Changes in Biochemical and Microbiological Quality of Silage Produced with the Use of Innovative Films

Round 1

Reviewer 1 Report

The introduction is very long and with little scientific information. 

Only from line 107 onwards is the content of interest to the article addressed.

In the material and methods it is not clear the number of repetitions per treatment. The statistical model seems to me inadequate. Perhaps the Dunnett's test is the most suitable.

The p-value is missing in the analyzed variables. 

The silage was made with a mixture of forage. This seems inadequate because the variation in the proportion of these forage species will provide a confounding effect. 

The discussion has to be based on statistics and the tables or graphs there is no information on differences between treatments. The p-value and the different letters ("a", "b"...) for the treatments that differ from each other are essential for understanding the article.

Author Response

We would like to express our gratitude for the review of our manuscript. We believe that the changes suggested by the Reviewer have made our manuscript better. We have addressed all of the concerns raised and provide answers on how we dealt with each comment (the answers are listed below in italics, point-by-point, after each comment). 

• The introduction is very long and with little scientific information. Only from line 107 onwards is the content of interest to the article addressed.

A: The introduction was rearranged and rewritten. Fragments of the text regarding the ensiling process were removed or significantly reduced. The whole paragraph dealing with ensiling film recycling was added, as expected. We hope that in the present form it will be of interest to the readers. Thank you.

• In the material and methods it is not clear the number of repetitions per treatment. The statistical model seems to me inadequate. Perhaps the Dunnett's test is the most suitable.

A: The information about the number of repetitions per treatment was added in the paragraph 2.1 (“Having transported to the storage place … in at least 16 repetitions for each film” and later “Every time of testing, for each experimental variant analyzed at both stages, four bales were opened for sample collection”).

Thank you for the suggestion about the Dunett’s test, but since the control film parameters were never evaluated in this type of experiment, we have chosen the Tukey’s test to be applied as it is here more adequate and enables to treat the control film as one of the tested variants. The Tukey test also provides information about differences between variants, which was indeed our intention. In addition, the Dunnet’s test compares every mean to the control mean, so it requires one mean value as a control (we could not compare the changes in time for all the variants including the control one). It may be used in cases when no significant changes in time are observed for the tested parameters (i.e. for total sugar (see t. 3), and one mean value for control can be indicated, but we decided to keep the same model and assumptions for analyses of the whole experiment.

• The p-value is missing in the analyzed variables. 

A: The information about p-value was previously mentioned only in the par. 2.5. Now, for each figure and table (whnever relevant) the information about the p-value was added. Thank you.

• The silage was made with a mixture of forage. This seems inadequate because the variation in the proportion of these forage species will provide a confounding effect. 

A: Thank you for your concern. The presented experiment was a part of complex field studies, composition of forage was dependent on the meadow condition, but still kept as constant as possible. The material was mixed carefully to provide similar conditions in each prepared bale, so that it would be possible to compare the parameters of silage between bales from the same stage of experiment. Because in the second season the botanic composition changed slightly we cannot compare the results of both seasons – we had to analyze the control sample parameters again.

• The discussion has to be based on statistics and the tables or graphs there is no information on differences between treatments. The p-value and the different letters ("a", "b"...) for the treatments that differ from each other are essential for understanding the article.

A: The statistical descriptors to the graphs and tables were calculated and added together with the information in figure descriptions. Only in single case we have left the data unmarked: where adding descriptors was less relevant and would negatively impact the readability of the chart (cf. the pH changes in stage one; in this graph the most important observation is that the pH did not exceed 6-7, so the conditions for proper ensiling were kept). Thank you.

We appreciate all the Reviewer’s suggestions and comments.  Thank you. We hope that the present, improved version has met the expectations of the Reviewer.

On behalf of the Authors

Yours sincerely,

Paulina Supel, PhD

Reviewer 2 Report

Dear Authors, 

Thank you for your contribution for science! The paper has significant content for the process of silage making but also for the environment, as the recycling part is of high importance. The biggest weakness of this paper is the statistical part. Explanation of statistics is insufficient throughout the manuscript and should be significanlty improved. The paper is written in good english and language wise, is very easy to follow. See below my comments in detail. 

 

INTRODUCTION

Introduction should be brief. In this case the introduction is too long, focusing too much on the basics of silages. Although this is nice, it loses purpose of this section, that is to BRIEFLY place the study in context, define purpose of work and hypothesis. I suggest that most of the theoretical content is removed or significantly shorten. Specially from line 36 to 138.

Suggest the intro focus on the actual silage problematic of this work: microorganisms, and plastic recycling. Leaving outside the processes of ensiling.

In the introduction I would like the authors to explicitly write the hypothesis, what are the authors’ expectations of this work. This will allow the writers to prepare for what is coming.

 Although bales are popular, bunker silos is significantly popular in a lot of countries, or the most common what of conservation. I suggest to re write the statement or specify the region/country you are referring to. (Line 12 in abstract and line 40 intro)

 

MATERIALS AND METHODS

Lacks explanation of number of bales, bales per treatment, etc

144- specify country

148: dried or fresh mass

151: was it wilted? Mention drying process

156- is a bit unclear the timeline of the analysis. Experiment 2 was modified based on experiment 1, but analysis of exp 2 were done in month 4 and of exp 1 on month 5? Is it months after exp 1 was finished? But be great if you could re write this part to be more clear for readers.

162- Include country

176- specify how the recycling process was done.

178-which layer is the recycled one in PR2.

205- specify the standard drying procedure, there are several standards.

214- statistical description is too short. Please describe in more detail. For example, treatments, repetitions, etc

 

RESULTS AND DISCUSSION

Also statistics need to be included, in text and figures. A lot of statements are done without statistical backup.

218- This paragraph belongs to materials and method. Also, specify what long-term storage means for the authors.

224- mentions studies in plural but only cites one. Please more citations if you state than more than one studied LAB.

225-232 authors state than “most studies”. Find this vague, as figure 1 shows a lot of treatments with no LAB in t3.

235-P6 is different from p7? If so, please use statistics in text and figures

235- why p3, p4, p5 have no LBA in t3 but authors stay that they have high LAB, does something happened to those bales at t3? Please explain

232- why there is no info (or zero) of p1 in t2 in figure 1, please explain

252- p1 shows no lactic acid bacteria in internal layer and low colonization on external, why authors state it as best result?

279- why does control have high pH in t2 and then down, evaluate if is it worth the mention

286- please use statistics in tables and text. Looking at table 1, is there not a difference I sugar between p1 and p3?

319- which is that treatment, specify code please, very hard to follow for the reader. By looking at the treatments, no one mentions type 3 micro cellulose in the internal payer. Regardless, it seems that none of the treatments showed a significant difference with the control (p7). Please explain your statement.

335- biofilm? Specify

 

LACKING:

Review of the recycling. In the introduction the authors mention the environmental impact of recycling. However, no mention of this topic in the results, which is of significant importance for the agricultural sector. An analysis of using of recycled from an environmental and economical perspective would add significance to the paper.  Also, it is important to discuss the recycling method under practical conditions. Is it viable?

Significance: A strong emphasis on the statistics should be include across the paper, and this include text, tables and figures. Please, include p values or codes (such as *) when there are significant differences. This is a strong requirement from my side.

Figures and Tables:

Suggest including treatment codes (what each treatment mean) under all tables and figures. It is very hard to follow many treatments only by the code.

Table 1 and 2: too much information and difficult to follow! Suggest merging them in a single table to evaluate both at the same time.

Citations: 8 out of 32 are less than 5 years old. Suggest to include newer papers. 

Author Response

We would like to express our gratitude for the review of our manuscript. We believe that the changes suggested by the Reviewer have made our manuscript better. We have addressed all of the concerns raised and provide answers on how we dealt with each comment (the answers are listed below in italics, point-by-point, after each comment). 

Thank you for your contribution for science! The paper has significant content for the process of silage making but also for the environment, as the recycling part is of high importance. The biggest weakness of this paper is the statistical part. Explanation of statistics is insufficient throughout the manuscript and should be significanlty improved. The paper is written in good english and language wise, is very easy to follow. See below my comments in detail. 

A: We are very thankful for this comment. We have made some improvements, and the details are listed below. Thank you.

• INTRODUCTION: Introduction should be brief. In this case the introduction is too long, focusing too much on the basics of silages. Although this is nice, it loses purpose of this section, that is to BRIEFLY place the study in context, define purpose of work and hypothesis. I suggest that most of the theoretical content is removed or significantly shorten. Specially from line 36 to 138.

A: The introduction was rearranged and rewritten. We hope that in this form it will be of much more interest to the readers. Thank you.

• Suggest the intro focus on the actual silage problematic of this work: microorganisms, and plastic recycling. Leaving outside the processes of ensiling.

A:. Fragments of the text about the ensiling process were removed or significantly reduced. The paragraph on the ensiling recycling was added. Thank you for this suggestion.

• In the introduction I would like the authors to explicitly write the hypothesis, what are the authors’ expectations of this work. This will allow the writers to prepare for what is coming.

A: Thank you for this advice. The Introduction was modified. Now the last paragraph brings aims and hypothesis: “The present study was focused on monitoring microbial population changes and silage parameters during the storage of ensiled forage in bales wrapped with modified PE film containing different innovative additives. Also, the possibility of use of the recycled PE was considered. The main research hypothesis was that the films containing tested additives might be used in silage fermentation, providing proper conditions for the process”.

• Although bales are popular, bunker silos is significantly popular in a lot of countries, or the most common what of conservation. I suggest to re write the statement or specify the region/country you are referring to. (Line 12 in abstract and line 40 intro)

A: The region was added in the Abstract. In the introduction there is information referring to the region (“The silage preparation has been gaining popularity in Europe”), so we have not change this part.

• MATERIALS AND METHODS: Lacks explanation of number of bales, bales per treatment, etc.

A: The information about the number of repetitions per treatment was added in the par. 2.1 (“Having transported to the storage place … in at least 16 repetitions for each film” and later “Every time of testing, for each experimental variant analyzed at both stages, four bales were opened for sample collection”). Thank you.

• 144- specify country

A: The information was added; also, later in par. 2.2 we specified the country while providing the names of companies. Thank you.

• 148: dried or fresh mass

A: The composition was assessed in the dry mass. The relevant information has been added to the text. Thank you.

• 151: was it wilted? Mention drying process

A: The information about the drying process has been complemented: “the forage was dried to about 50% content of dry mass (DM) with the method of wilting on swathes”. Thank you.

• 156- is a bit unclear the timeline of the analysis. Experiment 2 was modified based on experiment 1, but analysis of exp 2 were done in month 4 and of exp 1 on month 5? Is it months after exp 1 was finished? But be great if you could re write this part to be more clear for readers.

A: Thank you for this suggestion. In fact, these data needed to be specified. Now, within the text the relevant information is included: “The second experiment, conducted during the next season, was modified considering the previous results”

• 162- Include country

A: The information added. Thank you.

• 176- specify how the recycling process was done.

A: Since we have added a whole paragraph on the film recycling process, in the Introduction section, par. 2.2. we have kept the information: “The recycled PE of the middle film layer, used in the second experimental stage was prepared as described in the Introduction section.” We hope this will find your expectations. Thank you.

• 178-which layer is the recycled one in PR2.

A: Only PR1 variant contained the layer made of the recycled PE. The PR2 variant contained the addition of nanosilver, but no recycled PE. This way we were able to test all the possible combinations: with nanosilver in the external layer (PR1 and PR2) vs. without nanosliver admixture (PR0), with the recycled PE in the middle layer (PR1) vs. standard PE (PR0, PR2). We have changed the description of the variants since we agree that it might have been unclear in the previous version. Thank you.

 

• 205- specify the standard drying procedure, there are several standards.

A: The information about drying method was added in brackets: “(24h at 105°C)”

• 214- statistical description is too short. Please describe in more detail. For example, treatments, repetitions, etc

A: Thank you for this suggestion. The paragraph was rewritten, the information about repetitions was also added in the preceding paragraphs. Now, it reads: “The results were analyzed with Statistica 13.0 (Statsoft, USA) software. Two-way ANOVA followed by post-hoc Tukey test was used to evaluate differences at p< 0.05. If no statistical differences in changes of a parameter in time were observed for the sample, the “time” variable was neglected and one-way ANOVA was performed. Four biological repetitions (objects) were analyzed for each experimental combination (group).”

• RESULTS AND DISCUSSION: Also statistics need to be included, in text and figures. A lot of statements are done without statistical backup.

A: The statistical descriptors to the graphs and tables were added together with the information in figure descriptions. Only in single case we have left the data unmarked: where adding descriptors was less relevant and would negatively impact the readability of the chart (cf. the pH changes in stage one; in this graph the most important observation is that the pH did not exceed 6-7, so the conditions for proper ensiling were kept).Thank you.

• 218- This paragraph belongs to materials and method. Also, specify what long-term storage means for the authors.

A: Thank you for this suggestion. The paragraph was moved to the section Methods (par. 2.1.). The information about storage time was added: “ The ensiled material analyzed in this study was subjected to a long-term storage, i.e. 17 months for the first stage and 10 months in the second experiment, and all the obtained results are presented compared to the forage fermented with the use of a standard wrapping film.”

• 224- mentions studies in plural but only cites one. Please more citations if you state than more than one studied LAB.

A: The cited paper, Avila and Carvalho 2019, is a review article summarizing many years and experiments about silage fermentation, therefore “studies” were mentioned. To make this message clear, while citing Avila and Carvalho we have added [5 and the references therein].

• 225-232 authors state than “most studies”. Find this vague, as figure 1 shows a lot of treatments with no LAB in t3.

A: The information was clarified. Now it reads: “In our experiment we show that in most of the studied variants, i.e. P1, P5, P 6 and control (P7), the number of LAB still tended to increase during the storage.”

• 235-P6 is different from p7? If so, please use statistics in text and figures

A: The sentence has been rewritten and we hope now it sounds clearer: “Among the tested variants, formula P6 seems to fulfill the above requirements, especially due to the high LAB number after 17 months, although there were no statistical differences between P6 and the control sample, P7.”

• 235- why p3, p4, p5 have no LBA in t3 but authors stay that they have high LAB, does something happened to those bales at t3? Please explain

A: Since silage is a very difficult research material for microbial evaluation due to the problems with proper sample collecting, handling, storage and transport, many problems with analyses may occur. We have added this information in the text together with the relevant citation of Kung et al. (2018), where the authors deal with the subject in detail (“Silage review: Interpretation of chemical, microbial and organoleptic com-ponents of silages. J. Dairy Sci., 2018, 101): “For sample P1 after 10 months of storage no microbial presence in silage was observed, which may result from inappropriate sample maintenance before analysis. Silages are very often problematic samples with the high risk of compromising before the analysis in the laboratory. Therefore the possible differences between the result and real composition of the sample right after collection may occur and should be considered [29].”

• 232- why there is no info (or zero) of p1 in t2 in figure 1, please explain

A: Please see the comment above.

• 252- p1 shows no lactic acid bacteria in internal layer and low colonization on external, why authors state it as best result?

A: Thank you for this comment. In fact, in the previous version, the information we wanted to state was unclear. Now, the rewritten part is: “While analyzing the foil internal surface microbial colonization, the most important observed parameter was low frequency of molds combined with the presence of LAB in the silage. It was assumed that the innovative additives should inhibit the growth of unfavorable strains on the film and, at the same time, remain non-toxic to the lactic acid bacteria in the material.”

• 279- why does control have high pH in t2 and then down, evaluate if is it worth the mention

A: Thank you for this question. In fact, the pH value for sample P1 in t2 was statistically significantly higher than in other samples, although in this type of samples it is not important as long as acidic conditions are kept. The general change in time is more important, and in all the samples pH was kept acidic and still tended to decrease. Also, for the abovementioned reason, we have not included the statistical significance descriptors on the graph. We also did not want to decrease readability of the graph.

• 286- please use statistics in tables and text. Looking at table 1, is there not a difference I sugar between p1 and p3?

A: Thank you for this suggestion. The statistical descriptors were added to the values. In  fact, the only important difference in this table regards the sugar content between samples P1 and P3. However, both values are desired – we can see that for every sample a decrease in sugar content during the ensiling was observed from the initial level, 14,75%, as mentioned in the text.

• 319- which is that treatment, specify code please, very hard to follow for the reader. By looking at the treatments, no one mentions type 3 micro cellulose in the internal payer. Regardless, it seems that none of the treatments showed a significant difference with the control (p7). Please explain your statement.

A: Thank you for this comment. The mistake was corrected – the microcellulose was added to the middle, not internal layer. Also, following your suggestion we have changed descriptions of figures, tables and in the text (marked control in each stage, changed time descriptions). Now the information should be clear and easier to follow for the reader.
Note that we did not really want the tested formulae to be better than standard one – in fact we have expected them to be worse or similar; still applicable when combined with better recycling properties and parameters. The main aim of our research was to check if the additives anyhow negatively affect the fermentation process and post-fermentation biochemical changes in silage. During another part of the conducted Project, not described in this paper, physical parameters of films and possibilities of recycling after use were tested and compared with the results obtained in this research.

• 335- biofilm? Specify

A: Information was specified. Now it is: “any undesired microbe adhesion on the tested films should be limited”. Thank you.

• LACKING: Review of the recycling. In the introduction the authors mention the environmental impact of recycling. However, no mention of this topic in the results, which is of significant importance for the agricultural sector. An analysis of using of recycled from an environmental and economical perspective would add significance to the paper.  Also, it is important to discuss the recycling method under practical conditions. Is it viable?

A: Please check the Introduction section. Following your suggestions this section was rearranged, modified and rewritten. We hope that in presented form it will  find your expectations. Thank you for this valuable remark.

• Significance: A strong emphasis on the statistics should be include across the paper, and this include text, tables and figures. Please, include p values or codes (such as *) when there are significant differences. This is a strong requirement from my side.

A: Thank you for your suggestion. These changes have all been  made, we have added codes (descriptors) in the figures, tables and included these changes in the text. 

• Figures and Tables:Suggest including treatment codes (what each treatment mean) under all tables and figures. It is very hard to follow many treatments only by the code.

 

A: Thank you. We have made changes including specifying the control in each figure, table and text. We decided not to add full treatment information in captions of each table and figure since that would be too much text and we thought it would worsen the clarity of the paper. However, we decided to change the information about time and replace the “t” code into the actual “months”. We hope that now the information is clear to the reader and paper is easier to follow.

• Table 1 and 2: too much information and difficult to follow! Suggest merging them in a single table to evaluate both at the same time.

A: Thank you for this suggestion, the tables were merged into one (Table 1).

• Citations: 8 out of 32 are less than 5 years old. Suggest to include newer papers. 

A: Since the whole introduction section was rewritten, also the citation list has changed. Now it brings 33 relevant papers, while among them 15 were published after 2017. Thank you for this comment.

We greatly appreciate all the Reviewer’s suggestions and comments and are thankful for the effort leading us to improve the article. We hope that the current version has met the expectations of the Reviewer.

 

On behalf of the Authors

Yours sincerely,

Paulina Supel, PhD

Round 2

Reviewer 1 Report

Please delete the first paragraph of introduction. This information is unnecessary. 

All suggestions made previously have been carried out or justified.

Author Response

Dear Reviewer,

We are glad that our work and corrections have met the Reviewer's expectations. We appreciate all the comments and suggestions. Please find below our answers to the last issue raised by the Reviewer (the answers is in italics, after the comment). We hope that after the second round of the revision the manuscript is flawless and ready to be published.

On behalf of the Authors

Yours sincerely,

Paulina Supel, PhD

Answer to the Reviewer's comment:

Please delete the first paragraph of introduction. This information is unnecessary. 

A: We have added this information following the formal requirements for the authors participating in the IECAG conference, as listed in the instruction for authors (https://www.mdpi.com/journal/agronomy/instructions#preprints, p. 2). In the letter to the Editor we have pointed out the Reviewer’s suggestion and asked for final decision; if the Editor confirms this paragraph unnecessary we will delete it.

Reviewer 2 Report

Dear Authors, 

The paper has improved significantly. Thank you for taking the time to improve this considerably. However, there are still some items that can be improved. 

1.Abstract: Consider changing "the common" to "a common". as i said, many countried use bunker silos. 

2.Introduction: I appreciate the reduction, but still needs to be significantly reduced (cannot be longer than one page!). Remember that the introduction is for "introducing" the reader to the problem, not to review the whole silage making. Some suggestions on how to do it (hope this helps): 

- From line 48-76: reduce the long explanation, set the problem. 

-recycling process to materials and methods. in introduction just explain the recycling problematic

-93 to 102: could be places earlier (with lines 48-76) and reduced the whole explanation. 

 

-Very nice included hypothesis! thank you, this makes the reader ready to face the results. 

 

3. Materias and methods: 

- 121: does repetitions means bales; if so, cinlude bales in parenthesis. 

-now explanation on 1st and second experiment is clear to me, but as stated before, i will include the recycling process here and not in the intro. 

RESULTS: Table 1: still difficult to read. When i suggest the merging, I was hoping information will be merged and not just tables pasted together. Sugest to include in each cell the info on external and internal layer. for example: use * for external layer, and other icon for internal (f. ex. +). I think the reader will benefit of having the info together in one cell, as this is how it is dicussed in the paper. 

- significantly improved the recycling section. Thank you. 

 

CONCLUSION: 

-Compare results of the innovatives to control, is it actually worth it to have the innovations? or have the same results of control?

 

REFERENCES

Thank you for updating the references with newer papers!

 

I appreciate the changes and improvement, 

Reviewer 1

Author Response

Dear Reviewer,

We are glad that our work and corrections have met the Reviewer's expectations. We appreciate all the comments and suggestions. Please find below our answers to the last issues raised by the Reviewer (the answers are listed in italics, point-by-point, after each comment). We hope that after the second round of the revision the manuscript is flawless and ready to be published.

On behalf of the Authors

Yours sincerely

Paulina Supel, PhD

1.Abstract: Consider changing "the common" to "a common". as i said, many countried use bunker silos. 

A: The change was made, as suggested.

2.Introduction: I appreciate the reduction, but still needs to be significantly reduced (cannot be longer than one page!). Remember that the introduction is for "introducing" the reader to the problem, not to review the whole silage making. Some suggestions on how to do it (hope this helps): 

- From line 48-76: reduce the long explanation, set the problem. 

-recycling process to materials and methods. in introduction just explain the recycling problematic

-93 to 102: could be places earlier (with lines 48-76) and reduced the whole explanation. 

-Very nice included hypothesis! thank you, this makes the reader ready to face the results. 

 A: Thank you for pointing out to the above suggestions. In fact, reducing of the introduction was difficult since each paragraph was already significantly reduced and rephrased, earlier. Still, we have moved the details about recycling to the Materials and Methods section. We have also changed some sentences in order to shorten them. Now the Introduction chapter is only a few lines longer than one page of the manuscript (not including the first paragraph, which was mandatory).

3. Materias and methods: 

- 121: does repetitions means bales; if so, cinlude bales in parenthesis. 

-now explanation on 1st and second experiment is clear to me, but as stated before, i will include the recycling process here and not in the intro. 

A: The information about bales was included, and the part about recycling was moved from introduction to this section.

RESULTS: Table 1: still difficult to read. When i suggest the merging, I was hoping information will be merged and not just tables pasted together. Sugest to include in each cell the info on external and internal layer. for example: use * for external layer, and other icon for internal (f. ex. +). I think the reader will benefit of having the info together in one cell, as this is how it is dicussed in the paper. 

 A: We have finally managed to merge the tables as suggested, using different symbols as indicated. We hope the Reviewer will now confirm that the current version of data presentation looks better and is easier to follow. Thank you for this comment.

CONCLUSION: 

-Compare results of the innovatives to control, is it actually worth it to have the innovations? or have the same results of control?

 A: As we mentioned in the conclusions, the results obtained for innovative additives (esp. nanosilver) were similar or in some cases better than for control. For the recycled PE loss of quality is observed after longer storage, so we suggest to open and fed-out bales after no longer than few months. However, the reduced production costs and a positive environmental impact are strong arguments supporting the use of modified films in practice. Therefore, this last sentence was added as a last concluding statement at the end of conclusions.