Strategies for the Biotransformation of Tung Leaves in Bioethanol Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major comments:

1. The title of the article indicates the potential industrial use of tung leaves for the production of bioethanol, but the manuscript does not provide any economic/industrial information comparing the production of ethanol produced from tung leaves with other technologies. I suggest changing the title of the manuscript or supplementing the text with the indicated data.

2. The table indicated in the sentence "The results of this study are summarized in the following table" (2,64), is missing.

3. The description of the experimental methods is unclear. Proportions should be presented as mass or volume units, rather than mixed units.

4. I suggest present reducing sugar mass concentration during a fermentation as relative value instead of absolute, to make graph (1.a) more clear.

5. The manuscript lacks information about the statistical methods used in the experiments, which is extremely important in this kind of research. There are error bars on the graphs, but it is not known whether they refer to the uncertainty in repetitions of the value measured in the sample or in the experiments.

Minor comments:

1. In the "Introduction" paragraph, I suggest removing information about the bioethanol utilisation as obvious to readers.

2. Information about the type of chromatographic column is lack in experimental methods description.

Comments on the Quality of English Language

The quality of English is good and needs only correction of typos and linguistic slips.

Author Response

Dear Reviewers:

Thank you very much for your valuable comments and suggestions. We have revised our manuscript according to the reviewers’ comments. We have carefully addressed the issues (point by point) raised by the reviewers and prepared a “response to reviews” letter.

 

Reviewer #1:

Q1: 1. The title of the article indicates the potential industrial use of tung leaves for the production of bioethanol, but the manuscript does not provide any economic/industrial information comparing the production of ethanol produced from tung leaves with other technologies. I suggest changing the title of the manuscript or supplementing the text with the indicated data.

Response: Thanks for your comments. According to your suggestion, we have revised the title and abstract of the manuscript, which highlights that the manuscript is to ferment the tung leaves to produce bioethanol at the experimental level, so as to verify the feasibility and prospect of producing bioethanol from the leaves of tung tree.

 

Q2: The table indicated in the sentence "The results of this study are summarized in the following table" (2,64), is missing.

Response: Thanks for your comments. We are very sorry for some mistakes in the manuscript caused by our negligence and haste in submission. We have deleted the sentence here.

 

Q3: The description of the experimental methods is unclear. Proportions should be presented as mass or volume units, rather than mixed units.
Response: Thanks for your comments. We use liquid cellulase produced by Novozymes Biotechnology, and we have reformulated the expression here.

 

Q4: I suggest present reducing sugar mass concentration during a fermentation as relative value instead of absolute, to make graph (1.a) more clear.
Response: Thanks for your comments. Considering that we chose simultaneous saccharification and co-fermentation, the enzymatic hydrolysis of tung leaves in the early stage of fermentation will increase the content of reducing sugar in the solution, and the enzymatic hydrolysis process also exists in the decreasing stage of reducing sugar. Although the use of relative value would make the process of reducing sugar decline more intuitive, considering that the highest reducing sugar concentration occurs at 2-4 h during the fermentation process, in order to express the changes in reducing sugar concentration throughout the fermentation process, the choice of relative value would make the whole process more accurate.

 

Q5: The manuscript lacks information about the statistical methods used in the experiments, which is extremely important in this kind of research. There are error bars on the graphs, but it is not known whether they refer to the uncertainty in repetitions of the value measured in the sample or in the experiments.
Response: Thanks for your comments. We are sorry for missing the relevant description of the parallel experiment, and we have corrected it.

 

Minor comments:

Q1: . In the "Introduction" paragraph, I suggest removing information about the bioethanol utilisation as obvious to readers.

Response: Thanks for your comments. As you described, we have revised the part of bioethanol utilization in the "Introduction" paragraph to make the manuscript more concise.

 

Q2: Information about the type of chromatographic column is lack in experimental methods description.
Response: Thanks for your comments. We have already added in the manuscript. We also cite a paper we published earlier, which has a more specific detection procedure.

 

Reviewer 2 Report

Comments and Suggestions for Authors

 

1.     In the text, the reader can find fragments that completely do not fit the rest of the text, taken out of context, looking carelessly left in other texts:

a)     In L64, there is: "the following table", while this table is nowhere to be found.

b)     The sentence's meaning from L256-258 is entirely incomprehensible, and it looks like a carelessly left fragment of the original MDPI article format!

2.     Record from L113 "cellulose powder (...) is small in size" is imprecise because does it mean a small size of cellulose particles or a small amount"?

3.     The methodology described in the chapter "Experimental methods" is very poorly described and contains numerous omissions and understatements, which may prevent readers from repeating the experiment described in the article:

a)     In L119, it is stated that "First, 1, 10, 20, 30, and 40 g were weighed", but what was weighed is not written!

b)     Regarding the previous, the end of the sentence from L119 introduces the reader to even greater confusion. Namely, in L120 (and 367), it is stated that "oleander leaf" while the entire text is devoted to studying tung tree leaves! Do authors mean "oleaginous leaves"?

c)     In L127, it was stated that the leaves of the tung tree were (correctly this time) added to the next conical bottles "with the same substrate concentration" – what is the value of this concentration? This cannot be found in the text!

d)     The authors use the acronym ORP in the text, which they do not elaborate anywhere! Of course, a careful reader will guess that it is about REDOX potential, but the used abbreviations must be explained.

e)     L176, 179 and 183 used the term "substrate mass" with the unit g/L – it is probably the concept of "substrate mass concentration" used a dozen times in the rest of the text.

4.     What is this g/(L-gTS) unit that appears in L283?

5.     L205 mentions wine production! This is an article about the fermentation of tung tree leaves! It should be clearly explained  that this is a reference to literature to obtain an analogy with the process occurring in wine production.

6.     Charts from figures 1-4 are of small size and low resolution, which means that even enlarging them does not enable their detailed analysis!

7.     The chart in Fig. 5 is methodically incorrect! It cannot present two series of entirely different experiments on one axis! The efficiency of fermentation of tung tree leaves cannot be compared with the "substrate mass concentration" and "initial pH" because we do not know their substrate mass concentration for the variable pH experiment!

8.     The authors use the words interchangeably optimal/maximum(e.g. L383/389) in the text – these are not equivalent concepts! Moreover, they have different meanings!

9.     The conclusions are just lapidary! It must provide a reliable summary and provide conclusions that can be drawn from the text presented. Apropos L392 should contain conclusions and not "Discussion" because the discussion is already in L149.

a)     The statement from L405-406 does not follow from the text at all!

b)     The statement from L403 that "The results provided a theoretical and technical basis for the bioconversion of tung leaves" is untrue – the results of a relatively simple experiment are presented in the form of poorly readable graphs. The simple explanations given for the course of the curves presented cannot be considered a "theoretical and technical basis" for the complicated fermentation process of oleaginous leaves.

c)     What do the authors mean by the term "isotropic samples" from L400?

10.  As promised in L105," kinetic properties" were not analysed! Chapter 3 describes the temporal changes of selected values characterising the fermentation process of tung tree leaves. However, these descriptions are qualitative, and attempts to explain their temporal changes are poorly justified. They do not result from the obtained measurement results but from researchers' intuition, which is relatively poorly supported by the literature.

 

Summing up:
The text requires a thorough reconstruction! The experiment methodology should be accurately described. The statements and hypotheses used in the discussion should be justified quantitatively, not just intuitively. A reliable summary and conclusions must be presented.

Comments on the Quality of English Language

-

Author Response

Reviewer #2:

Q1: In the text, the reader can find fragments that completely do not fit the rest of the text, taken out of context, looking carelessly left in other texts:

1. a) In L64, there is: "the following table", while this table is nowhere to be found.
2. b) The sentence's meaning from L256-258 is entirely incomprehensible, and it looks like a carelessly left fragment of the original MDPI article format!
   Response: Thanks for your comments. We are very sorry for some mistakes in the manuscript due to our negligence in work and haste in submission. We appreciate your suggestions very much. In accordance with your request, we have revised the manuscript, put the table in the appropriate position, and deleted the sentence in L256-258.

 

Q2: Record from L113 "cellulose powder (...) is small in size" is imprecise because does it mean a small size of cellulose particles or a small amount"?

Response: Thanks for your comments. We are sorry that we carelessly wrote the “tung leaf powder” as “cellulose powder”. After re-examination, we found that this sentence was not rigorous, so it was deleted from the manuscript.

Q3: The methodology described in the chapter "Experimental methods" is very poorly described and contains numerous omissions and understatements, which may prevent readers from repeating the experiment described in the article:

1. a) In L119, it is stated that "First, 1, 10, 20, 30, and 40 g were weighed", but what was weighed is not written!
2. b) Regarding the previous, the end of the sentence from L119 introduces the reader to even greater confusion. Namely, in L120 (and 367), it is stated that "oleander leaf" while the entire text is devoted to studying tung tree leaves! Do authors mean "oleaginous leaves"?
3. c) In L127, it was stated that the leaves of the tung tree were (correctly this time) added to the next conical bottles "with the same substrate concentration" – what is the value of this concentration? This cannot be found in the text!
4. d) The authors use the acronym ORP in the text, which they do not elaborate anywhere! Of course, a careful reader will guess that it is about REDOX potential, but the used abbreviations must be explained.
5. e) L176, 179 and 183 used the term "substrate mass" with the unit g/L – it is probably the concept of "substrate mass concentration" used a dozen times in the rest of the text.
   Response: Thanks for your comments. a) We have corrected it. Please look at L115.
6. b) Sorry for the confusion caused by the unprofessionalism of our manuscript, we have revised "oleaginous leaves" to tung tree leaves.
7. c) When we explored the effect of pH on the ethanol fermentation of tung leaves, the concentration we chose was based on the first set of experiments, and we chose the optimal substrate mass concentration of 100 g/L. Sorry for not describing it carefully, we have added it in the manuscript, please see L123 and L304-305.
8. d) We've corrected it. Please see L135.
9. e) We have corrected this in the manuscript. Please see L182, L186, 190 and L192.

 

Q4: What is this g/(L-gTS) unit that appears in L283.
Response: Thanks for your comments. We initially wanted to express the amount of ethanol produced by 1g of tung leaves, but we incorrectly did not define what TS is, which we have corrected in the manuscript. Please see L288.

 

Q5: L205 mentions wine production! This is an article about the fermentation of tung tree leaves! It should be clearly explained  that this is a reference to literature to obtain an analogy with the process occurring in wine production.
Response: Thanks for your comments. We have revised this description and added a new paper to supplement the accuracy of our description above.

 

Q6: Charts from figures 1-4 are of small size and low resolution, which means that even enlarging them does not enable their detailed analysis!
Response: Thanks for your comments. We apologize for the unprofessional addition of figures to the manuscript; we have re-added new clear figures for your review.

 

Q7: The chart in Fig. 5 is methodically incorrect! It cannot present two series of entirely different experiments on one axis! The efficiency of fermentation of tung tree leaves cannot be compared with the "substrate mass concentration" and "initial pH" because we do not know their substrate mass concentration for the variable pH experiment!
Response: Thanks for your comments. As you were concerned in Q3 c), we have redescribed the experimental procedure in the manuscript, and we hope that this revision of ours will address this issue.

 

Q8: The authors use the words interchangeably optimal/maximum(e.g. L383/389) in the text – these are not equivalent concepts! Moreover, they have different meanings!
Response: Thanks for your comments. We have corrected inaccurate statements in the manuscript, please see L397.

 

Q9: The conclusions are just lapidary! It must provide a reliable summary and provide conclusions that can be drawn from the text presented. Apropos L392 should contain conclusions and not "Discussion" because the discussion is already in L149.

1. a) The statement from L405-406 does not follow from the text at all!
2. b) The statement from L403 that "The results provided a theoretical and technical basis for the bioconversion of tung leaves" is untrue – the results of a relatively simple experiment are presented in the form of poorly readable graphs. The simple explanations given for the course of the curves presented cannot be considered a "theoretical and technical basis" for the complicated fermentation process of oleaginous leaves.
3. c) What do the authors mean by the term "isotropic samples" from L400?
   Response: Thanks for your comments. a) We have reorganized the “conclusions” of the manuscript and removed sentences that do not fit with what is supported by the experiments in the manuscript, which makes the conclusions more concise and precise.
4. b) As you mentioned, we made inaccurate descriptions of our conclusions, which we have revised.
5. c) We have removed inaccurate and redundant sentences.

 

Q10: As promised in L105," kinetic properties" were not analyzed! Chapter 3 describes the temporal changes of selected values characterizing the fermentation process of tung tree leaves. However, these descriptions are qualitative, and attempts to explain their temporal changes are poorly justified. They do not result from the obtained measurement results but from researchers' intuition, which is relatively poorly supported by the literature.
Response: Thanks for your comments. We have added a kinetic description section. Regarding the description of the figures, our team has been working on anaerobic fermentation for biohydrogen, biomethane and ethanol for a long time, so we have revised our description and re-added some literature to support our description.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors addressed most of the comments significantly increasing the scientific value of the manuscript.

Regarding the comment (question 4), after the authors' explanations, I agree that changes in reduced sugar content are better to show as absolute values.

Author Response

Dear Reviewers:

Thank you very much for your valuable comments and suggestions. We have revised our manuscript according to the reviewers’ comments and prepared a “response to reviews” letter. All the changes we have made in the manuscript are marked in red.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In the revised version, the authors took into account almost all indicated comments and suggestions.

One comment may be made regarding the current chapter 4 entitled "Discussion": there do exist already chapter, with discussion in the title (3. Results and Discussion). Current chapter. 4 should be devoted to summary and conclusions - currently it is too short and even concise. It should contain scientific conclusions and an indication of their application in science and practice.

After taking these comments into account, the text will be suitable for publication.

Author Response

Reviewer #1:

Comments and Suggestions: Authors addressed most of the comments significantly increasing the scientific value of the manuscript.

Regarding the comment (question 4), after the authors' explanations, I agree that changes in reduced sugar content are better to show as absolute values.

Response: Thank you for your valuable suggestions that have improved our manuscript. We are glad that you have accepted our suggestion regarding the comment (question 4).