A Preliminary Investigation on Bacterial Diversity and Fermentation Quality of High-Moisture Alfalfa Silage Prepared with Biochar

Round 1

Reviewer 1 Report

Overall the manuscript is well-readable, interesting and present a possibility to improve the alfalfa silage quality. However, somewhere, the manuscript should be improved. Furthermore, the manuscript several types and grammatical mistakes which should be double checked.  Specific comments follow.

Abstract

1. 1, line 19-20: please rephrase

 The relative abundance of Lactobacillus species, Enterococcus species and bacteria…..

Results

In Materials and methods the authors presented how the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined, but this is not relevant for the paper because no content comparacy was presented between fresh and silage alfalfa (with or without biochar).

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Research carried out by the author seems to be important  to the development and enhancement of existing information on this subject. The paper can be accepted for publication after the corrections have been made.

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1. Please be sure that a manuscript thoroughly establishes how this work is fundamentally novel. Specific comparisons should be made to previously published materials that have a similar purpose. Please present a strong case for how this work is a major advance. This needs to be done in the manuscript itself, not just in the response to review comments. This is a very important point in terms of which I will further consider the manuscript.

A novel information you can find in the following papers:

Gorovtsov, Andrey V., et al. "The mechanisms of biochar interactions with microorganisms in soil." Environmental Geochemistry and Health 42.8 (2020): 2495-2518.

Wolna-Maruwka, Agnieszka, et al. "The effect of biochar-based organic amendments on the structure of soil bacterial community and yield of maize (Zea mays L.)." Agronomy 11.7 (2021): 1286.

Ajeng, A. A., Abdullah, R., Ling, T. C., Ismail, S., Lau, B. F., Ong, H. C., ... & Chang, J. S. (2020). Bioformulation of biochar as a potential inoculant carrier for sustainable agriculture. Environmental Technology & Innovation, 101168.

2. Please be sure that the abstract and the conclusions section not only summarize the key findings of the work but also explain the specific ways in which this work fundamentally advances the field relative to prior literature.
3. The introduction chapter should be expanded with information on the biochar influences the microbial community structure during fermentation process of the plant wastes.
4. The LAB and coliform bacteria culture- no reference to the literature from which the methodology was taken.
5. Analyses of bacterial communities (,Material and methods’ chapter) should be described in more detail.
6. In the ,Material and methods’ chapter there is no description of the bioinformatics methods used in the data analysis.
7. How can you explain the decrease in the number of mold during fermentation compared to the starting material, while decrease of the pH value.
8. The length of the paper has to be increased significantly, increase also the detail of the conclusions, inserting more quantitative data (especially ,Results and discussion; chapter).
9. Make sure the references are added correctly according to the journal's instructions.
10. The language correctness should be verified by a native speaker.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

REVIEW REPORT for the article: “Bacterial diversity and fermentation quality of high-moisture alfalfa silage prepared with biochar“

• The article name suitable represents the information contained in the work, because it is principally focused on a forage methodology, especially in alfalfa ensilaging in a mixture with biochar to improve nutrient content and beneficial microbial communities;
• The paper extension is in more or less form appropriated in relation to the analyzed problematics, therefore it would be recommended some extension of the work in the introduction part, where it would be good to add some results of important works at bibliographic resources to show the relevance of the provided research;
• For a better understanding, the work should be include the highlights of the work;
• The abstract in the form of the presented work is well elaborated to introduce and to understand the work. It is relevant and the keywords presented are appropriately chosen;
• All the figures and tables presented fulfill the task to make the fundamental concepts and the communicated results more understandable, but it would be good to comment more in a little extend the conclusions of the presented data using comparisons with other results in the thematic area, because some extrapolation of the results would be good realizing for reasons of short evaluation period, because the years present some differences and they can be influence a little the result of ensilaging;
• It should be good to elaborate and add some table where it would be clearly presented the different variants used in the trial for better comprehension;
• The work is presented in a good quality using good English and the highest technical standards for the evaluation of the presented results;
• The result and discussion part should be extended, adding necessarily other citations to compare the own results and analyze the relation of them to be able the correct conclusions in the sense of the own results;
• The conclusion part should have interesting findings that are important for the advance of the research in the given area;
• The cited literature sources are sufficient for the support of the presented original work.

Adjustments.

• Please to arrange the following formal mistakes:
• Page 2, line 65: improve the expression … package machine were prepared…. and change to … package machine and that were prepared….

• The necessity for a deep simplified explaining and extension of the presented and analyzed forage method, explaining why the selection of the presented mixtures;
• It should be important to do a little extension with a better and detailed explaining of the tables and figures presenting the scope of the results revised in terms of the proposed comparisons;
• It should be necessary to add some comments in relation to the future necessary research to provide in the field of the work;

Conclusions.

The article has the character of an original paper that promotes the advanced critical thinking at the practice on the application of agriculturally oriented selected methods in forage production and ensilaging for the assessment of the biochar influence when applied into alfalfa ensilaging on the bacterial diversity and fermentation quality for high-moisture substrate. The work provided research with three different variants for the study with a control variant, and the variants with the addition of 1% and 2 % of biochar in a mixture with chopped alfalfa. The fermentation was provided in controlled 15 and 30 days. The results of the research showed that the mixture of alfalfa with biochar presented a decreased pH, presence of coliform bacteria, nonprotein-nitrogen and ammonia-nitrogen content. Conversely, this addition showed increased values of dry matter, lactic acid and true protein content. In reference to the bacterial community, the addition of biochar had influence on the distribution of the bacterial community presenting an abundant raise of Lactobacillus species, Enterococcus species and bacteria of the phylum Firmicutes, and the decreasing level of undesirable bacteria. Therefore, the mixture of alfalfa with biochar is an opportunity to improve the fermentation quality and bacterial diversity of alfalfa silage, because the importance of nutrient content at feeding crops is one of the major concerns in agriculture related to the Livestock systems. Here, the presented work “Bacterial diversity and fermentation quality of high-moisture alfalfa silage prepared with biochar“, belongs to the complex assessments provided to determine if the factor of biochar addition can have some influence at the level of beneficial bacterial communities and the quality of silage (DM content, ammonia content, etc.…). The work presents the idea to use the advantages presented of a biochar produced from wood pyrolysis in mixture with chopped alfalfa to a better process of ensilaging. The mixture presented a positive assessments at 15 and 30 days, where it was verified the presented bacterial populations, produced organic acids and the resulted chemical composition of the mixture (before and after fermentation). The trial methodology at this study presents significant findings that can show an alternative to improve the nutrient content in alfalfa silage especially at the high level of nutrient, low level of inhibitors and high level of beneficial microorganisms. The data resulted of the study and the selected analyzed parameters with sufficient warranty permit to conclude about the perspective of biochar application at alfalfa ensilaging. In relation to the results, I do not see them with surprise. Since based on other studies carried out, it is known that the biochar properties can serve as a material to improve the nutrient retention and bacterial development influencing the quality of fermentation course, and this factor is reinforced by the optimal pH level and environment stabilization (acetate production). Therefore, the selected application of the presented forage method shows a very suitable way to increase the silage quality from alfalfa avoiding the inhibition presented by undesirable microorganisms, determined chemical substances (ammonia) and the losses of nutrients in relation to the other presented alternatives. Of course, it is necessary to emphasize that the work can provide only an extrapolation of the results that can be statistically assessed, because the small group of examples tested and the short testing time provided. Therefore, it is evident to suppose that in different years the result can be different according to the conditions presented at cultivation and the corresponding whether (dry, normal rainfall and very rainy year). However, if we can base our conclusions on the information found in world literature in this related field, the findings would be similar to the presented in this work confirming the correctness of them. For this, it would be good to continue with the research and to present results in a period covering from 5 to 10 years of research and presenting the combination variants but comparing ensilaging process with different cultivation, mixtures and processing methods. 

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Point 3: The introduction chapter should be expanded with information on the biochar influences the microbial community structure during fermentation process of the plant wastes.

The description still does not provide the information on the problem of the research undertaken

Point 4: The LAB and coliform bacteria culture- no reference to the literature from which the methodology was taken.

The original literature was not provided

Point 7: How can you explain the decrease in the number of mold during fermentation compared to the starting material, while decrease of the pH value.

Response 7: After silage, the number of lactic acid bacteria in alfalfa increased, and the resulting lactic acid content also increased, so the pH value decreased, which inhibited the growth and reproduction of mold.

I do not think this is the correct answer, as molds prefer acidic conditions (optimal growth at pH = 5-6).

Point 8: The length of the paper has to be increased significantly, increase also the detail of the conclusions, inserting more quantitative data (especially, Results and discussion; chapter).

No changes were made to the ,,Results and discussion’’ section.

Point 9: Make sure the references are added correctly according to the journal's instructions.

Author Response

Please see the attachment.

Author Response File: Author Response.docx