Simple Sugars Alter the Odorant Composition of Dairy Cow Manure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper presents evidence of a potential odor reduction in manure by adding sugars to manure slurry.

While the idea of influencing gut microbiota through diet is not new, this application is original. However, the authors discuss "odor" and "malodor" without providing direct evidence, as they only measure odorants, chemical compounds. In all the manuscript, there are no indications of olfactory thresholds of compounds analysed or olfactometric data. For these reasons, I do not think the manuscript is acceptable in its present form. It suggests a potential odor reduction without supporting data.

The manuscript should be completely revised considering this issue, including the title, or olfactometric data should be included.

Author Response

We are sorry if we gave the impression that we reduced the odor of the manure. Rather we feel that addition of sugar to the manure altered the odor by reduction in the concentrations of typical manure malodorants and increases in the concentrations of volatile organic compounds such as ethyl butyrate and propyl propanoate. We give no olfactory data. We did not measure the concentrations of the aroma compounds in the gas phase but in the aqueous phase. The aroma of the manure was improved somewhat by the addition of the sugars having a distinctive fruity note and as per Reviewer three’s comments, we performed statistical analyses on the aqueous phase concentrations of the volatile compounds.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe the potential of reducing malodours from cows when adding sugar to the manure. The paper is well written and certainly interesting for readers. The manuscript can be improved further adressing the following points:

1. The incubation period of four weeks needs more explanation.

2. All tables show a partially non-linear dependance between  the concentrations of added sugars and the other variables. This is not well discussed. Are these complex dependancies expected? Can they be explained?

3. Structure of the manuscript: The short Section 4 should be entitled "Conclusions" instead of "Discussion". A discussion section should be included clarifying the issues raised under point 2. The current section 4 contains one sub-section. Were there other sub-sections planned?

Author Response

1. We added a justification for the incubation period of four weeks to the Material and Methods section (L. 117-121)
2. We can’t explain the non-linear dependence for the effects of sugar on variables such as pH, production of SCFA and concentrations of short chain fatty acids except to speculate that it was due to shifts in the microbiota of the slurries. At this point this would be speculative, however, we do speculate on this in the Conclusion section (L 298-304).
3. We changed the title of the Discussion section to Conclusions as the ‘Results’ section did have a good deal of discussion in it and of course changed the title of section 3 to Results and Discussion.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes an experiment evaluating the addition of simple sugars to cattle manure on the production of malodorants in vitro. An interesting experiment although I am not sure of practicality of adding sugars to manure at scale.

I have 2 major concerns that must be addressed before this article can be published.

 

1. The description of the incubation conditions and preparation of the manure is inadequate for the reader to fully understand the results and for other researchers to repeat the experiment.

2. There is no statistical analysis of the data. An appropriate statistical analysis must be completed.

Specific comments are in the attached PDF.

Comments for author File: Comments.pdf

Author Response

1. We added more experimental details on lines 117-121.
2. We performed statistical analysis, mean comparisons of the volatile odor compounds, for Tables 3-5. We did not perform mean comparisons for the final pH, biogas production, or short chain fatty acids because we feel these are of limited scientific value due to these effects of sugar fermentations have been known for many years.

Regarding comments made on the manuscript by reviewer #3:

 

Originally, we had the pH data in table 1 before making Figure 1. We regret the error and have corrected the caption.

We added the fact that the lines represented a polynomial regression (exponential decay) model and added the square of the correlation coefficient for each fit.

On lines 227 we talk of p-cresol and skatole because this is where the most obvious changes occurred, we do mention the effect of glucose addition on dimethyl disulfide concentrations And added a sentence noting that glucose addition had little effect on phenol concentrations (l. 243).  

We define the abbreviation LAB on line 220 for its first use and have included in the list of abbreviations at the end of the manuscript.

Neither indole nor skatole were detected in manure amended with 26.3 or 65.8 g/L of lactose.

On lines 273 I added percentages of ester production for ethyl butyrate and propyl propionate.

Regarding the question on line 196, we have no explanation for the variation between trial controls but have acknowledged the fact the different manure samples we used for each experiment. (lines 119-121).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The response of the author is not satisfactory. I understand that they did not perform olfactory analysis, it is clear. But they still refer to “odors” in several points in the manuscript.

“We are sorry if we gave the impression that we reduced the odor of the manure. Rather we feel that addition of sugar to the manure altered the odor by reduction in the concentrations of typical manure malodorants and increases in the concentrations of volatile organic compounds such as ethyl butyrate and propyl propanoate. We give no olfactory data. We did not measure the concentrations of the aroma compounds in the gas phase but in the aqueous phase. The aroma of the manure was improved somewhat by the addition of the sugars having a distinctive fruity note and as per Reviewer three’s comments, we performed statistical analyses on the aqueous phase concentrations of the volatile compounds.”

 

My main point was in the use of the word “odor” in the text. I attach here, as an example, an extract from a letter to Chem. Senses 25: 693, 2000, that I fully support, in order to better explain my position:

 

“Odorants are molecules, properties of the external world objectively definable in terms of their physico-chemical characteristics and capable of being transposed by particular nervous systems into odors. Odors, on the other hand, are the products (constructs if one will) of nervous systems and thus potentially open to the many modulating influences of what might be broadly thought of as ‘mind’. Thus, when referring to olfactory phenomena prior to and including reception of molecules at the sensory surface we are dealing with odorants and in the chain of subsequent events, whether consciously perceived or not, with odors.”

 

As an example, in this reference the authors deal with the same points in this manuscript, I suggest to cite it, and they use the correct wordings when referring to the hedonic characteristic: “Correlation of odorant concentration was closely associated with the origin of the odorant in the diet.”

Swine odor analyzed by odor panels and chemical techniques. DOI: 10.2134/jeq2010.0522

 

For these reasons I still think the manuscript is not acceptable for publication in its present form.

 

 

Other points:

 

The Title:

Improve the Aroma of Manure …  I am not I feel that improving the manure “aroma” is not reasonable. May be  “I'm not a native English speaker, but it seems to me that improving the aroma of manure is an unreasonable phrase. Perhaps “changing” the aroma fits the scenario better?

 

Legend to fig. 1 Data represent the mean of four determinations ± standard deviation of the mean. Within rows, means followed by the sample letter are not significantly different by a Duncan’s multiple range test at p = 0.05.

It is not clear. What is the “sample letter”? Which are the data significantly different?

 

Short Chain Fatty Acids. Why formic acid has not been detected?  In this publication http://dx.doi.org/10.1016/j.biosystemseng.2013.11.004,  formic acid was dominant in the manure. The authors should discuss this.

The manure samples are solid or liquid? The authors refer to mL or L ? It has been diluted with water? How much? Or is just undiluted manure slurry. It should be clarified in the text.

Line 249: This, accompanied by the decreases in manure malodorants, imparted a much less offensive odor to the manure slurries.

 

How did the authors defined that the odor is less offensive if no olfactory data has been collected? Suggest to remove the sentence and modify the text.

Line 274: Sucrose also greatly affected the odor profile.

Same as before.

Line 294: It was noted that the manure slurries amended with higher concentrations of all three sugars had a less intense manure like odor and more of a fruit like character.

 

Same as before

 

Author Response

We included the editorial reference by Hudson to address that the semantic difference between odor and odorant and also included the article by Trabue et al. as reference #8. We also used the term odor less often and used the terms odor, odorant and malodorant instead. In some cases, odor was used colloquially as commonly used in speech and text and in most cases used the expression odor (composed of individual odorants) profile in addressing the fact the odor profile of the manure was undeniably altered while trying to be less subjective.  

We hope that we are more clearly saying that the addition of sugar to manure alters the composition of odorants emitted by the manure.

We discussed hedonic tone and odor thresholds of the compounds somewhat on lines 308-321. This discussion is somewhat superficial, but given the nature of the study, hope it will be sufficient.

 

Other points:

 

The Title:

Improve the Aroma of Manure …  I am not I feel that improving the manure “aroma” is not reasonable. May be  “I'm not a native English speaker, but it seems to me that improving the aroma of manure is an unreasonable phrase. Perhaps “changing” the aroma fits the scenario better?

 We agree, improve is subjective. We changed it to “alter”.

 

Legend to fig. 1 Data represent the mean of four determinations ± standard deviation of the mean. Within rows, means followed by the sample letter are not significantly different by a Duncan’s multiple range test at p = 0.05.

It is not clear. What is the “sample letter”? Which are the data significantly different?

 We deleted the word sample in the table footnote in tables 3,4, and 5 and replaced in by the word same.

Short Chain Fatty Acids. Why formic acid has not been detected?  In this publication http://dx.doi.org/10.1016/j.biosystemseng.2013.11.004,  formic acid was dominant in the manure. The authors should discuss this.

Formic acid was not detected in our experiments nor is it  product of bacterial fermentation per se but more usually formed methylotrophs or by the cleavage of pyruvate to produce formate and acetyl-CoA. Also, per definition, does a one carbon compound form a chain.  We added that we found no formate in our manure slurries (Line 207).

The manure samples are solid or liquid? The authors refer to mL or L ? It has been diluted with water? How much? Or is just undiluted manure slurry. It should be clarified in the text.

Lines 116-117 show that 50 milliliters of solid cow manure were diluted to a final volume of 100 mL. We rewrote this slightly to make it clearer.

Line 249: This, accompanied by the decreases in manure malodorants, imparted a much less offensive odor to the manure slurries.

 

We reworded the sentence to make it less subjective.

 

How did the authors defined that the odor is less offensive if no olfactory data has been collected? Suggest to remove the sentence and modify the text.

We modified it by simply saying it altered the odor. As in the reference by Trabue et al. it would seem reasonable to assume to have lowered the offensiveness of the manure by lowering the concentrations of compounds such as skatole.

Line 274: Sucrose also greatly affected the odor profile.

We changed the expression odor profile to odorant profiles and made more qualitative statements about the perception of the sugar amended manure. We also changed most instances of the word aroma to odor.

Same as before.

Line 294: It was noted that the manure slurries amended with higher concentrations of all three sugars had a less intense manure like odor and more of a fruit like character.

 

Same as before

We changed the wording to simply state that the sugar amended slurries produced less of the semi-volatile compounds usually associated with manure and more of the semi-volatiles normally associated with fruit (line 309).

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all of my concerns.

Author Response

Thank you for your help on this manuscript. 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

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