know, as to why the selected examples from the literature were cited and discussed.

 

 

Comment: Line 784 – 787: The authors should better differentiate between “enzyme engineering” and “metabolic engineering”

Response: The word metabolic has been replaced with enzyme in the revised manuscript.

Round 2

Reviewer 4 Report

The authors provided a thorough revision of their submitted review article. They have addressed all my major concerns and they added and discussed previously missing topics in a meaningful way. In my opinion, the quality and comprehensiveness did strongly increase.

However, I still have some (but only minor) concerns that need to be addressed. Please see below. Finally, I would like to suggest a final language editing and polishing step to the authors.

 

Line 164: spelling error: omics technology

Line 175: “activity” à change to “activities”

Line 202: CAZy: this abbreviation has already been used earlier in the text

Line 309: The authors describe LPMOs belonging to family AA10 and AA15 as “bacterial enzymes”. While this is true for family AA10, this is not correct for AA15. AA15 LPMOs have so far only been characterized from the eukaryotic organism (from arthropods (doi: 10.1038/s41467-018-03142-x) and oomycetes (doi: 10.3390/biom11081180))

Line 474: The authors write: “They use oxygen as an electron acceptor and copper’s redox capacities to oxidize phenolic compounds in lignin because of low redox potential (0.4-0.8 V) compared to other ligninolytic enzymes (>1 V)” – I would not consider laccase with redox potential of up to more than 800 mV as “low redox potential”. Also, the meaning of the sentence is still not completely clear to me because laccases are indeed able to oxidize aromatic lignin model compounds. Only the non-phenolic moieties of lignin cannot be oxidized directly. Please check e.g.: https://doi.org/10.1016/j.biotechadv.2014.12.008

Furthermore, it would be helpful to state that the redox potentials given relative to NHE.

Line 705: “relating” à change to “related”

Line 706: check grammar and spelling. Delete “in”, correct “microbial”

Line 711: “cell-surface yeast strains“ is not the correct scientific term. Please check and correct!

Line 717: The authors write “Consequently, understanding the genetic architecture of the microorganisms is also crucial factor for effective implementation and success of the lignocelluloisc biorefineries.” – In my opinion this statement is true but not related to the examples described before in the text. Furthermore, please correct grammar and spelling (“is also a crucial factor…” and “lignocellulosic”)

Line 799: I think this sentence need to be improved. Do the authors mean “minimal enzyme costs” instead of “maximal enzyme costs”? This sentence is not clear for me.

Line 893: “…potential application in 2G bioethanol production” – I think the authors mean “second generation” by 2G. However, they do not explain this abbreviation nor do they describe first, second and third generation biofuels in the text.

Comments for author File: Comments.pdf

Author Response

Reviewer’s comments

The authors provided a thorough revision of their submitted review article. They have addressed all my major concerns and they added and discussed previously missing topics in a meaningful way. In my opinion, the quality and comprehensiveness did strongly increase.

However, I still have some (but only minor) concerns that need to be addressed. Please see below. Finally, I would like to suggest a final language editing and polishing step to the authors.

Response: Thank you so much for your comments and suggestions for improving the contents of the present Manuscript (MS). We have taken care of each comment and suggestion to make MS more informative. We have thoroughly read the MS and made the grammatical changes wherever required.

 

Comment: Line 164: spelling error: omics technology

Response: The correction has been made in the revised manuscript as per your suggestion.

 

Comment: Line 175: “activity” à change to “activities”

Response: As per your suggestion, the correction has been made in the revised manuscript.

 

Comment: Line 202: CAZy: this abbreviation has already been used earlier in the text

Response: We have removed the expansion of the abbreviation from this line in the revised manuscript.

 

Comment: Line 309: The authors describe LPMOs belonging to family AA10 and AA15 as “bacterial enzymes”. While this is true for family AA10, this is not correct for AA15. AA15 LPMOs have so far only been characterized from the eukaryotic organism (from arthropods (doi: 10.1038/s41467-018-03142-x) and oomycetes (doi: 10.3390/biom11081180))

Response: We have corrected the statement in the revised manuscript.

 

Comment: Line 474: The authors write: “They use oxygen as an electron acceptor and copper’s redox capacities to oxidize phenolic compounds in lignin because of low redox potential (0.4-0.8 V) compared to other ligninolytic enzymes (>1 V)” – I would not consider laccase with redox potential of up to more than 800 mV as “low redox potential”. Also, the meaning of the sentence is still not completely clear to me because laccases are indeed able to oxidize aromatic lignin model compounds. Only the non-phenolic moieties of lignin cannot be oxidized directly. Please check e.g.: https://doi.org/10.1016/j.biotechadv.2014.12.008. Furthermore, it would be helpful to state that the redox potentials given relative to NHE.

Response: We have modified and rewritten the statement in the revised MS to avoid any ambiguity and make it clearer.  

 

Comment: Line 705: “relating” à change to “related”

Response: As per your suggestion, the correction has been made in the revised manuscript.

 

Comment: Line 706: check grammar and spelling. Delete “in”, correct “microbial”

Response: As per your suggestion, the correction has been made in the revised manuscript.

 

Comment: Line 711: “cell-surface yeast strains“ is not the correct scientific term. Please check and correct!

Response: We have corrected and rewritten the statement as per the reference cited in the revised MS.

 

Comment: Line 717: The authors write “Consequently, understanding the genetic architecture of the microorganisms is also crucial factor for effective implementation and success of the lignocelluloisc biorefineries.” – In my opinion this statement is true but not related to the examples described before in the text. Furthermore, please correct grammar and spelling (“is also a crucial factor…” and “lignocellulosic”)

Response: We have replaced the statement with “Consequently, the use of recombinant cellulolytic enzymes from microbial species is also a crucial factor for the effective implementation and success of lignocellulosic biorefineries.” in the revised manuscript.

 

Comment: Line 799: I think this sentence need to be improved. Do the authors mean “minimal enzyme costs” instead of “maximal enzyme costs”? This sentence is not clear for me.

Response: We have corrected and rewritten the sentence in the revised manuscript.

 

Comment: Line 893: “…potential application in 2G bioethanol production” – I think the authors mean “second generation” by 2G. However, they do not explain this abbreviation nor do they describe first, second and third generation biofuels in the text.

Response: We have removed “2G” from the sentence and corrected the sentence as per your suggestion.