Effect of Low-Temperature-Tolerant Lactic Acid Bacteria on the Fermentation Quality and Bacterial Community of Oat Silage at 5 °C vs. 15 °C

Round 1

Reviewer 1 Report

Dear Authors,

You did an interesting study on LAB strains potentially suitable as ensiling additives for low temperature conditions. However, unfortunately, you did not describe how you selected those. The M&M part has to be improved and the results extended. The second point is, as you selected for oat forage, which you ensiled at extremely low DM contents, the application of LABs is questionable per se. Please look up literature. Usually, you then have to use chemical preservatives to prevent butyric acid fermentation. And you produce a lot of silage effluent. Please justify your approach. Have a look at further comments in the text.

Kind regards.

 

Comments for author File: Comments.pdf

Author Response

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Reviewer 2 Report

Line 63: The species belonging to these are the ones indicated lately in lines 82-83?

Line 74: I don't understand this data not shown, as title of a paragraph!!!!!!!

Lines 75-83: I think that this paragraph, as previous story of "2.1. Isolation of LAB from Oat Silage " should be included in the pargraph 2.1.

Lines 89-90: DNA Extracted with a kit or other methods, please explain this part.

Lines 94-99: I think that the DNA was used as template for the amplification......etc. But authors should explicate what they amplified by PCR, and to what primers they refer.....

Lines 114-115: In line 63 you affirmed that the concentration was 1x10⁵ CFU/g, here is 1x10⁶. Please check it.

Lines 139-140: Why only 20 microliters, and which media? you talk about MRS before, are there other media?

Lines 142-143: Why 37°C and not 30? And why 28 °C and 37 °C? I don’t understand

Lines 157-158: Butyric acid content is very variable among the different treatments. Authors should explain why? Or postulate it.

Line 178: Table 2, maybe?

Table 1: Here in some values the upper letters are missing, eve n with very different numbers, such as PA, BA, NDF, and ADF

Line 193-194: How do you explain this behavior?

Figure 2: At 5 °C there is less biodiversity that at 15 °C. And another question, how do you explain that the strains of Leuconostoc mesenteroides inoculated, are not present anymore?

Line 220: Weissella cilbaria: Are you sure about the correlation of this strain with temperature?

Figure 4: Considering the heatmap, it would be interesting to offer a hypothesis about the positive and negative correlations.

Line 243: FO3 is a single strain or a mix of strains, sincerely this passage it is not clear to me. From the line 63, it was supposed that FO3 belongs the L. mesenteroides species.

Lines 270-274: My curiosity is still about L. mesenteroides. Indeed, considering that FO3, FO5 and FO8 were identified as the aforementioned species, it is at least strange that this species totally disappeared in figure 2. Could you please explain it?

Line 293-297: same question as before, no L. mesenteroides anymore?

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The aim of the study was to investigate the effects of some LAB inoculants on the fermentation quality and bacterial community of oat silage. The selection of bacterial strains chosen for the study was not clearly presented but authors claim that strains tested were low temperature - tolerant microorganisms. All of them were classified in Leuconostoc genera. Additionally L. plantarum was also investigated. Although the aim of the study is clear and worth investigation, I see some basic not understandable choices. I do not know why only Leuconostoc strains were used? What for was applying L. plantarum?  And mainly - why silage after 60 days of fermentation contained mainly Lactobacillus species and this was not significantly influenced by bacterial inoculation on the start? Did you manage to describe the microbiota of oat before ensiling? I think it could be crutial.

Morover, I have some other comments:

1. It does not look well when showing unclassified species in absract, it is better to write Lactobacillus sp.
2. There is a new nomenclature for LAB species, please revise it.
3. In what form bacteria were added to the silage?
4. line 141 - De Man ... not Deman
5. Please describe in materials and methods section what is Shannon index, the Simpson index etc.
6. Describe abbreviation OTU
7. Figure 1 - please describe time of ensiling
8. Table 2 - please explain "Alpha diversity".
9. Please write species names in italics
10. Figure 4 - delete heading "Spearman correlation .." above the figure.
11. Please improve conclusions relating to changes in genera subpopulations in silage.

Author Response

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Round 2

Reviewer 1 Report

Dear Authors,

although you have addressed most of the comments which you found in the manuscript you have not yet considered the overall statement which I gave last time. As this wet oat silage is highly prone to butyric acid fermentation you have to take into account all aspects around very low dry matter silages and not just the low temperature! You have to characterize the fresh forage material before ensiling. Consider for example the equation of Weissbach et al. (1974) on when to expect a anaerobically stable silage: DM = 450-80*WaterSolubleCarbohydrates*BufferingCapacity (G. Pahlow, R. E. Muck, F. Driehuis, S. J. W. H. Elferink, and S. F. Spoelstra. Microbiology of Ensiling. In: Silage Science and Technology, edited by D. R. Buxton, R. E. Muck, and J. H. Harrison, Madison, Wisconsin, USA:American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., 2003, p. 31-93). Please also look up articles on butyric:lactic acid ratio in silage and evaluate your results accordingly. Please reflect your results in a broader context.
The language must be revised by a native English speaker with agricultural background.

Kind regards

 

Comments for author File: Comments.pdf

Author Response

Thanks for your kind attention! 

We have revised this manuscript according to your suggestions, please see the red words in the attachment.

Best regards!

Author Response File: Author Response.docx

Reviewer 2 Report

I think that this version of the manuscript, and due to the replies of the authors, better responds to my inquiries.

Author Response

Thanks for your attention!

Reviewer 3 Report

thank you for providing all my suggestions

Author Response

Thanks for your attention!