PONE-D-19-35214

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal extract offers a promising alternative for lignocellulose pretreatment of oil palm residues

PLOS ONE

Dear Dr. Doucet,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers believe that you are addressing an important and timely topic.  However, all felt that the manuscript would benefit from significant revision. In particular, there were questions about statistical analyses and interpretation of some data. There were also concerns about organization of the manuscript, missing information and excessive use of abbreviations.  See the 3 reviews for more detailed suggestions.

We would appreciate receiving your revised manuscript by May 30, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Katherine A. Borkovich, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.Thank you for stating in your Funding Statement:

"This work was partially supported by a grant from Universidad Industrial de Santander, Vicerrectoria de Investigación y Extension (Grant number 5199) (to C.I.S. and D.M.V.), Industrias Acuña LTDA, and a Natural Sciences and Engineering Research Council of Canada (NSERC), via Discovery Grant RGPIN-2016-05557 (to N.D.). A.T.V. received a Postdoctoral Fellowship from NSERC. A.M.R. was supported by a doctoral scholarship from the Colombian Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) (PhD scholarship 567, 2012), and was the recipient of a scholarship from the Emerging Leaders in the Americas Program (ELAP) from the Government of Canada. F.V. and N.D. hold Fonds de Recherche Québec-Santé (FRQS) Research Scholar Junior 1 and Junior 2 Career Awards, respectively (numbers 35038 and 32743)."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. Thank you for stating the following in the Financial Disclosure section:

"This work was partially supported by a grant from Universidad Industrial de Santander, Vicerrectoria de Investigación y Extension (Grant number 5199) (to C.I.S. and D.M.V.), Industrias Acuña LTDA, and a Natural Sciences and Engineering Research Council of Canada (NSERC), via Discovery Grant RGPIN-2016-05557 (to N.D.). A.T.V. received a Postdoctoral Fellowship from NSERC. A.M.R. was supported by a doctoral scholarship from the Colombian Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) (PhD scholarship 567, 2012), and was the recipient of a scholarship from the Emerging Leaders in the Americas Program (ELAP) from the Government of Canada. F.V. and N.D. hold Fonds de Recherche Québec-Santé (FRQS) Research Scholar Junior 1 and Junior 2 Career Awards, respectively (numbers 35038 and 32743)."

We note that you received funding from a commercial source: Industrias Acuña LTDA

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please respond by return email with your amended Competing Interests Statement and we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper entitled “Genome sequencing and functional characterization of a Dictyopanus pusillus fungal extract offers a promising alternative for lignocellulose pretreatment of oil palm residues” gives an insight into the isolation, identification of potent ligninolytic enzyme producing strains form different isolates, functional characterization and genomic study of the highly potent strains was performed. The manuscript is nicely written and is technically sound.

However, there are certain points which the authors need to explain or revise before the paper may be accepted for publication. Author must carefully revise the MS in light of below given comments.

General Comments

1. Throughout the text phrase“fungal enzymatic extract” is used in title it’s just fungal extract please check if the “fungal enzymatic extract” can be used.

2. In Page no 4 line “We found that laccase activity was the main enzymatic contributor to our fungal extracts, which included a highly active isolate from D. pusillus LMB4.” must be moved to the result section

3. Please elaborate the methodology for preparation of wheat bran extract

4. In Page 17-18, “These results are also in agreement with prior observations suggesting that basic pH is a desirable property for laccase used in biotechnological processes, since low pH values were linked to increased enzyme degradation” The authors are suggesting basic pH has positive effect where the results shows otherwise. The enzyme is highly active in pH range of 3-4 during production (Fig 3A) and produced enzyme was highly stable in pH range of the 4 and 5 (Fig 3B) . If possible the author must try higher pH then 5 such as 7-9 for claiming enzyme to be active in basic environment or must stick to the observations obtained and refrain from using such statements contradicting their results.

5. In fig 6 components A, B, C, D, E were explained in figure title but what does each image suggest is not represented . You have used small “a” and “b” in figure to denote both figure however in the marking for table it is denoted by capital A and B further capital A, B was used to represent different parameters Its is confusing for reader so please represent what does fig a) and b) or may use Fig 6 (i) or (ii)

6. Regarding the submitted manuscript to adhere with author guidelines of the MS. (Reviewer have checked with recently published paper in PloSOne)

(a) Results is published separately from discussion but in present paper the Resutls and Discussion is one section, Please check with author guidelines once

(b) Referencing pattern in the text is not is in line with the PlosOne

Please check and revise manuscript after meeting the author guideline

Reviewer #2: The information on genome sequencing is scanty. The statistical data/ p values in Figures 3, 4, 5 are missing. Information on statistical optimization cellulolytic digestion is not available. The manuscript can be perhaps split into two seperate manuscripts with more relevant data and sent for publication.

Reviewer #3: The manuscript submitted by Rueda et al. focuses on biomass delignification using fungal strain to do pretreatment, the topic is very important, since biomass utilization, especially the agriculture waste ,including the oil palm residues the authors are interested in, is still a problem at the moment.

The study isolates a strain of Dictyopanus pusillus with highly laccases activity, and the author also show the strain they identified could be a good candidate for biomass (the oil palm residues here) pretreatment. The released sugar got significantly increased with the fungus pretreatment compare the without fungus.

In order to make the paper more easy reading and understanding, I have some suggestions before the paper could be accepted for publication.

1.It would be better to have a figure to show the whole experiment design, to show the whole process of the study, including fungal strain isolation, sequencing, enzymes determination, biomass pretreatment and so on.

2.There are a lot of uncommon abbreviations, such as EFB, SPS, very hard to following, it is better just write the full name.

3.In the page 19, the author said, the genome heterozygosity at 13,53%, please explain more about this heterozygosity, not every reader are familiar what this really means, it come from the sampling or it should be, because this fungus strain…

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal enzymatic extract offers a promising alternative for lignocellulose pretreatment of oil palm residues

PONE-D-19-35214R1

Dear Dr. Doucet,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Katherine A. Borkovich, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have revised the MS of the paper entitled “Genome sequencing and functional characterization of a Dictyopanus pusillus fungal extract offers a promising alternative for lignocellulose pretreatment of oil palm residues” satisfactorily as per suggested comments in my earlier review. The paper can be considered for publication. Also, during the further processing I suggest the author must make sure the format of referencing is in line with the PLosOne.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Pradeep Verma