Analysis on Fermentation Quality, Chemical Composition and Bacterial Communities of Corn Straw and Soybean Straw Mixed Silage

Round 1

Reviewer 1 Report

the work is interesting but presents serious inconsistencies.

in one of them in the text it mentions that. it works with soybean straw and then. that it is rice straw. with which. i don't know if it is a copy paste of some other article or not. do you know the difference between. soybean and rice?
in the sampling. in the text it mentions that. samples are taken on days 1,7,14,60, in the first image it is noted that. samples are taken on day 30 and it is not mentioned, if. each silo is opened for sampling. it does not fit because it mentions day zero and never. it is seen on day 1 and day 14 ?
When. the. chemical. composition. of. the. samples. before. ensiling. is. shown,. it. shows. a. dry. matter. content. of. 18 and. 40 %. The. straw. according. to. the. NRC,. INRA. AFRC must be. above 88 %, so. it. is. not. silage. straw,. then. it. says. that. it. adds. water. in. sufficient. quantity. to. ensile. the. sample. D; of 20, 25 and 30 %, which means that each treatment starts with different amounts of moisture and it is not logical that SBM increases. DM from. 30 to 44 % in 7 d, this as explained by the statistical analysis is wrongly stated, it should be 2 main effect analyses. treatment and time in one row and. in the case of time the analysis should be repeated measurements with respect to time, because it is a consequence of time and cannot be analysed with a factorial, x time effect.

the chemical composition should be checked because if they are not straws, a maize will have. 5 % of CP. but not 9 %. that e sun maiz. whole plant and. should. check. in. detail. the. content. of. moisture. dry. matter. and. it. is. missing. to. analyse. lignin,. also. if. it. says. that. it. counts. by. difference. hemicellulose,. this. is. never. seen. in. the. manuscript. 

In the case of NH3 it is recommended. to present the data in mg/L. and VFAs in mmol/l. 

should revise their conclusions. I do not agree. The results showed that the quality of soybean straw alone fermented silage was poor, while the quality of mixed fermentation silage was relatively high due to its high crude protein content, lactic acid content, and low ammonia nitrogen content, because. the only presursor of. fat in milk is acetic acid with which. SBMs traw. would. increase. milk. fat. production. compared. to. the. other. treatments. and. it. has. higher. PC and the pH. below 4.2. 

What about the whole bacterial study ? combined analysis of the metabolome and microbial community in the fermented silage ? It does not conclude anything  


What about the whole bacterial study ? combined analysis of the metabolome and microbial community in the fermented silage ? It does not conclude anything  

Author Response

Response to the reviewer 1#：

Q1: The work is interesting but presents serious inconsistencies.In one of them in the text it mentions that. it works with soybean straw and then. that it is rice straw with which I don't know if it is a copy paste of some other article or not. do you know the difference between. soybean and rice?

Response:

Dear reviewer, thank you for your prompt feedback on our manuscript. We sincerely apologize for any inappropriate or erroneous usage of words or phrases that may have compromised the quality of the paper. In the revised version, we have made every effort to address the concerns you raised as well as any other areas of potential confusion for the readership.

While conducting this experiment, we also conducted research on rice straw. Therefore, during the preparation of the paper, there were some confusions in the Materials and Methods section, including the use of “rice straw” in Q1 and issues related to subsequent sample collection and experimental procedures. We have now thoroughly reviewed and corrected each part of the experimental methods and protocols.

Changes to the manuscript:

“rice straw” have been changed to “soybean straw” in the revised manuscript.

Q2: In the sampling. in the text it mentions that samples are taken on days 1,7,14,60, in the first image it is noted that samples are taken on day 30 and it is not mentioned, if each silo is opened for sampling. it does not fit because it mentions day zero and never it is seen on day 1 and day 14?

Response:

Thank you for bringing up this issue. We also recognize that the previous description regarding the sampling time point was incorrect. Regarding the "day 1" samples mentioned earlier, they were actually fresh samples that were just mixed on the day of preparation without fermentation. Following your guidance, we should revise the labeling from "day 1" to "day 0".

In order to better detect pH changes in the mixed silage, samples were also collected on the 30th day of the experiment, but no other indicators were measured.

Changes to the manuscript:

Now the sentence has been changed to “Samples collected on day 0,7,14,30 and 60 of the experiment were used to determine the pH value, and samples collected on day 7,14, and 60 of the experiment were used to determine the nutritional composition of the feed.”

Q3: When the chemical composition of the samples before ensiling. is. shown, it shows a dry matter content of 18 and 40 %. The straw according to the NRC, INRA AFRC must be above 88 %, so it is not silage straw, then it says that it adds water in sufficient quantity to ensile the sample D of 20, 25 and 30 %, which means that each treatment starts with different amounts of moisture and it is not logical that SBM increases.

Response:

We apologize for this mistake. The statement "Clean drinking water was added during the mixing process to achieve a dry matter content of about 65% in the mixed silage" was our plan when we were fermenting rice straw. This sentence has now been deleted in the revised manuscript.

In southern China, due to the warm and humid climate, the bulk straw resources including corn straw have low dry matter content at the time of harvest. This is the main reason for the difficulty in producing high-quality silage and short storage time in the region. On the other hand, another main goal of this study is to utilize soybean straw, which has a high local yield but is currently treated as field waste, as feed through fermentation for local beef or goat farming, thus addressing the shortage of forage resources.

Q4: DM from. 30 to 44 % in 7 d, this as explained by the statistical analysis is wrongly stated, it should be 2 main effect analyses. treatment and time in one row and in the case of time the analysis should be repeated measurements with respect to time, because it is a consequence of time and cannot be analysed with a factorial x time effect.

Response: Thank you for your valuable suggestions which have helped to improve this paper. The description of the data analysis has been revised in accordance with your advice.

Changes to the manuscript:

The sentence in Result 3.2 “There was also an effect of treatment by time interaction on dry matter, ADF, NDF, and WSC (p<0.05).” has been deleted.

Q5: The chemical composition should be checked because if they are not straws, a maize will have5 % of CP but not 9 % that esun maiz whole plant and should check in detail. The content of moisture dry matter and it is missing to analyse lignin. also if it says that it counts by difference hemicellulose, this is never seen in the manuscript.

Response:

We apologize that we are unable to make substantial revisions to the paper regarding this suggestion. We have confirmed that the determined crude protein content (on a dry matter basis) is indeed around 9%, which we attribute to the low dry matter content of the corn stover used in the experiment.

Q6: In the case of NH3 it is recommended. to present the data in mg/L. and VFAs in mmol/l.

Response:

Dear reviewer, thank you for your suggestion. However, we have doubts about changing the units for NH3-N and VFAs. So far, all the data or literature we have encountered for measuring NH3-N and VFAs in silage feed are in "g/kg DM" units. The current expression is obviously more conducive to evaluating the quality of silage feed, whether compared with the results of this paper or with other relevant research results. Therefore, we did not make changes according to your suggestion, and we hope you can understand.

Q7: Should revise their conclusions. I do not agree. The results showed that the quality of soybean straw alone fermented silage was poor, while the quality of mixed fermentation silage was relatively high due to its high crude protein content, lactic acid content, and low ammonia nitrogen content, because the only presursor of fat in milk is acetic acid with which. SBM straw would increase milk fat production. compared to the other treatments and it has higher PC and the pH below 4.2.

Response:

The quality of silage includes two aspects: one is the quality of fermentation during the ensiling process, which refers to the fermentation quality; the other is the nutritional value of the silage, which is the feed value. In this study, the corn silage group was used as a control group with good fermentation quality during ensiling, while the mixed silage group and the single soybean straw group were compared to evaluate the quality of the mixed silage. From the perspective of feed value, in fact, none of the three silage groups can meet the demand for high-quality silage for dairy cows, but they can be consumed by beef cattle or goats. The nutritional components available for ruminants in silage are very limited, and relatively speaking, the nutritional value of high-protein feed is higher. As for the indicators such as lactic acid, short-chain acids, and ammonia nitrogen in silage, they are used to evaluate the fermentation quality of silage. Generally, a high lactic acid content indicates that lactic acid fermentation dominates during ensiling, and the growth of miscellaneous bacteria and harmful bacteria is well suppressed. Acetic acid in silage is produced by microbial action from lactic acid, and can reduce the number of spoilage yeasts, thereby improving the aerobic stability of silage. The ammonia nitrogen index can translate the degree of protein breakdown in silage, and the higher the relative content, the more protein is degraded and the poorer the quality of silage.

Taking all these factors into account, the mixed silage group has a higher quality than the single soybean straw group, despite its lower crude protein content, due to its higher lactic acid content, lower ammonia nitrogen content, and stable pH value below 4.2 after 60 days of fermentation. Therefore, we conclude that the quality of mixed silage is higher than that of soybean straw group.

Q8: What about the whole bacterial study ? combined analysis of the metabolome and microbial community in the fermented silage ? It does not conclude anything

Response: We included in our conclusion the results of the joint analysis of microbial communities and metabolite profiles. That is “By conducting a combined analysis of the metabolome and microbial community in the fermented silage, a positive correlation was found between manihotivorans, brevis, zeae and significantly down-regulated metabolites such as Pyrocatechol and N-Benzoyltyramine; and a positive correlation was also found between dublinensis, marcescens, agglomerans and up-regulated metabolites such as D-Xylonic acid and Neochlorogenic acid. These identi-fied microbial-metabolite regulatory networks can help in developing better utilization technologies for soybean straw as feed resources.” .

Allow us to explain the reasons behind how we arrived at this conclusion.

The development of efficient silage fermentation bacteria is crucial in silage fermentation. The selection of fermentation bacteria varies greatly depending on different requirements, such as those that can promote fermentation better or those that can improve the aerobic stability of silage. Currently, most of the available fermentation agents were isolated from silage. In the latter part of this study, we aimed to identify one or more beneficial bacteria that could aid in the successful fermentation of soybean straw. As mentioned in the conclusion section, by analyzing metabolite functions and bacterial communities, we have screened several potential target bacterial groups that may have isolatable value (the selection criteria are mentioned in the discussion section). However, since we have not yet completed the experiments on the isolation, identification, enrichment culture, and effect verification of the target bacterial colonies, we have only briefly described our findings in the conclusion section.

Thank you again for your valuable suggestions on improving the quality of this paper.

Reviewer 2 Report

Analysis on Fermentation Quality, Chemical Composition and Bacterial Communities of Corn Straw and Soybean Straw Mixed Silage

This work addresses an important issue and the benefit of using local resources to address the shortage of forage resources. The Introduction well addressed the hypothesis and the necessity of the study. However, the last few line in the introduction is more of M&M section and should be relocated in the corresponding section but not the Introduction (L64-69). Other than this, other sections provided enough details of how the experiment was conducted and the obtained results were discussed in full detail.

The M&M section is presented in details and the statistical approach is logic.

The results are significant interesting and supported by clear figures.

The obtained results are clearly discussed.

The conclusion is to the point.

Please proof-read your text by asking a scientific native speaker.

Author Response

Thank you for your professional suggestion and recognition of this paper. At the end of the introduction, we made some modifications.

The sentence “Single fermentation of corn straw or soybean straw was used as a control, and the changes in nutrient composition were analyzed after 7, 14, and 60 days of mixed fermentation of corn straw and soybean straw at a 1:1 ratio.” has been deleted.

Reviewer 3 Report

#

Soybeans can be harvested as a hay or silage crop. This option is often considered either when forage is short or when the soybean crop is damaged for harvest as a grain crop. Soybean forage, like most legumes, tends to be high in protein and low in fiber (relative to grasses) making soybeans an excellent forage if harvested properly. The use of soybeans in mixtures with corn as ensiling material seems a very good solution. In the work presented for review, the results show the dominance of the bacteria responsible for proper ensiling (Lactobacillus, Klebsiella, Citrobacter, Weissella) in the silages. The simultaneous positive correlation of labeled bacterial genera and species with selected fermentation end products allows to consider silage from a mixture of corn and soybeans to be of good quality. In terms of editing, the work is developed in a very correct manner. The selection and citation of literature is appropriate. The description of tables and figures is appropriate. The discussion of results and discussion are correct.

The authors have not shied away from minor errors, hence my inquiry below:

In paragraph Introduction and 2.1 Silage preparation:

-please specify the method of sampling for analysis, sampling technique and sample weight

-line 66 and 74 is there no error? The word rice or soybean?

In my opinion, with minor corrections, the work can be published in the journal Fermentation.

Author Response

Thank you for your valuable suggestions and recognition of this paper.

(1) The method of sampling for analysis has been supplymented.

“When sampling, the silage bag is completely opened, the silage is fully and evenly mixed in the shortest possible time, the evenly mixed silage is piled into a cone shape and then flattened, the silage is divided into four equal parts from the center of a circle, two parts at any diagonal angle are randomly discarded, the remaining two parts are evenly mixed, and the mixture is continuously evenly mixed and shrunk according to the method until the amount of the remaining sample is close to the required amount for measurement.”

(2) “rice straw” has been changed to “soybean straw”.