Effect of Monensin Supplementation in the Bovine Diet on the Composition and Anaerobic Digestion of Manure with and without Screening

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript deals with the effect of monensin supplementation in the bovine diet on the composition and anaerobic digestion of manure. The subject is important for environmental application. There are some comments that the authors should consider before publication.

1. The knowledge gap needs to be clearly addressed in the Introduction.

2. The authors should compare their results with the results obtained by antibiotics (monensin and others antibiotics) in the literature.

3. The authors should add the discussion of the obtained results. For example, the authors should comment the reasons for increasing the biogas production and decreasing the methane concentration by monensin dosage up to cca 3 mg/kg DMI (Fig. 5).

4.  The Conclusion should be extended. The ideas for further research work should be added.

Author Response

General comments: Manuscript deals with the effect of monensin supplementation in the bovine diet on the composition and anaerobic digestion of manure. The subject is important for environmental application. There are some comments that the authors should consider before publication.

Response: Thank you for the feedback provided. We have made every effort to incorporate the reviewer's suggestions into the manuscript and believe we have addressed all the requests. Should there be any additional modifications required, we are prepared to make further adjustments.

Comments 1. The knowledge gap needs to be clearly addressed in the Introduction.

Response 1: We appreciate the suggestion and have made an effort to include additional information and references to further research results in order to provide greater consistency in the information presented.

Comments 2. The authors should compare their results with the results obtained by antibiotics (monensin and other antibiotics) in the literature.

Response 2: Additional comparisons with the literature have been incorporated into the manuscript. The references used focus on the ionophore employed in our study. Research on ionophores specifically for waste management is still relatively scarce. Different active compounds, even within the ionophore group, exhibit varying actions or resistance to waste treatment due to their physical and chemical properties. Because of this in the initial submission, we referenced studies directed at the use of monensin.

Comments 3. The authors should add the discussion of the obtained results. For example, the authors should comment the reasons for increasing the biogas production and decreasing the methane concentration by monensin dosage up to cca 3 mg/kg DMI (Fig. 5).

Response 3: We have made efforts to enhance our discussion and include additional references to better clarify the results in our manuscript. Thank you for the comments.

Comments 4.  The Conclusion should be extended. The ideas for further research work should be added.

Response 4: We appreciate the reviewer's suggestion and have made this adjustment in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents the results of a study where the effect of increasing doses of monensin on the compositional characteristics of cattle manure and subsequent behavior of the waste in anaerobic digestion was investigated. In my opinion the paper presents some drawbacks which make it not completely mature to deserve publication.

First, the paper does not seem very original, it seems that previous papers (see, for example, reference n.7) already treated the same topic with a similar approach. The novelty of the study over the present state of the art is not highlighted in the introduction.

The introduction is not clear, for example it is not clearly said why monensis is (is that an antibiotic? why the use of antibiotics is cited at line 37?).

The section of materials and methods must be improved:

How the doses of monesin were fixed?

How was the duration of the adaptation period (line 107) fixed?

Was the TS content of the feedstock for the anaerobic digestion obtained from the original waste by dilution?

How the reactors for the AD were stated up? Which was the origin of the inoculum?

Which was the duration of the AD tests?

The experimental points obtained from the tests must be clearly indicated in the diagrams and also the evolution of the feedstock fed to the digesters and the digestate extracted from them, the evolution of the production of biogas and methane during the AD tests.

From an economic perspective, increasing the HRT to buffer the effect of the monesin does not seem a feasible option. What about applying pre-treatments?

The results obtained with HRTs of 20 and 30 days must be reported together, in order to make a comparison between the two situations possible. In this way it is quite difficult following the description of the two trends.

Conclusions must be expanded.

Finally, English is not my first language, but the paper needs an intense revision concerning the choice of the words and the structure of the sentences by a mother-tongue editor.

Comments on the Quality of English Language

English is not my first language, but the paper needs an intense revision concerning the choice of the words and the structure of the sentences by a mother-tongue editor.

Author Response

Comments 1: The paper presents the results of a study where the effect of increasing doses of monensin on the compositional characteristics of cattle manure and subsequent behavior of the waste in anaerobic digestion was investigated. In my opinion the paper presents some drawbacks which make it not completely mature to deserve publication.

Response 1: We understand that the reviewer may have had some concerns regarding the readiness and scientific innovation of our manuscript (as noted in comment 2). In our resubmission, we have carefully revised the manuscript to address these concerns and close the identified gaps. Additionally, we have provided further explanations where clarity may be needed across different areas of expertise

Comments 2: First, the paper does not seem very original, it seems that previous papers (see, for example, reference n.7) already treated the same topic with a similar approach. The novelty of the study over the present state of the art is not highlighted in the introduction. The introduction is not clear, for example it is not clearly said why monensin is (is that an antibiotic? why the use of antibiotics is cited at line 37?).

Response 2: In our study, we did not merely perform anaerobic digestion of the waste (as broadly mentioned in reference 7). We carefully controlled the process since the beginning by administering specific doses of monensin to the cattle, collecting and quantifying the waste produced, and closely monitoring the anaerobic digestion. We also tested two physical separation conditions for the substrates and two retention times. This comprehensive approach allowed us to directly link waste treatment efficiency and energy generation with the monensin dosage.

 Regarding monensin, because it is technically an ionophore antibiotic, we initially referred to it as an antibiotic. However, recognizing that this term may be less familiar to those outside the agricultural field, we chose to use the term 'medication' to enhance clarity.

The section of materials and methods must be improved:

Comments 3: How the doses of monensin were fixed?

Response 3: The doses were established based on data from the literature, with the main recommendations derived from the studies by Rezaei Ahvanooei et al. (2023) (doi.org/10.1038/s41598-023-27395-9) and Appuhamy et al. (2013) (doi.org/10.1038/s41598-023-27395-9). Additionally, if the calculation of the monensin dosage is also under question, we can clarify the following: in animal nutrition, supplement doses are typically determined based on the dry matter (DM) intake of the animals. For example, if a bovine consumes, on average, 2% of its body weight (BW) in DM daily, and its BW is 400 kg, it would ingest 8 kg of DM per day. Therefore, if the recommended dose is 5 mg of monensin per kg of DM intake per day, this animal should consume 40 mg of monensin per day.

Comments 4: How was the duration of the adaptation period (line 107) fixed?

Response 4: We had not previously cited the reference on which we based our recommendation for the adaptation period, and we appreciate the reviewer's observation. The reference has now been included in the text. If the reviewer deems it necessary, we can further elaborate on what constitutes an adaptation period to the diet.

Comments 5: Was the TS content of the feedstock for the anaerobic digestion obtained from the original waste by dilution?

Response 5: The total solids (TS) content in the substrates used to feed the biodigesters was adjusted based on the TS content of the manure, as shown in Table 1. In this table, we present the TS content of the manure, the experimental substrates without screening (adjusted to 2.5%), and the screened substrates. The manure was diluted using only water.

Comments 6: How the reactors for the AD were stated up? Which was the origin of the inoculum?

Response 6: The biodigesters were initially started with cattle manure (without monensin) and water for dilution, maintaining the daily loads until stable biogas and methane production were achieved. At this point, feeding with manure from cattle fed with the experimental doses of monensin, marking the start of the experimental period, which lasted 18 weeks. This period included 4 weeks until the biogas and methane production stabilized, and 14 weeks for monitoring the results. The information regarding the duration of the experimental period has been added to the manuscript.

Comments 7: Which was the duration of the AD tests?

Response 7: The response to comment 6 contains this information.

Comments 8: The experimental points obtained from the tests must be clearly indicated in the diagrams and also the evolution of the feedstock fed to the digesters and the digestate extracted from them, the evolution of the production of biogas and methane during the AD tests.

Response 8: We kindly request that the reviewer provide further clarification on their request, as we were unable to understand the comment.

Comments 9: From an economic perspective, increasing the HRT to buffer the effect of the monesin does not seem a feasible option. What about applying pre-treatments?

Response 9: Waste treatment should be considered from an economic perspective; however, it should not be the only consideration. Extending the retention time of substrates or implementing pre-treatment can enhance both energy recovery and the quality of the digestate. The quality of the digestate, in terms of organic matter removal during digestion and nutrient concentration, can directly impact the reduction of synthetic fertilizer use, resulting in economic benefits. Additionally, in many dairy farms, phase separation, which can be considered a pre-treatment, is already a commonly employed technique. It helps to reduce the retention time of substrates and, allows for the coarse residue to be composted, thus reducing the volume of digestate produced and enabling the compost to be stored or even sold outside the farm.

Comments 10: The results obtained with HRTs of 20 and 30 days must be reported together, in order to make a comparison between the two situations possible. In this way it is quite difficult following the description of the two trends.

Response 10: Hydraulic retention times (HRT) of 20 and 30 days are commonly used for the digestion of cattle manure, primarily due to the dilution level of the waste. Our objective in conducting this study is not to compare the HRTs with each other, as it is already well established that the longer the substrate remains in digestion, the greater the degradation and, consequently, the production of biogas and methane. Instead, our aim is to assess the effectiveness of screening within each tested RT, as well as the impact of monensin according to these times. Therefore, we suggest that the evaluation of the results should remain independent.

Comments 11: Conclusions must be expanded.

Response 11: We appreciate the reviewer's suggestion and have made this adjustment in the manuscript.

Comments 12: Finally, English is not my first language, but the paper needs an intense revision concerning the choice of the words and the structure of the sentences by a mother-tongue editor.

Response 12: Our article was reviewed by a company specialized in the revision of scientific papers for publication in English (we are attaching the certificate with this resubmission). This reviewer is a native speaker and has academic training and experience with the terminology required. As authors with multiple publications in this field, we did not notice any inappropriate terms in the manuscript. However, if the reviewer has identified any issues, we kindly request that they point them out so we can address them with the language reviewer and improve our presentation. We appreciate the feedback and attention to detail.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Great work! Some few comments:

LL. 125-127 Can you explain the difference between unscreend material and waste?

LL. 149-150: What kind of Gasometer?

LL. 153: Please add city and country

2.3 Can you describe the used inoculum? Can you determine the organic loading rate please?

LL. 152-263: Do you think it is usefull to choose also 40 days or 50 days retention times?

LL. 365-382: Add a new headline „impact of seperation“

Author Response

General comments: Dear Authors, Great work! Some few comments:

Response: We appreciate your feedback. We hope to have addressed the concerns and improved our manuscript accordingly.

Comments 1: LL. 125-127 Can you explain the difference between unscreend material and waste?

Response 1: We appreciate your comments. Waste refers to our raw material employed for anaerobic digestion, specifically fresh manure. Getting the anaerobic digestion substrates, we just diluted them with water, resulting in a total solids concentration of 2.5%. The substrates prepared in this way were described as 'unscreened,' and those subjected to solid-liquid separation through screening were described as 'screened.' To improve clarity, we changed 'waste' to 'fresh manure' in Table 2.

Comments 2: LL. 149-150: What kind of Gasometer?

Response 2: The gasometers used in our study consist of two cylindrical PVC pipes: one serves as a water seal and the other as a gasometer, where the produced biogas is stored, connected to the biodigesters, as described in our Materials and Methods section. These gasometers resemble batch-type biodigesters; however, in our setup, the gas is retained solely by the water in the lower pipe. For better visualization, we recommend referring to the article by Oliveira et al. (2022) (doi.org/10.1590/0103-8478cr20200760), which includes an illustration of the water seal, although it pertains to a batch system.

Comments 3: LL. 153: Please add city and country.

Response 3: Our first paragraph in the Materials and Methods section includes the city (Dourados) and the country (Brazil).

Comments 4: Can you describe the used inoculum? Can you determine the organic loading rate please?

Response 4: In this study, we did not use an inoculum. The biodigesters were initially started with cattle manure (without monensin) and water for dilution, maintaining the daily loads until stable biogas and methane production were achieved. At this point, feeding with manure from cattle fed with the experimental doses of monensin, marking the start of the experimental period, which lasted 18 weeks. This period included 4 weeks until the biogas and methane production stabilized, and 14 weeks for monitoring the results. The information regarding the duration of the experimental period has been added to the manuscript.

Comments 5: LL. 152-263: Do you think it is usefull to choose also 40 days or 50 days retention times?

Response 5: Hydraulic retention times of 20 or 30 days are commonly used for anaerobic digestion, and even shorter retention times are employed. Retention times of 40 or 50 days may not be necessary to ensure process efficiency. It is also important to consider the time required to achieve high-quality digestate and optimal biogas and methane production.

Comments 6: LL. 365-382: Add a new headline “impact of separation”

Response 6: We considered your suggestion. Our sincere appreciation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved to warrant publication in Fermentation.

Author Response

We thank you for all the contributions made to our manuscript

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed in a quite satisfactory way all my comments, except comment n.8, which seems it was not completely understood.

I try to better explain my comment. All the figures of the paper do not report the results of each single experimental test, but curves which are obtained from the application of a model which interpolates the final result of each test (that is each test carried out for a certain dose of monesin). This does not allow the reader to have information on the evolution/performance of each single test. This is quite uncommon (and not acceptable) in scientific papers.

At least in the Supplementary materials, the authors should provide detailed results of their tests, that is the amount of the feedstock fed to the digesters and the digestate extracted from them, the evolution of the production of biogas and methane during the AD tests, which are the common results of AD tests.

Author Response

General comments: The authors addressed in a quite satisfactory way all my comments, except comment n.8, which seems it was not completely understood.

Response: We appreciate your recognition of our efforts to address the Reviewer’s comments, and we will make every effort to resolve the outstanding issue related to Comment 8.

 

Comments 1: I try to better explain my comment. All the figures of the paper do not report the results of each single experimental test, but curves which are obtained from the application of a model which interpolates the final result of each test (that is each test carried out for a certain dose of monesin). This does not allow the reader to have information on the evolution/performance of each single test. This is quite uncommon (and not acceptable) in scientific papers.

At least in the Supplementary materials, the authors should provide detailed results of their tests, that is the amount of the feedstock fed to the digesters and the digestate extracted from them, the evolution of the production of biogas and methane during the AD tests, which are the common results of AD tests.

 

Response 1: We appreciate your suggestions for improving the presentation of our results. Attached is the table to be used as supplementary material, which provides a detailed description of the volumes of the digesters used in our study, including their respective loads, discharges, and average daily production of biogas, methane, and methane concentration for each experimental treatment applied. We hope that the data presented will contribute to a more comprehensive understanding of our work.

Table 1. Volume, loads, discharges, and average daily production of semi-continuous experimental-scale digesters operated with manure from cattle fed with monensin doses (mg kg^-1 of dry matter intake).

Experimental treatment	Average digester volume (liters)	Daily load (influent, mL/day)	Daily discharge (effluent, mL/day)	Average biogas production (liters/day)	Average methane content in biogas (%)	Average methane production (liters/day)
20 - HRT
US - Control	7.721	386.05	374.70	0.885	68.79	0.609
US - 1.8	7.701	385.05	379.90	0.911	65.05	0.592
US - 3.6	7.906	395.30	379.80	0.968	62.48	0.605
US - 5.4	8.078	403.90	374.56	0.864	60.44	0.522
US - 7.2	8.295	414.75	413.41	0.534	58.67	0.313
SC - Control	7.698	384.90	373.83	0.799	74.84	0.598
SC - 1.8	7.816	390.80	376.17	0.885	70.82	0.627
SC - 3.6	8.193	409.65	405.57	0.777	68.48	0.532
SC - 5.4	8.010	400.50	380.24	0.612	66.52	0.407
SC - 7.2	8.607	430.35	390.02	0.329	59.57	0.196
30 - HRT
US - Control	8.169	272.30	266.40	0.939	72.53	0.681
US - 1.8	7.848	261.60	242.90	0.914	70.06	0.640
US - 3.6	7.546	251.53	242.71	0.918	69.75	0.640
US - 5.4	7.908	263.60	257.74	0.772	69.03	0.533
US - 7.2	8.030	267.67	243.67	0.761	67.42	0.513
SC - Control	8.104	270.13	224.39	0.793	74.48	0.591
SC - 1.8	8.119	270.63	257.92	0.715	73.30	0.524
SC - 3.6	7.996	266.53	266.48	0.721	71.25	0.513
SC - 5.4	7.546	251.53	245.85	0.595	70.09	0.417
SC - 7.2	8.182	272.73	247.84	0.500	69.17	0.346

^{HRT: hydraulic retention time; US: unscreened substrate; SC: screened substrate.}

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Please add your comments in the text.

Response 5: For manure retention time longer than 20-30 days are recommended and usefull.

 

Author Response

Comments: Dear Authors,

Please add your comments in the text.

Response 5: For manure retention time longer than 20-30 days are recommended and usefull.

 Response: We appreciate the suggestions, and our response to Comment 5 has already been incorporated into the concluding remarks in our manuscript.