Proficient Lignocellulolytic Novel Bacterial Isolates from Diversified Galiyat Forests of Lower Himalaya

Round 1

Reviewer 1 Report

The work forests-2376703 showed data involving isolates of bacteria producing enzymes of interest to convert lignocellulosic biomass into biofuels. It is necessary to discuss what this study presented as new and different from others that isolated enzyme-producing bacteria. Below are other comments to improve the work.

- Line 19: change “neutral pH” to “pH between 6.7 and 7.0”, because pH has values. The medium is who can be neutral, basic or acidic;

- Line 26: “taxonomic bacteria”???????

- It is important to discuss in the introduction how the three enzymes studied (cellulase, xylanase and laccases) are applied in the production of biofuels. It is important to cite and discuss the articles of Ramos et al. (https://doi.org/10.1016/j.esd.2022.03.007) and Curran et al. (https://doi.org/10.1016/j.biotechadv.2021.107809);

- Line 49: sorbitol is not produced from hemicelluloses. Rephrase the sentence;

- Line 51: lignin is not a polysaccharide. Rephrase the sentence;

- Line 66-67: This sentence is contradictory. The authors need to review this aspect;

- Line 69-72: This sentence is misspelled;

- This is not true. Biological pretreatment is not yet an established technology. Rephrase the sentence;

- Line 208-209: The medium is who can be neutral. Rephrase the sentence;

- In nature, decomposed woods are most commonly colonized by filamentous fungi. Rephrase the sentence;

- It is not appropriate to use the term “purification” for microorganisms. The ideal is to use the word “isolation”;

- Lines 244, 294: correct to “cellulase”;

- Figure 3a: enzymatic activity must be reported in IU/mg of sample (on a dry basis) or IU/volume of water (moisture) of the sample;

- Table 2: there are some typos for the word “xylanase”;

- Line 344: correct to “acidic medium conditions”;

- Line 346: change “acidification” to “reduction”;

- Table 3: write the names of the species in italics;

- line 432: Change “cellulose degradation” to “cellulase production”;

- line 437: Change “corncob xylose degradation” to “xylanase activity”;

- Line 459: References must be cited in accordance to Guide to authors.

Minor editing of English language required.

Author Response

The work forests-2376703 showed data involving isolates of bacteria producing enzymes of interest to convert lignocellulosic biomass into biofuels. It is necessary to discuss what this study presented as new and different from others that isolated enzyme-producing bacteria.

Lignocellulosic biomass enriched Galiyat forests - lower Himalaya - represent a potential ecosystem with huge floral diversity with potential lytic bacteria. And, study-bacteria are proven more efficient than the ones reported earlier in the literature. These aspects are further lighlighted in the revised version of the manuscript. Please see the revised submission.

Below are other comments to improve the work.

- Line 19: change “neutral pH” to “pH between 6.7 and 7.0”, because pH has values. The medium is who can be neutral, basic or acidic;

The change is made accordingly, Please see the revised submission.  

- Line 26: “taxonomic bacteria”???????

We meant bacterial diversity; now changed into ‘taxonomically varied bacteria’.

- It is important to discuss in the introduction how the three enzymes studied (cellulase, xylanase and laccases) are applied in the production of biofuels. It is important to cite and discuss the articles of Ramos et al. (https://doi.org/10.1016/j.esd.2022.03.007) and Curran et al. (https://doi.org/10.1016/j.biotechadv.2021.107809);

Thanks for the suggestion; citations and recommended modifications are made accordingly. See L47, L61, and L75 of the revised submission.

- Line 49: sorbitol is not produced from hemicelluloses. Rephrase the sentence;

The suggested correction is made, thanks for the suggestion. See L53 of the revision.

- Line 51: lignin is not a polysaccharide. Rephrase the sentence;

Sorry for the confusion; we meant that polysaccharides are bound in plant biomass via bonding between cellulose and hemicellulose. Anyways, the sentence is now modified as “bonding within and between cellulose, hemicellulose, and lignin forms a composite biopolymer structure in plant biomass”.

- Line 66-67: This sentence is contradictory. The authors need to review this aspect;

Though literature varied enzymatic activities for bacteria and fungi, we omited the word fungi to make the sentence simple and to avoid any possible contradiction. The sentence is now read as “Recent analysis of a microbial community showed that bacteria are significant to lignocellulolytic enzyme activity”.

- Line 69-72: This sentence is misspelled;

The sentence is now modified; with better comprehension – hopefully. New sentence is read as “The biofuel production from lignocellulosic biomass has potential, but efficient enzymatic hydrolysis is a barrier because the bioconversion requires efficient synergetic actions among released enzymes i.e., cellulases, xylanases, and laccases”.

- This is not true. Biological pretreatment is not yet an established technology. Rephrase the sentence;

The word established is changed with promising; hopefully it will better serve the purpose.

- Line 208-209: The medium is who can be neutral. Rephrase the sentence;

The sentence is rephrased; thanks.

- It is not appropriate to use the term “purification” for microorganisms. The ideal is to use the word “isolation”;

Thanks for the suggestion; the term has been appropriately dealt with. See L159, L244, L314, L426.

- Lines 244, 294: correct to “cellulase”;

Sorry for these typos, corrected now.

- Figure 3a: enzymatic activity must be reported in IU/mg of sample (on a dry basis) or IU/volume of water (moisture) of the sample;

The suggested modifications are incorporated in the revised manuscript.

- Table 2: there are some typos for the word “xylanase”;

Sorry for these typos; corrections are made accordingly.

- Line 344: correct to “acidic medium conditions”;

Done.

- Line 346: change “acidification” to “reduction”;

Done.

- Table 3: write the names of the species in italics;

Done.

- line 432: Change “cellulose degradation” to “cellulase production”;

Done.

- line 437: Change “corncob xylose degradation” to “xylanase activity”;

Done.

- Line 459: References must be cited in accordance to Guide to authors.

Reviewer 2 Report

Overall, I think the article is good, but it still needs to be carefully revised, as the manuscript is full of grammatical, spilling, and spacing errors. For example, see a few comments below.

   Please revised the title its is longer,

Please revised line 40-42, 43-45, 54-57

Revised figure 2,3b its resolution is very low

Please revised line 245-250, 412-415

Line 37 change “biofuels, has increased” to “biofuels has increased”

needs improvement

Author Response

Overall, I think the article is good, but it still needs to be carefully revised, as the manuscript is full of grammatical, spilling, and spacing errors. For example, see a few comments below.

Thanks for esteemed reviewer’s valuable suggestions; manuscript is now accordingly revised.

Please revised the title it is longer,

The title is revised to shorten as suggested; if all respected reviewers and journal’s editorial feel ok, we present the manuscript as “Proficient Lignocellulolytic Novel Bacterial Isolates from diversified Galiyat Forests of Lower Himalaya”.

Please revised line 40-42, 43-45, 54-57

Suggested corrections are accordingly done, please see the revised manuscript.

Revise figure 2, 3b its resolution is very low.

The figures’ quality is enhanced; hopefully the resolution is okay now.

Please revised line 245-250, 412-415

The above mentioned text is corrected, as suggested.

Line 37 change “biofuels, has increased” to “biofuels has increased”

The comma is removed, as suggested.

Reviewer 3 Report

Forest comprises of very diverse tree species constructed by mainly weather, climate and soil characters, and the tree species are specified by different structures of lignocelluloses. Forests are native natural ecology to conserve unique biodiversity of living things including bacteria especially lignocellulytic bacteria. Thus, the soils and humus from forest are value resources for isolate bacteria producing not only lignocellulytic enzymes but also for many purposes such as antibiotic production. In this study, authors harvested four soil samples and four degraded wood samples from two sites in Thandiani and two sites in Nathiagali belonging to Galihat forest, lower Himalya that is very famous biodiversity. Thus, the samples are very good for mining lignocellolytic bacteria. Authors showed some characters of the soils and trees in the samples selected sites; investigated total bacteria in the samples, isolated 90 bacterial isolates by nutrient agar plates, of them 46 isolates were potential for cellulase, xylanase and laccase producers. The 16S rDNA of 36 out of the 46 isolates were sequenced for classification for assessment of diversity. In my opinion, the samples were very good, authors also hold numbers of lignocellulolytic isolates, but authors have just analyzed in too simple way to show strong point of this research. Thus, the new findings in this manuscript are not clear. In scientific logic thinking, if you want to isolate the lignocellulolytic potential strains, authors can isolate strains by nutrient agar plates, but for assessment of the lignocellulolytic potential strains, authors must culture strains in the medium containing inducers as the carbon sources such as xylan, avicel, … or crude biomass such as rice straw or corn stover,… In theory, if authors cultured the isolates overnight in LB medium, the strains don’t need to secrete enzymes for hydrolyzing any substrate for carbon sources, thus enzymes usually are not secreted. By many generations, the strains will loss ability to secret enzymes. This also is problem in this study. Thus you have to find a way to explain for culturing isolates in LB medium for finding lignocellulolytic potential strains in this study. Besides, authors have to deeply analyze your obtained results for further consideration.

The major comments:

-       English along the manuscript is so difficult and poor; many concepts were not scientifically proper, for example in the page 1: you wrote: “The first generation of bioethanol (produced from direct fermentation of sucrose) has been widely used particularly in the US and Brazil. Among bioenergy sources, biomass of second generation…. Why do you mean “second generation” including biomass? Biomass is bio-renewable source to produce the second generation of bioethanol. Thus, many sentences and concepts in manuscripts should be seriously revised.

-       For deep analysis of your results: (1) after isolated the bacterial strains, please summary total isolates, unique morphological isolates in each site, ratio of morphotypes versus total isolated bacteria in each site. Thus, based on the ratio, you can compare for assessment of the diversity. (2) After having the lignocellulose isolates, you can compare the distribution of cellulase, xylanase, laccase producing bacteria between 8 samples, try to link with characters of soils, humus or trees species, weather,… to understand your results. By Vent chart, you can show how many strains produced 3 enzymes, 2 enzymes or only one enzyme clearly for each site. This analysis is very clear for choosing lignocellulolytic potential strains. (3) After identification, classification of your isolates, you have to analyze diversity of the lignocellulolytic bacteria from different phylogeny taxons (phyla, classes, families, genera) to learn distribution of phylotypes producing the enzymes, distribution of extracellular enzyme activity among the isolates, extracellular hydrolytic enzyme activity by the isolates and try to link with the characters of soils, humus or trees species, weather,… to show what is new finding in this research. All the deep analysis I recommend above, authors make in tables and figures (if over than 14 tables +figures/1 manuscript, you can contribute as supplementary tables or figures).

-       In the introduction, you please add more information about diversity of bacteria, lignocellulolytic bacteria at least in forest and in Himalayan Forest, show advantages in lignocellulose degradation in this ecology. Then please indicate what has not been investigated up to now, that are reasons for conducting this research. Base on the research questions you give a way for resolve in this manuscript.  

-       In Materials and Methods, please describe very clearly, exact and details, avoid use “i.e.” here and in the result section. The materials as chemicals, kits you should integrate into methods with information of producers or origin. All the name of tables and figures should be check to make more detail and meaning accuracy and add captions for all. The media need to be added composition.   Accordingly, all the methods must be revised.

-       In the result section, besides deep analysis as I recommended above, you must add assessment and explain your results. Figure 3B is not good, thus must improve quality and make a separated Figure for this. Section 3.4. I dont think the result in this section exhibit for the validation of lignocellulolytic enzymes secreted from the isolates. The results expressed in this section just simple to analyze results in Figure 3A by another way. So in my opinion, you should not make a separated section like this to create a confusion for readers. Table 2 must be added the accession number of 16S rDNA sequences of the strains and use the names of the sites for samples selection in agreed names along the MS. This table should be above the figure 5.

-       In discussion section: Please show all the new findings clearly. Normally, each new finding will be discussed to make valuable of your finding. In each paragraph, you please briefly describe what is the new finding in this study, what is in agreement with previous studies, what are different? From these, please show what is value of your research! Please don’t discuss outside your research.

The minor comments:

Please check along the manuscript: to use italic characters for all Latin names of genera and species of isolates, note that the higher taxon level for example families, orders, classes and phyla names are not italic; (2) Many minor mistakes, typo mistakes must be revised. 

Comments for author File: Comments.pdf

 English along the manuscript is so difficult and poor; many concepts were not scientifically proper, thus authors must extesively edit. 

Author Response

Forest comprises of very diverse tree species constructed by mainly weather, climate and soil characters, and the tree species are specified by different structures of lignocelluloses. Forests are native natural ecology to conserve unique biodiversity of living things including bacteria especially lignocellulytic bacteria. Thus, the soils and humus from forest are value resources for isolate bacteria producing not only lignocellulytic enzymes but also for many purposes such as antibiotic production. In this study, authors harvested four soil samples and four degraded wood samples from two sites in Thandiani and two sites in Nathiagali belonging to Galihat forest, lower Himalya that is very famous biodiversity. Thus, the samples are very good for mining lignocellolytic bacteria. Authors showed some characters of the soils and trees in the samples selected sites; investigated total bacteria in the samples, isolated 90 bacterial isolates by nutrient agar plates, of them 46 isolates were potential for cellulase, xylanase and laccase producers. The 16S rDNA of 36 out of the 46 isolates were sequenced for classification for assessment of diversity.

Thanks a lot for your valuable comments; your detailed feedback has certainly improved the quality of our manuscript.

In my opinion, the samples were very good, authors also hold numbers of lignocellulolytic isolates, but authors have just analyzed in too simple way to show strong point of this research. Thus, the new findings in this manuscript are not clear.

As you mentioned above, the forests are native natural ecology to conserve unique biodiversity of living things including lignocellulytic bacteria. On the same lines, diversified forests of lower Himalaya must contain proficient lignocellulytic bacteria that laid the presumption of this particular study. We have further highlighted this aspect as manuscript’s novelty in introduction section from L82 to L92 and onward.

In scientific logic thinking, if you want to isolate the lignocellulolytic potential strains, authors can isolate strains by nutrient agar plates, but for assessment of the lignocellulolytic potential strains, authors must culture strains in the medium containing inducers as the carbon sources such as xylan, avicel or crude biomass such as rice straw or corn stover. In theory, if authors cultured the isolates overnight in LB medium, the strains don’t need to secrete enzymes for hydrolyzing any substrate for carbon sources, thus enzymes usually are not secreted. By many generations, the strains will loss ability to secret enzymes. This also is problem in this study. Thus you have to find a way to explain for culturing isolates in LB medium for finding lignocellulolytic potential strains in this study.

Esteemed reviewer is right that enzyme induction in required for such analyses and we did the way needed. The text was not clear in “Material and Methods” and we corrected it now. Please see the section 2.5 of the revised manuscript. In brief, LB was used to enrich and isolate the potential bacterial strains; afterwards the isolated bacteria were subsequently grown (again on LB) to screen for the potential lytic enzyme producers. After this screening, potential lignocellulolytic bacteria were grown in nutrient broth supplemented with CMC, cob corn xylan and ABTS as inducers for cellulase, xylanase and laccase respectively.

Besides, authors have to deeply analyze your obtained results for further consideration.

Thanks for your valuable suggestion; your recommendations will certainly improve the quality of our manuscript. But, for the time being, we are moving forward with current presentation and ‘ll adopt your analyses for our further future submissions.

The major comments:

-       English along the manuscript is so difficult and poor; many concepts were not scientifically proper, for example in the page 1: you wrote: “The first generation of bioethanol (produced from direct fermentation of sucrose) has been widely used particularly in the US and Brazil. Among bioenergy sources, biomass of second generation…. Why do you mean “second generation” including biomass? Biomass is bio-renewable source to produce the second generation of bioethanol. Thus, many sentences and concepts in manuscripts should be seriously revised.

Thanks for the suggestion; the manuscript has now been linguistically revised by English speaker, Dr. Emilie Widemann ([email protected]).

-       For deep analysis of your results:

(1) after isolated the bacterial strains, please summary total isolates, unique morphological isolates in each site, ratio of morphotypes versus total isolated bacteria in each site. Thus, based on the ratio, you can compare for assessment of the diversity.

Some of these observations are already reported like total bacterial isolates, bacteria from varied locations and sampling medium. Please see section 3.3 of the manuscript. Other detailed analyses would take time and thus delay the publication and thesis dissertation of the PhD student. We want to move forward with current presentation and ‘ll adopt your detailed suggested analyses for our further future submissions.

(2) After having the lignocellulose isolates, you can compare the distribution of cellulase, xylanase, laccase producing bacteria between 8 samples, try to link with characters of soils, humus or trees species, weather, to understand your results. By Vent chart, you can show how many strains produced 3 enzymes, 2 enzymes or only one enzyme clearly for each site. This analysis is very clear for choosing lignocellulolytic potential strains.

As per figure 4, proficient lytic bacteria are compiled for efficient cellulase, xylanase, laccase producing bacteria from two different locations and sampling media. Best enzyme producers (3, 2 or only one) are identified in this figure; please see L962-968 of section 3.4 in the revised version of the manuscript. Vent chart, may though represent better but the current presentation also suffices the importance of the message presented in this work. As other four reviewers favor the current presentation, we request to consider the revised manuscript for publication.

(3) After identification, classification of your isolates, you have to analyze diversity of the lignocellulolytic bacteria from different phylogeny taxons (phyla, classes, families, genera) to learn distribution of phylotypes producing the enzymes, distribution of extracellular enzyme activity among the isolates, extracellular hydrolytic enzyme activity by the isolates and try to link with the characters of soils, humus or trees species, weather,… to show what is new finding in this research. All the deep analysis I recommend above, authors make in tables and figures (if over than 14 tables +figures/1 manuscript, you can contribute as supplementary tables or figures).

Thanks for your valuable suggestion; your recommendations will certainly improve the quality of our manuscript. But, for the time being, we are moving forward with current presentation and ‘ll adopt your analyses for our further future submissions.

-       In the introduction, you please add more information about diversity of bacteria, lignocellulolytic bacteria at least in forest and in Himalayan Forest, show advantages in lignocellulose degradation in this ecology. Then please indicate what has not been investigated up to now, that are reasons for conducting this research. Based on the research questions you give a way for resolve in this manuscript.  

Two more studies have been included in the introduction, regarding bacterial diversity in Himalayas.

Thakur et al., 2018 - Canadian Journal of Microbiology. 64. Doi: 10.1139/cjm-2017-0754 &

Venkatachalam et al., 2014 - Microbial Ecology DOI: 10.1007/s00248-014-0476-4.

-       In Materials and Methods, please describe very clearly, exact and details, avoid use “i.e.” here and in the result section. The materials as chemicals, kits you should integrate into methods with information of producers or origin. All the name of tables and figures should be check to make more detail and meaning accuracy and add captions for all. The media need to be added composition. Accordingly, all the methods must be revised.

The whole section has been updated as per the recommendations, please see the revised version of the manuscript.

-       In the result section, besides deep analysis as I recommended above, you must add assessment and explain your results. Figure 3B is not good, thus must improve quality and make a separated Figure for this.

The figure legend and some confusing points from the figure have been removed for better clarity of the above-mentioned figure 3b.

Section 3.4. I dont think the result in this section exhibit for the validation of lignocellulolytic enzymes secreted from the isolates. The results expressed in this section just simple to analyze results in Figure 3A by another way. So in my opinion, you should not make a separated section like this to create a confusion for readers.

With due apology, there is some confusion for this comment. Figure 3A is presumptive screening for isolated Himalayan bacteria (grown in LB) while figure 4 (section 3.4) demonstrating the lignocellulolytic enzyme potential of selected/screened bacteria (grown in nutrient broth assisted with CMC, xylan and ABTS). This way, substrate-induced lytic enzymes production was the validation of LB-initiated enzyme assessments.

Table 2 must be added the accession number of 16S rDNA sequences of the strains and use the names of the sites for samples selection in agreed names along the MS. This table should be above the figure 5.

The sequences are submitted to NCBI’s database and so the accession numbers will accordingly be added in the manuscript before final publication. Moreover, the table-2 and figure-5 are re-arranged as suggested by the esteemed reviewer.

-       In discussion section: Please show all the new findings clearly. Normally, each new finding will be discussed to make valuable of your finding. In each paragraph, you please briefly describe what is the new finding in this study, what is in agreement with previous studies, what are different? From these, please show what is value of your research! Please don’t discuss outside your research.

Thanks for your valuable suggestions; the discussion is modified as per above mentioned recommendations. Hopefully, the revised version of the manuscript is improved overall, particularly for discussion as well.

The minor comments:

• Please check along the manuscript: to use italic characters for all Latin names of genera and species of isolates, note that the higher taxon level for example families, orders, classes and phyla names are not italic;

The needful corrections are made accordingly; all technical names are italicized throughout the manuscript.

• Many minor mistakes, typo mistakes must be revised. 

The manuscript is revised for linguistic errors and typo mistakes; the proof reading is done by English speaker, Dr. Emilie Widemann ([email protected]).

Reviewer 4 Report

Dear Authors:

I am glad to review your manuscript and provide the following comments:

Why did you collect soil and wood samples? Does soil can be the lignocellulose biomass as well?

Table 1, the format and fonts of the Table should be improved.

Figure 1, the quality of map should be improved.

For the ANOVA, please introduce the methods and p-value using this suggested reference.

Modeling and Simulation of the Mixed Mode Ventilation Strategies with Heat Recovery and Energy Recovery Wheels for Energy Conservation and IAQ Improvement in the Commercial Buildings. Recent Researches in Urban Sustainability. Architecture and Structures, 209-215.

Qian, X. (2019). Statistical Analysis and Evaluation of the Advanced Biomass and Natural Gas Co-Combustion Performance (Doctoral dissertation, Morgan State University).

In Figure 3, bacterial isolates are not very clear. Please 

Use either "Cellulase" and "Xylanase" or "Xylose degradation" and "Cellulose degradation" to make a consistent description.

In Figure 2, what is the white bar? B. diversity? Please make a new y-axis to label them.

In Figure 3b, there are only soil and wood points in the PCoA analysis. Black dots or triangle were missing in the Figure. Please use the correct labels.

In Figure 4, the words are too small and what are the units for the bars?

For the methods, please use the SI units and provide sources (model, city, and brand) of facilities.

 Overall, your Figures and Tables should be revised that readers are readable and contents are correct.

I am looking forward to review the revised version.

Reviewer

 

Author Response

Dear Authors:

I am glad to review your manuscript and provide the following comments: Why did you collect soil and wood samples? Does soil can be the lignocellulose biomass as well?

Thanks for your valuable comments; your feedback certainly improved the quality of our manuscript. As for the sampling, soil is a cosmopolitan habitat of diverse microbiota while the decaying wood was presumptively the most enriched habitat for lignocellulolytic microbes. Systematic and Applied Microbiology, 37(1), 60–67 & Microbial biotechnology, 9(2), 149-156. Moreover, the organic layer of forest soils is enriched with humus, thus representing the lignocellulose biomass (organic matter) present therein; please see Forests 12(840):1-15.

DOI:10.3390/f12070840.

Table 1, the format and fonts of the Table should be improved.

Suggested changes are made accordingly, please see the revised manuscript.

Figure 1, the quality of map should be improved.

The figure resolutions is improved; but a little clarification for your consideration is that the fade yellow color indicate the decreased/marginal vegetation. It is not quality but the system’s output for green segment of the region.

For the ANOVA, please introduce the methods and p-value using this suggested reference.

Modeling and Simulation of the Mixed Mode Ventilation Strategies with Heat Recovery and Energy Recovery Wheels for Energy Conservation and IAQ Improvement in the Commercial Buildings. Recent Researches in Urban Sustainability. Architecture and Structures, 209-215.

Qian, X. (2019). Statistical Analysis and Evaluation of the Advanced Biomass and Natural Gas Co-Combustion Performance (Doctoral dissertation, Morgan State University).

The reference has been added; thanks for the recommendation.

In Figure 3, bacterial isolates are not very clear.

As the bacterial numbers are >90, so the names are somehow confined at minimum space; but if you zoom the page/figure, the names are clearly readable.

Please Use either "Cellulase" and "Xylanase" or "Xylose degradation" and "Cellulose degradation" to make a consistent description.

For enzyme activity, we largely used the terms cellulase, xylanase and laccase but just at some points (introduction/discussion) the word cellulose/xylose degradation is used to avoid monotonous feeling during the reading. As the use is not very common, we therefore request to keep the way it is.

In Figure 2, what is the white bar? B. diversity? Please make a new y-axis to label them.

White bars are the bacterial diversity (morphotypes); the figure legend is modified now. Please see the revised version of the manuscript.

In Figure 3b, there are only soil and wood points in the PCoA analysis. Black dots or triangle were missing in the Figure. Please use the correct labels.

Sorry for the confusion; color (red, green) indicates the sample-medium i.e., soil or wood while the shape (asterisk, triangle) indicates sample-location i.e., Nathigali, Thandiani. The legend has been changed accordingly.

In Figure 4, the words are too small and what are the units for the bars?

The units & scales are now added and figure quality was improved for better resolution.

For the methods, please use the SI units and provide sources (model, city, and brand) of facilities.

Thanks for the suggestions; both aspects are dealt appropriately. Please see the MM section of the revised manuscript.

Overall, your Figures and Tables should be revised that readers are readable and contents are correct.

All the tables and figures (except phylogenetic tree that was already ok) are updated; please see the revised version of the manuscript.

Reviewer 5 Report

This paper is well-written and these results could be useful for readers. 

Author Response

This paper is well-written and these results could be useful for readers. 

Thanks a lot for your encouragement; its really appreciable.

Round 2

Reviewer 1 Report

The article forests-2376703 presented some improvements, but some corrections still need to be made.

- It is important to discuss in the introduction how each enzyme (cellulase, xylanase, laccase) can be applied in the production of biofuels. It is necessary to describe separately the effect of each enzyme on the process;

- Figures 3a, 5: enzymatic activity must be reported in IU/mg of sample (on a dry basis) or IU/volume of water (moisture) of the sample. Authors need to clarify in the text how enzyme activity data is being reported.

Minor editing of English language is required.

Author Response

Reviewer 1

The article forests-2376703 presented some improvements,

Thanks a lot for your encouragement.

but some corrections still need to be made.

- It is important to discuss in the introduction how each enzyme (cellulase, xylanase, laccase) can be applied in the production of biofuels. It is necessary to describe separately the effect of each enzyme on the process;

The suggestions are important and the text in revised manuscript (L76-L82) deals with the potential role of these enzymes. But, we avoid the dedicated description of these enzymes for biofuel because, in our study, we did not produced biofuel rather reported proficient microbial strains having the capacity to produce these enzymes. We doubt that the elaborative roles of each enzyme on the biofuel production process, may distract the readers.

 

- Figures 3a, 5: enzymatic activity must be reported in IU/mg of sample (on a dry basis) or IU/volume of water (moisture) of the sample.

Necessary corrections are made accordingly; please see the updated version of the manuscript.

 

Authors need to clarify in the text how enzyme activity data is being reported.

All these clarifications are made accordingly in the section 2.5 (particularly L200-L202, L301-L303, L311-L314) of the revised version.

 

Minor editing of English language is required.

The manuscript is revised again for linguistic errors and typo mistakes; the proof reading is done by English speaker, Dr. Emilie Widemann ([email protected]).

Reviewer 2 Report

 The author made the necessary changes to the article, which improved its overall quality by addressing important issues. This is a good step for publication.

Author Response

Reviewer 2

The author made the necessary changes to the article, which improved its overall quality by addressing important issues. This is a good step for publication.

Thanks a lot for your encouragement; it’s really appreciable.

Reviewer 3 Report

The manuscript was much improved but I still am not absolutely satisfied with this revised version, because the obtained results were not deep analyzed. However, some main and important points I showed in the round one but authors did not consider to correct as following:

-           In Materials and Methods, please describe more detail methods for building standard curves of glucose, xylose and for laccase activity that help for readers believe your results (volume of glucose or xylose,...solutions must be the same with volume of sample reaction, volume of DNS must be the same, and condition is in correspondance). In all three methods, the conditions for enzyme-substrate reaction were not indicated. In the method for cellulase activity, the enzyme-substrate reaction was carried out at pH7 with unknown temperature, but definition of one cellulase unit was at pH8, 35^oC. The same problems appeared in the others. I really dont know why you have this mistakes, I really wonder about this mistake, then what I should believe!

-           The unit for enzyme activity, authors can use U (unit) or IU (international unit), not UI, I really dont know why you use UI for enzyme activity. You dont explain, but dont revise!

-           Electrophoresis of DNA on agarose gel could not confirm the purity, quantity and size of DNA. You just adjudge relative specificity of PCR products, relative quantity and size;

-           Figure and table are the separated units from text, thus please add captions with description of abbreviated words.

-           In table 2: Use italic letters for the genera and species names. Add caption to indicate what are the bold, underlined words!

-           Many typho mistakes and English grammar should be checked and revised.

All the problems I showed above, authors must seriously consider and revise because it really scientific problems. 

Comments for author File: Comments.pdf

Should be check and improved!

Author Response

Reviewer 3

The manuscript was much improved

Thanks a lot for your encouragement.

 

but I am still not absolutely satisfied --- important points I showed in the round one but authors did not consider to correct as following:

- In Materials and Methods, please describe more detail methods for building standard curves of glucose, xylose and for laccase activity that help for readers believe your results (volume of glucose or xylose,...solutions must be the same with volume of sample reaction, volume of DNS must be the same, and condition is in correspondence). In all three methods, the conditions for enzyme-substrate reaction were not indicated. In the method for cellulase activity, the enzyme-substrate reaction was carried out at pH7 with unknown temperature, but definition of one cellulase unit was at pH8, 35^oC. The same problems appeared in the others. I really dont know why you have these mistakes, I really wonder about this mistake, then what I should believe!

Sorry for any earlier ambiguity; the recommendation inclusions are made accordingly in the section 2.5 (particularly L200-L202, L301-L303, L311-L314) of the revised version. We used Sethi et al. 2013 (https://doi.org/10.5402/2013/985685), Kamble and Jadhav 2012 (doi:10.1155/2012/683193) and Bagewadhi et al. 2017 (https://doi.org/10.1007/s40093-017-0184-4) for cellulase, xylanase and Laccase activity assay respectively.

 

- The unit for enzyme activity, authors can use U (unit) or IU (international unit), not UI, I really dont know why you use UI for enzyme activity.

Sorry for this oversight; originally, we used unit enzyme that was modified to unit/mL in R1 but totally overlooked the unit to be IU and not UI. Necessary corrections are now made accordingly; please see the revised version of the manuscript.

 

- Electrophoresis of DNA on agarose gel could not confirm the purity, quantity and size of DNA. You just adjudge relative specificity of PCR products, relative quantity and size;

Agreed; the agarose gel - for PCR products - is used to evaluate the relative size of the amplicons. The sentence is therefore modified; reading as “The amplified PCR products were observed on 0.8% agarose gel.” L235 of the revised manuscript.

 

- Figure and table are the separated units from text, thus please add captions with description of abbreviated words.

Footnotes are added accordingly; please see the revised version of the manuscript.

 

- In table 2: Use italic letters for the genera and species names. Add caption to indicate what are the bold, underlined words!

The technical names are italicized now; bold, underlined were the proficient strains in our study that are separately mentioned in table-3, so removed these highlights from here. Please see the revised manuscript.

 

- Many typo mistakes and English grammar should be checked and revised.

The manuscript is revised again for linguistic errors and typo mistakes; the proof reading is done by English speaker, Dr. Emilie Widemann ([email protected]).

 

All the problems I showed above, authors must seriously consider and revise because it really scientific problems. 

We appreciate the valuable feedback; it certainly helped us to enhance the quality of our manuscript. We really hope the current version will satisfy the comments.

Reviewer 4 Report

Dear Authors:

I believe you made several changes based on previous comments.
Below is my additional comments:

1.  Quality of Figure 1 is not good. Please improve the resolution of the Figure.

2.  You need to add labels for the Figures (e.g., Figure 2).

3.  Formats of the Manuscript should be checked. Such as Title, Authors, etc.

4. Double check the grammar, writing styles, etc.

I hope you will modify based on these comments.

Best regards,

Reviewer

Dear Authors:

Please get help from professional writers to review and double check the grammar, writing styles, etc.

Best regards,

Reviewer

Author Response

Reviewer 4

Dear Authors:

I believe you made several changes based on previous comments.

Thanks a lot for your encouragement; it’s really appreciable.

Below is my additional comments:

1. Quality of Figure 1 is not good. Please improve the resolution of the Figure.

A new diagram with enhanced quality is prepared; please see the updated version of the manuscript.

 

2. You need to add labels for the Figures (e.g., Figure 2).

Necessary corrections are made accordingly; please see the updated version of the manuscript.

 

3. Formats of the Manuscript should be checked. Such as Title, Authors, etc.

Manuscript format (e.g. Title, Authors, etc.) is corrected as the journal’s sample; we request the copy-editors of the journal to further help in identifying and correcting any such mistake.

 

4. Double check the grammar, writing styles, etc.

The manuscript is revised again for linguistic errors and typo mistakes; the proof reading is done by English speaker, Dr. Emilie Widemann ([email protected]).

I hope you will modify based on these comments.

We appreciate the feedback; it certainly helped to enhance the manuscript quality. We hope the current version will satisfy the comments.

 

Please get help from professional writers to review and double check the grammar, writing styles, etc. Best regards,

The manuscript is revised again for linguistic errors and typo mistakes; the proof reading is done by English speaker, Dr. Emilie Widemann ([email protected]).