Effects of Sucrose, Glucose and Molasses on Fermentation Quality and Bacterial Community of Stylo Silage

Round 1

Reviewer 1 Report

Review of Manuscript Fermentation-1629052

The aim of the manuscript was to evaluate the effect of adding different sugars on the fermentation parameters and microbial community of stylo silage. I would like to request the authors to consider the following major and specific comments and remarks:

Major comments

Abstract: treatments have to be better explained: dosage of sugar, was done in mini silos in laboratory? Time points? Give at least the CP, NDF and sugar content of the grass used for ensiling. Abstract is not informative enough and does not reflect the content of the manuscript.

The introduction is too general, and authors did not put efforts to really justify the realization of the experiment. In general, I could not see a structure and convincing story in the introduction. Molasses is mostly used (and cheap) to supply with sugar when ensiling legume forages. Why sucrose or glucose? This must be shortly justified and explained here. Authors dis not use relevant literature related to the topic. What about other studies where sugar was added to other legume forages. Finish the introduction with some hypothesis

M&M: some weaknesses of the experiment are: (1) silage was evaluated only after 30 d, and other ensiling times are recommended for this kind of lab experiment like 60 and 90 days; (2) only one quality of forage legume was used and did not consider variability expected in the practice (different DM and CP content and cutting time); (3) I am not agreeing with using vacuumed bags which are not representative of the practice, laboratory methods are normally based on using container were material is compacted and some air remains in which normally challenges the LAB; (4) full chemical composition was unfortunately not done in silages (and different time points) and only in the fresh material. Therefore, the results of this study are of limited use in the practice. These limitans have to be discussed in the respective chapter.

Results were well presented and scientifically discussed

The chapter conclusion is just about a summary of the results. Authors have to give general conclusions and implications of the study

Minor comments

L17-18: For better comparison give also the values of the control and show only the average of the other treatments.

L25: But under this specific dosage of sugar. Add this comment. Do you have further recommendations based on these results?

L31: on DM or FM basis?

L32: With this CP content I will not expect problems by ensiling. Problems arises by materials with CP contents > 20% in DM basis

L41L molasses are widely used, but about sucrose and glucose I would doubt. Rewrite the sentence

L43: that enhances the quality of silage is too general and superficial. BE more specific!

L44-45: Here also! Be more specific. Widely clarify your thoughts

L53: When was harvested? What was the age of the material after planted or last cut?

L55: Why this dosage? Must be somewhere shortly explained

L68-69: It think this belongs to results chapter

L65-74: Procedure of handling silages after sampling have to be clearly explained. Mention the method used for analysis of acids

L104-107: What about the interaction of sugars and time points? This was presented but methods for running the statistics are not explained here.

L115: But the pH of the control also ensured well preservation

L189-194: It is not relevant the comparison of CP and DM

L195: But most important is the ratio between WSC and CP content

L198: What was “failed” here? Mention some values to understand

L202: But pH alone is not of relevance. Most important is the consideration of together pH and DM content

L205-206: What were these additives and materials?

L211: I think butyric acid is more related to clostridium than acetic acid. Check this sentence

Author Response

Dear Editor and Reviewers:

Thank you for your response and for the reviewers’ and editorial board members’ comments regarding our manuscript.The comments were valuable and helpful to improve our manuscript as well as the significance of the study results. We have further edited the manuscript, and the revised portions are marked yellow in the text. Responses to the editor's and reviewers’ comments are listed below.

We believe that we have improved the manuscript based on the reviewers’ and editor’s comments. These changes do not influence the content and framework of our study. We also hope that with these improvements, our manuscript is now acceptable for publishing.

Thank you very much for your work concerning our paper.

Sincerely yours,

Mao Li

 

Major comments

Abstract: treatments have to be better explained: dosage of sugar, was done in mini silos in laboratory? Time points? Give at least the CP, NDF and sugar content of the grass used for ensiling. Abstract is not informative enough and does not reflect the content of the manuscript.

We have rewrote this part at L16-34.

The introduction is too general, and authors did not put efforts to really justify the realization of the experiment. In general, I could not see a structure and convincing story in the introduction. Molasses is mostly used (and cheap) to supply with sugar when ensiling legume forages. Why sucrose or glucose? This must be shortly justified and explained here. Authors dis not use relevant literature related to the topic. What about other studies where sugar was added to other legume forages. Finish the introduction with some hypothesis

We have rewrote this part at L44-78.

M&M: some weaknesses of the experiment are: (1) silage was evaluated only after 30 d, and other ensiling times are recommended for this kind of lab experiment like 60 and 90 days; (2) only one quality of forage legume was used and did not consider variability expected in the practice (different DM and CP content and cutting time); (3) I am not agreeing with using vacuumed bags which are not representative of the practice, laboratory methods are normally based on using container were material is compacted and some air remains in which normally challenges the LAB; (4) full chemical composition was unfortunately not done in silages (and different time points) and only in the fresh material. Therefore, the results of this study are of limited use in the practice. These limitans have to be discussed in the respective chapter.

(1)Thank you for your professional comment. It has been shown that fermentation duration have major effects on the microbial community and the silage composition. Refer to the characteristics of tropical forage and some previous studies, the stylo silage fermented for 30 d is acceptable. In future studies, we will compare them to determine the appropriate fermentation time. 

Mka B , Fk A . Effect of wilting organic pineapple by-products and jack bean ( Canavalia ensiformis ) foliage inclusion on silage fermentation and its nutritive value[J]. Animal Feed Science and Technology, 258(C):114303-114303

Waroon, Khota, Suradej, et al. Natural lactic acid bacteria population of tropical grasses and their fermentation factor analysis of silage prepared with cellulase and inoculant[J]. Journal of Dairy Science, 2016, 99(12):9768-9781.

Zhao X , Liu J , Liu J , et al. Effect of ensiling and silage additives on biogas production and microbial community dynamics during anaerobic digestion of switchgrass[J]. Bioresource Technology, 2017:349-359.

(2)Thank you for your professional comment. In future studies, we will compare them to determine silage of the different DM, CP content and cutting time.

(3)Thank you for your professional comment. In the future we will try the tools you mentioned for silage studies.

(4)Your are right, the full chemical composition of silage also was very important. Unfortunately, in the early stage of the COVID-19 pandemic outbreak in 2020, refrigerators in our lab were power off  and samples were damaged, so subsequent analysis could not be completed. This paper only discussed the role of WSC from the perspective of fermentation quality and microorganisms. We have added the related content at the end of the discussion.

Results were well presented and scientifically discussed

The chapter conclusion is just about a summary of the results. Authors have to give general conclusions and implications of the study

We have added the information at L 304-315.

 

Minor comments

L17-18: For better comparison give also the values of the control and show only the average of the other treatments.

We have added the information at L24-25.

L25: But under this specific dosage of sugar. Add this comment. Do you have further recommendations based on these results?

We have added the information at L34-35.

L31: on DM or FM basis?

We have corrected as FM basis.

L32: With this CP content I will not expect problems by ensiling. Problems arises by materials with CP contents > 20% in DM basis.

Thank you for your professional advice. For common forage material, your judgment is accurate. But tropical forages are a little different,it is difficult to convert to good quality silage because they often have coarse and stemmy structures with low sugar and high fiber contents. Stylo is a typical tropical forage with relatively low contents of water-soluble carbohydrates (WSC), which makes it hard to make high quality silage.

L41L molasses are widely used, but about sucrose and glucose I would doubt. Rewrite the sentence

L43: that enhances the quality of silage is too general and superficial. BE more specific!

L44-45: Here also! Be more specific. Widely clarify your thoughts

We have rewrote this part at L50-64.

L53: When was harvested? What was the age of the material after planted or last cut?

We have added the information at L82-85.

L55: Why this dosage? Must be somewhere shortly explained

We have added the information at L88.

L68-69: It think this belongs to results chapter

We have added it in the results chapter.

L65-74: Procedure of handling silages after sampling have to be clearly explained. Mention the method used for analysis of acids

We have added the information at L97-108.

L104-107: What about the interaction of sugars and time points? This was presented but methods for running the statistics are not explained here.

We have added the information 2.4. Statistics

L115: But the pH of the control also ensured well preservation

You are right, from the pH index, the quality of stylo silage without additives is acceptable, but considering organic acid index such as lactic acid, the fermentation quality of stylo silage without additives is not good enough. WSC obviously improved the fermentation quality.

L189-194: It is not relevant the comparison of CP and DM

L195: But most important is the ratio between WSC and CP content

L198: What was “failed” here? Mention some values to understand

We have rewrote this part at L226-233.

L202: But pH alone is not of relevance. Most important is the consideration of together pH and DM content

Your are right, the DM of silage also was very important. Unfortunately, Because the samples were damaged, we did not carry out chemical composition analysis of the silage. However, according to the dry matter content of raw materials, it is speculated that the dry matter of silage may also be higher.

L205-206: What were these additives and materials?

We have corrected it at L239-241.

L211: I think butyric acid is more related to clostridium than acetic acid. Check this sentence.

We have corrected it at L247.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

• In the Introduction section, mention this study's novelty.
• Use an impersonal form of writing, and the text format should be uniformized.

• Please use the abbreviations g. or spp. in the text for microorganisms indicated in the manuscript - e.g., Megamonas spp.- when species are not mentioned.
• Lines 66, 72. Please briefly present the methods or specify the standards used for the DM, CP, WSC, NDF, and ADF. For the organic acids, provide more information, e.g., the method used is chromatographic? The reference used indicates another reference Liu et al. (2012).
• Line 77. Why were the extracts incubated at 4 °C for 24 h for microbial identification?
• Line 98. Principal Component Analysis (PCA) or Principal Coordinate Analysis (PCoA)?
• Principal Coordinate Analysis (PCoA) was performed with SAS 9.3?
• Lines 132. Please mention the ACE index to the material and methods
• Line 213. Clostridia is a Class of microorganisms. Please rephrase.
• In conclusion, mention clearly how the study advances the knowledge in the field.

Author Response

Dear Editor and Reviewers:

Thank you for your response and for the reviewers’ and editorial board members’ comments regarding our manuscript. The comments were valuable and helpful to improve our manuscript as well as the significance of the study results. We have further edited the manuscript, and the revised portions are marked yellow in the text. Responses to the editor's and reviewers’ comments are listed below.

We believe that we have improved the manuscript based on the reviewers’ and editor’s comments. These changes do not influence the content and framework of our study. We also hope that with these improvements, our manuscript is now acceptable for publishing.

 

Thank you very much for your work concerning our paper.

Sincerely yours,

Mao Li

 

• In the Introduction section, mention this study's novelty.

We have rewrote this part at L44-78.

• Use an impersonal form of writing, and the text format should be uniformized.

We have rewrote this it.

• Please use the abbreviations g. or spp. in the text for microorganisms indicated in the manuscript - e.g., Megamonas spp.-when species are not mentioned.

I’m sorry, I don’t understand your means. By convention, genus level microorganisms are listed directly without  spp. Maybe you could give an examples of where was wrong and how to correct it, so many thanks.

Ogunade, I.M., Jiang, Y., Pech Cervantes, A.A., Kim, D.H., Oliveira, A.S., Vyas, D., Weinberg, Z.G., Jeong, K.C., Adesogan, A.T., 2018. Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: effects of Escherichia coli O157:H7 and silage additives. J. Dairy Sci. 101, 2048–2059.

He L, Lv H, Xing Y, Wang C, You X, Chen X, Zhang Q, 2020a.The nutrients in Moringa oleifera leaf contribute to the improvement of stylo and alfalfa silage: Fermentation, nutrition and bacterial community. Bioresour Technol 301:122733.

 

Lines 66, 72. Please briefly present the methods or specify the standards used for the DM, CP, WSC, NDF, and ADF. For the organic acids, provide more information, e.g., the method used is chromatographic? The reference used indicates another reference Liu et al. (2012).

We have rewrote the Chemical assay part.

• Line 77. Why were the extracts incubated at 4 °C for 24 h for microbial identification?

We have added the information at L102-103.

• Line 98. Principal Component Analysis (PCA) or Principal Coordinate Analysis (PCoA)?

We have corrected it at L131-133.

• Principal Coordinate Analysis (PCoA) was performed with SAS 9.3?

We added the reference  at L131-133.

• Lines 132. Please mention the ACE index to the material and methods

We have added the information  at L128-131.

• Line 213.Clostridia is a Class of microorganisms. Please rephrase.

We have corrected it.

• In conclusion, mention clearly how the study advances the knowledge in the field.

We have rewrote the conclusion.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Review of Manuscript Fermentation-1629052.R1

I have read the revised version of the paper that aimed at evaluating the effect of different sugars on fermentation characteristics and bacterial community of stylo silage. Thanks to the authors for answering my questions in very detail and following my suggestions. I think authors made enough efforts to improve the quality of this paper. I have no more questions or comments to this revised version of the manuscript