Pleurotus ostreatus Can Significantly Improve the Nutritive Value of Lignocellulosic Crop Residues

Round 1

Reviewer 1 Report

Dear authors,

please find my comments in word document.

Comments for author File: Comments.docx

Author Response

Reviewer #1

Dear authors,

It was my great pleasure to read and review your manuscript. Please find below my suggestions how to improve your paper.

English language: your English is perfect (since you are from USA). However, I found several technical errors (for instance line 43) bit I am sure that technical MDPI support will correct that before publishing.

Response

Authors would like to thank the reviewer for giving us the opportunity to revise and improve our manuscript. Authors are not entirely sure of the technical error on line 43.

 

Reviewer #1

INTRODUCTION:

“Although using novel feeds to provide alternative sources was suggested [13], the potential of such feeds is unknown.” This sentence is not clear to me. Please rewrite it. Research is being done every day, new results are published.

Response

Authors have revised the sentence to read…”the potential of such novel feeds will need to be confirmed”. The statement does not refer to a particular feed but generally to novel feeds that have not been extensively evaluated.

 

Reviewer #1

MATERIAL AND METHODS:

Why did you choose to do SSF for only 3 weeks? It appears that you would gain better results if the incubation was longer. Did you base your decision on some preliminary results? Add this explanation in text.

Response

The incubation period was based on previous studies from our lab. Longer incubation period does not necessarily guarantee better results. Yes, there will be more breakdown of lignin, but the white-rot fungi will then start utilizing the cellulose which will further reduce the nutritive value of the substrate.

 

Reviewer #1

RESULTS AND DISCUSSION:

This comment applies to all tables: Please add that the results are average value of how many replicates. My personal choice would be to arrange columns so that the control sample is first and then treated samples. As a reader I find it easier to follow.

Response

Authors appreciate the suggestions by the reviewer, and we have included the number of replicates (n) and rearranged all the Tables.

 

Reviewer #1

Line 224: P ≤ 0.05, why did you write that it was P ≤ 0.10?

Response

You are correct and we have revised the part to reflect that the P value was significant.

Reviewer #1

Line 290: You cannot start sentence with number of reference. If you used some reference manager I know from personal experience that the software might have done that, but please make sure that it is corrected in the final version.

Response

Authors have corrected this to read…“Additionally, [58] and [63] reported …”

 

Reviewer #1

Line 322: “(57.5 - 282%)” what these numbers in bracket refers to? Please rewrite the sentence.

Response

The values refer to the percentage differences in total gas between the 2 treatments, but we have deleted it to avoid any confusion.

 

Reviewer #1

Line 367: DMd is not appearing in Table 3, rather IVADDM and IVTDDM. Please correct this so it is clear what you are commenting.

Response

Done.

 

Reviewer #1

Table 4: add below table what is A:P

Response

Authors have included the acetate:propionate ratio below Table 4.

 

Reviewer #1

CONCLUSION:

Line 454: what WRF stands for?

Response

Authors have deleted the acronym.

 

Reviewer #1

It appears that you have missed some references that are important for your study. For instance:

Improving the feeding value of straws with Pleurotus ostreatus - Animal Production Science, 2015, 55, 241–245. Please include this paper in your commenting. Also make sure that you have included all relevant references in manuscript.

Response

Thank you for bringing this reference to our attention. We believe that there is no way we could include all relevant references in our manuscript. Our reference list is 72 and one could argue that it is a lot for a research article.

 

 

Reviewer 2 Report

Attached notes and considerations

Comments for author File: Comments.pdf

Author Response

Reviewer #2

I corroborate that the mycelium can increase the availability of nutrients existing in the biomass. However, I disagree that the increase in DM was caused by the mycelium. It would be interesting to verify the MS composition of corn straw without any treatment.

Response

Authors would like to thank the reviewer for giving us the opportunity to revise and improve our manuscript. The increase in DM was likely driven by an increase in microbial mass.

 

Reviewer #2

I recommend entering the values of the chemical composition of the corn plant before any treatment. The increase in DM and ash in maize straw needs to be better explained. Theoretically, the fungus will decompose part of the DM components and this decomposition activity does not imply an increase in ash, which represents mineral composition

Response

Authors have revised the Tables as suggested.

 

Reviewer #2

Considering that all interactions were significant, it is necessary to evaluate, through regression, the incubation times in relation to treatment and non-treatment with Pleurotus.

Make for all meaningful interactions.

Response

Authors agree with the reviewer and are currently working on multiple incubation times.

 

Reviewer #2

It is not part of the conclusion. These are assumptions to be used in the introduction.

Response

Authors have revised the conclusion.

 

Reviewer #2

It is important to emphasize that the delignification hypothesis was refuted, since it had no effect on lignin.

Response

Done.

Reviewer 3 Report

Dear authors, it seems to me that this is a good manuscript and it has relevance in the scientific world. However, many points affect the quality of the manuscript.

 

My main concern and observation is the low number of observations. In addition to the low number of observations added to the SEM values, they indicate to me that there is a risk that a Type I error has been committed. It is necessary for the authors to add a power test to demonstrate that the number of observations is the minimum necessary to this studio.

 

Abstract: The abstract is the main face of your study. You must show all your study here and on this form share your funds. At this level, your abstract has not relevant or useful information. Improve it the abstract.

 

Lines 14-15: “The 14 increase in crude protein (52.9%) and ash (31.9%)…” Whose? Digestibility? Degradability? Passage? Of the stover?

 

Lines 17-22: The abstract needs to be rewritten. The data is very generic and difficult to understand. E.g.: Increase in protein... increase in protein, where? And this is just one line, almost all the results in the abstract have the same format.

 

Lines 23-24: This conclusion is not in accordance with the results and the objective of the manuscript.

 

Introduction: In the present form, the introduction is a generic review that no describes correctly the study. You need to add data about how the FDN, FDA and lignin contents decreased with the use of lignocellulolytic enzymes or fungi. The proportion of fungi used to improve the degragability of 1kg MS of wastes and corn stover, etc. Improve it the introduction.

 

Line 30: Please, avoid plagiarism. Plagiarism is an unethical factor and it is legislated. Manuscript with the same text:  https://pubmed.ncbi.nlm.nih.gov/29198265/

 

Lines 43: Correct: Sugggest

 

Lines 58-59: What biological treatments?

 

Line 74: Fermentation … In this sense, is it possible to affirm that you studied corn stover silage?

 

Material and methods: The material and methods topic needs to be rewritten.

 

Line 80: Shredded approximately what size?

 

Line 81: How long was the stover soaked? An overnight is not a time measure.

 

Line 83: Remove “leftover”.

 

Line 86: Milled approximately what size?

 

Line 95: “Two runs”? This description is not technical. 2x3 factorial, What were the factors?

 

Line 103: Four replicates are equal to 4 bags per bottle, or 4 bottles per treatment, or what? Improved the description.

 

Lines 109-110: How the collection was made? Be more specific. You use warm bottles? CO₂? Rumen areas collected? etc

 

Line 111: Why 4 hours? The usual time used is 2 hours.

 

Line 197: Add the statistical model.

 

Results and discussion: The description of the results is well written; however, the discussion is very generic and speculative. The use of comparative data is fine, but it is not specifically a discussion. Also, the use of data that is contradictory to your results is not well used, which decreases the quality of your manuscript. I wrote some suggestions but the discussion on several points needs to be rewritten.

 

Lines 210-213: This theory is incomplete. Rewrite it to complete the idea.

 

Lines 216-218: If it is important to describe a report contrary to yours; How does this text improve your discussion?

 

Line 225: Why is increasing the ash content an improvement?

 

Lines 229-232: Re-evaluate this comparison considering an error in the interpretation of the data.

 

Line 234: How does this process happen?

 

Lines 243-244: Comment similar to lines 216-218.

 

Line 248: How does this depression happen?

 

Lines 253-261: I think you tried to better describe the loss of organic matter; however, this text is very generic and very speculative. Rewrite it or merge it with the previous subtopic.

 

Line 271: What does SSF mean? Describe the initials the first time they appear, here and throughout the text.

 

Lines 288-290: Comment similar to lines 216-218.

 

Lines 297-300: Other speculative lines.

 

Lines 300-301: Do you mean that your experiment design is incorrect because a short SSF period was considered before starting the experiment?

 

Lines 307-320: How? Generic and speculative text. Rewrite it.

 

Line 357: 109% digestibility? Explain this.

 

Conclusion: Try to be more objective in your conclusion and avoid describing the results here.

 

Lines 449-451: I thought the study was to demonstrate the importance of the fungus in improving the nutritional quality of corn stover for ruminant feeding.

Author Response

Reviewer #3

Dear authors, it seems to me that this is a good manuscript and it has relevance in the scientific world. However, many points affect the quality of the manuscript.

Response

           Authors would like to thank the reviewer for giving us the opportunity to revise and improve our manuscript.

 

Reviewer #3

My main concern and observation is the low number of observations. In addition to the low number of observations added to the SEM values, they indicate to me that there is a risk that a Type I error has been committed. It is necessary for the authors to add a power test to demonstrate that the number of observations is the minimum necessary to this studio.

Response

We have established an in vitro batch culture protocol in our lab and our power test shows that a minimum of 4/5 observations/reps are needed. Authors have also included the number of replicates (n=8) used in the present study in all the Tables.

 

Reviewer #3

Abstract: The abstract is the main face of your study. You must show all your study here and on this form share your funds. At this level, your abstract has not relevant or useful information. Improve it the abstract.

Response

Based on the instruction from the journal on Abstract presentation, authors disagree with the reviewer that our abstract has no relevance or useful information. Authors believe that the current abstract adheres to the journal’s abstract requirement stated below:  

Abstract: The abstract should be a total of about 200 words maximum. The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used; 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations. The abstract should be an objective representation of the article: it must not contain results which are not presented and substantiated in the main text and should not exaggerate the main conclusions.

 

Reviewer #3

Lines 14-15: “The 14 increase in crude protein (52.9%) and ash (31.9%)…” Whose? Digestibility? Degradability? Passage? Of the stover?

Response

Authors have added some text to clarify that the figures refer to corn stover.

 

Reviewer #3

Lines 17-22: The abstract needs to be rewritten. The data is very generic and difficult to understand. E.g.: Increase in protein... increase in protein, where? And this is just one line, almost all the results in the abstract have the same format.

Response

Authors have revised the abstract to differentiate between the chemical composition of corn stover and in vitro batch culture results.

 

Reviewer #3

Lines 23-24: This conclusion is not in accordance with the results and the objective of the manuscript.

Response

Authors have revised the conclusion to focus strictly on the current results.

 

Reviewer #3

Introduction: In the present form, the introduction is a generic review that no describes correctly the study. You need to add data about how the FDN, FDA and lignin contents decreased with the use of lignocellulolytic enzymes or fungi. The proportion of fungi used to improve the degragability of 1kg MS of wastes and corn stover, etc. Improve it the introduction.

Response

Authors have revised parts of the Introduction.

 

Reviewer #3

Line 30: Please, avoid plagiarism. Plagiarism is an unethical factor and it is legislated. Manuscript with the same text:  https://pubmed.ncbi.nlm.nih.gov/29198265/

Response

Authors have revised the sentence, but the information/author was appropriately cited.

 

Reviewer #3

Lines 43: Correct: Sugggest

Response

Done.

 

Reviewer #3

Lines 58-59: What biological treatments?

Response

Authors have included white-rot fungi in the manuscript.

 

Reviewer #3

Line 74: Fermentation … In this sense, is it possible to affirm that you studied corn stover silage?

Response

Just to clarify, fermentation refers to solid-state fermentation which was clearly mentioned in the next sentence. There is a huge difference between ensiling to make silage and solid-state fermentation.

 

Reviewer #3

Material and methods: The material and methods topic needs to be rewritten.

Response

Done.

 

Reviewer #3

Line 80: Shredded approximately what size?

Response

The corn stover were shredded to 5 cm length. Authors have included that in the manuscript.

 

Reviewer #3

Line 81: How long was the stover soaked? An overnight is not a time measure.

Response

The samples were soaked for 18 h. Authors have added that in the manuscript.

 

Reviewer #3

Line 83: Remove “leftover”.

Response

Done.

 

Reviewer #3

Line 86: Milled approximately what size?

Response

Done.

 

Reviewer #3

Line 95: “Two runs”? This description is not technical. 2x3 factorial, What were the factors?

Response

The factors are the 3 incubation periods. Authors have deleted “two runs” and defined the 2 x 3 factorial.

 

Reviewer #3

Line 103: Four replicates are equal to 4 bags per bottle, or 4 bottles per treatment, or what? Improved the description.

Response

The standard protocol is one bag/bottle. Authors have added “(bottles)” in the revised manuscript.

 

Reviewer #3

Lines 109-110: How the collection was made? Be more specific. You use warm bottles? CO₂? Rumen areas collected? etc

Response

Authors have revised this part to read…“Rumen fluid (RF) was sampled from the cranial, ventral and dorsal regions of the rumen. Thereafter, the solid part of the rumen fluid was filtered out using four layers of cheese cloth. The liquid part was funneled into a pre-warmed insulated thermoflask.”

 

Reviewer #3

Line 111: Why 4 hours? The usual time used is 2 hours.

Response

Time of collection varies widely in journals from a couple of hours prior to feeding to a couple of hours after feeding. Authors would have preferred to collect rumen fluid 2 h before feeding but since the cows were with the milking herd, they were fed very early in the morning and the only possible time was 4 h after feeding and this ensures that rumen fluid were collected when the microbes are at the log/exponential phase of growth.

 

Reviewer #3

Line 197: Add the statistical model.

Response

Done.

 

Reviewer #3

Results and discussion: The description of the results is well written; however, the discussion is very generic and speculative. The use of comparative data is fine, but it is not specifically a discussion. Also, the use of data that is contradictory to your results is not well used, which decreases the quality of your manuscript. I wrote some suggestions but the discussion on several points needs to be rewritten.

Response

Authors have revised several parts of the discussion.

 

Reviewer #3

Lines 210-213: This theory is incomplete. Rewrite it to complete the idea.

Response

Authors have revised the sentence as suggested by the reviewer.

 

Reviewer #3

Lines 216-218: If it is important to describe a report contrary to yours; How does this text improve your discussion?

Response

It is always good to highlight the diverse reports in literature. Comparing one’s results with literature could be regarded as a norm as long as that is not the focus of the discussion.

 

Reviewer #3

Line 225: Why is increasing the ash content an improvement?

Response

Authors have replaced the word “improvement” with “increase” which is more appropriate. Thank you for pointing that out.

 

Reviewer #3

Lines 229-232: Re-evaluate this comparison considering an error in the interpretation of the data.

Response

The revision above has addressed this comment.

 

Reviewer #3

Line 234: How does this process happen?

Response

The nitrogen in the corn stover will be used by P. ostreatus to accumulate microbial mass resulting in increased CP content of the treated corn stover.

 

Reviewer #3

Lines 243-244: Comment similar to lines 216-218.

Response

Authors did not see any problem with the two sentences. One was on DM and the other on CP.

 

Reviewer #3

Line 248: How does this depression happen?

Response

By inhibiting the actions of 3-hydroxy-3-methylglutaryl-coenzyme A and by extension, it disrupts fat synthesis.

 

Reviewer #3

Lines 253-261: I think you tried to better describe the loss of organic matter; however, this text is very generic and very speculative. Rewrite it or merge it with the previous subtopic.

Response

Authors respect the opinion of the reviewer, but our extrapolation of the OM result is supported by results in Tables 5.

 

Reviewer #3

Line 271: What does SSF mean? Describe the initials the first time they appear, here and throughout the text.

Response

Authors have added the abbreviation at first mention in the “Introduction”.

 

Reviewer #3

Lines 288-290: Comment similar to lines 216-218.

Response

Authors did not see any reason to revise the sentence.

 

Reviewer #3

Lines 297-300: Other speculative lines.

Response

Authors have deleted these lines.

 

Reviewer #3

Lines 300-301: Do you mean that your experiment design is incorrect because a short SSF period was considered before starting the experiment?

Response

No, the experimental design is fine. There is no way to achieve full degradation of corn stover in 3 weeks and full degradation will result in reduced nutrient value of corn stover. It is all about finding the fine balance.

 

Reviewer #3

Lines 307-320: How? Generic and speculative text. Rewrite it.

Response

Authors have revised this section.

 

Reviewer #3

Line 357: 109% digestibility? Explain this.

Response

That is the rate of change between treated and untreated corn stover at 6 h of in vitro batch fermentation.

 

Reviewer #3

Conclusion: Try to be more objective in your conclusion and avoid describing the results here.

Response

Authors have revised the conclusion.

 

Reviewer #3

Lines 449-451: I thought the study was to demonstrate the importance of the fungus in improving the nutritional quality of corn stover for ruminant feeding.

Response

Authors have revised the conclusion.

Round 2

Reviewer 3 Report

Observations have been made before and sometimes the authors disagree with them. In my case, I describe these observations to show you the ambiguity of these parts and to make them clearer for the readers of the manuscript after its publication.

The authors made changes according to the reviewers' suggestions; however, I do have a few observations/suggestions:

1. The format of the manuscript needs correction.

2. Referring to using contradictory data; my point is not not to use that. My point is that just posting the contradictory idea is not enough. Example: I studied my running distance and the average value is 5 km, a value that was also observed by José. On the other hand, Luis runs 4 km. Do you understand the many ideas that appear and should be explained in the manuscript? You do not need to explain the results of another author, but you do need to explain his result. If someone found a different observation, why is the result different from yours? What is it about your experiment that made the result different?

3. Lines 484-487: In the rewritten conclusion, these lines look more like a sentence pertaining to the topic of the discussion and not a conclusion sentence. Use the same idea but rewrite it as a conclusion format.

 

 

Author Response

Reviewer #3

Observations have been made before and sometimes the authors disagree with them. In my case, I describe these observations to show you the ambiguity of these parts and to make them clearer for the readers of the manuscript after its publication.

The authors made changes according to the reviewers' suggestions; however, I do have a few observations/suggestions:

1. The format of the manuscript needs correction.

Response

Authors would like to thank the reviewer for additional feedback. Authors could not address this part as it is vague and may be related to #2 below.

 

Reviewer #3

2. Referring to using contradictory data; my point is not not to use that. My point is that just posting the contradictory idea is not enough. Example: I studied my running distance and the average value is 5 km, a value that was also observed by José. On the other hand, Luis runs 4 km. Do you understand the many ideas that appear and should be explained in the manuscript? You do not need to explain the results of another author, but you do need to explain his result. If someone found a different observation, why is the result different from yours? What is it about your experiment that made the result different?

Response

Authors have included additional text on what could have be responsible for the differences between our results and those studies/results.

 

Reviewer #3

3. Lines 484-487: In the rewritten conclusion, these lines look more like a sentence pertaining to the topic of the discussion and not a conclusion sentence. Use the same idea but rewrite it as a conclusion format.

Response

Authors have revised part of the conclusion.