Adding Digestive Enzymes to Anaerobic Co-Digestion of Cattle Manure and Industrial Corn Grain Waste^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors proposed a study on the addition of hydrolytic enzymes to a semi-continuous reactor in psychrophilic conditions. The reactor operated in monodigestion of cattle manure or co-digestion of cattle manure and industrial corn grain waste (70:30, on a volatile solids basis). The effect of the enzyme mixture (cellulase, xylanase, and pectinase) was evaluated in both mono- and co-digestion. This is a hot topic because, although the practice of adding enzymes to anaerobic digesters is widespread and well established, scientific evidence of its potential benefits is lacking. Indeed, the results in the literature are highly conflicting, primarily due to inconsistency and experimental designs.

The authors propose a well-conducted study with an adequate experimental design. The lack of efficacy of in situ enzyme activity is supported by robust data.

I would ask the Authors to further investigate the evaluation of Table 3, from which much information can be drawn for the subsequent discussion (L270-303), which needs to be further explored and less generic. First of all, the Authors should also include the results of the statistical analysis in Table 3. Although it is true that intermediates (notably non-structural carbohydrates) deriving from the hydrolysis of hemicellulose were not used for biogas production, it is also true that there is a clear difference in the reduction efficiency between NDF and ADF in the comparison between CM+CG and CM+CG+E. In my opinion, there is no difference in the reduction of NDF (43.59 vs. 47.41), while a clear reduction of ADF is evident in the CM+CG treatment compared to CM+CG+E (32.57 vs. 6.84). This is the key point. In fact, NDF is composed of holocellulose and lignin, while ADF is composed of cellulose + lignin. It is known that cellulose has a far higher specific methane yield than hemicellulose (https://doi.org/10.1016/j.apenergy.2018.05.055). Such a large difference in ADF consumption (i.e., cellulose, for obvious reasons) led to a significant difference in CH4 production, despite the fact that the addition of the enzyme was much more effective in removing hemicellulose.

Minor adjustments are required before the publication:

L78 Please replace “That said” with “Thus” and “trial” with “study”

L98 Please specify temperature and duration

L100 Please specify temperatures

L106 Please replace “biofertilizer” with “digestate”

Figure 1: Please replace “affluent” with “influent” and “Biofertilizer” with “effluent”

Please remove “dimensionless” from all the tables, as well as “ratio” after C/N

L161-163 Please add the reference in the correct format and report it in the reference list.

Table 3: Please add the letters for significance and replace the comma with a dot. Moreover, add “%” after “Red.”

Author Response

Manuscript ID: fermentation-3991477

Type of manuscript: Article

Title: Adding digestive enzymes to anaerobic co-digestion of cattle manure and industrial corn grain waste

 

COMMENTS – REVIEWER 1

 

Comments 1: I would ask the Authors to further investigate the evaluation of Table 3, from which much information can be drawn for the subsequent discussion (L270-303), which needs to be further explored and less generic. First of all, the Authors should also include the results of the statistical analysis in Table 3.

Response 1:

In Table 3, the letters for statistical significance were added for the following data: Total solids (TS, g L⁻¹) and Volatile solids (VS, g L⁻¹). At the base of Table 3, the statistical significances for minor and major letters were also added (L270-272).

As for the chemical composition data, analysis was performed in composed treatment samples (pool samples for the treatments), thus, it was not possible to run statistical analysis on these data. It was added (%) for values of “Red”, directly into the text – highlighted in yellow.  

 

Comments 2: Although it is true that intermediates (notably non-structural carbohydrates) deriving from the hydrolysis of hemicellulose were not used for biogas production, it is also true that there is a clear difference in the reduction efficiency between NDF and ADF in the comparison between CM+CG and CM+CG+E. In my opinion, there is no difference in the reduction of NDF (43.59 vs. 47.41), while a clear reduction of ADF is evident in the CM+CG treatment compared to CM+CG+E (32.57 vs. 6.84). This is the key point. In fact, NDF is composed of holocellulose and lignin, while ADF is composed of cellulose + lignin. It is known that cellulose has a far higher specific methane yield than hemicellulose (https://doi.org/10.1016/j.apenergy.2018.05.055). Such a large difference in ADF consumption (i.e., cellulose, for obvious reasons) led to a significant difference in CH4 production, despite the fact that the addition of the enzyme was much more effective in removing hemicellulose.

Response 2:

For the chemical composition data, as explained earlier, the analysis was performed on composed samples (pool) of the treatments, therefore, it was not possible to apply statistical analysis on the observed data. However, considering the numerical values observed, the authors included two additional paragraphs of discussion, as suggested by the Reviewer, directly into the text (L292-313), highlighted in yellow.

 

Comments 3: Minor adjustments are required before the publication:

- L78 Please replace “That said” with “Thus” and “trial” with “study”

Response: replacements suggested were done and are highlighted in yellow.

- L98 Please specify temperature and duration

Response: The industrial residues of CG were initially dried naturally for removal of excess humidity, for 4 (four) days at room temperature, which varied from 25 to 28^oC (L99-100) –highlighted in yellow.

The drying of the residues of grain corn was performed with the aim of allowing grinding on a knife mill, to prevent clogging of the material in the sieves and blades of the mill.  

- L100 Please specify temperatures

Response: The freezer thermostat was configured to -18°C, adequate temperature to guarantee that the materials freeze up quickly and safely, preventing bacterial proliferation and excessive formation of ice crystals (L102)

- L106 Please replace “biofertilizer” with “digestate”

Response: Replacement was done directly into the text, highlighted in yellow (L108).

- Figure 1: Please replace “affluent” with “influent” and “Biofertilizer” with “effluent”

- Please remove “dimensionless” from all the tables,

- Please remove “ratio” after C/N from all the tables

Response: Changes suggested were made into the Figures and Tables.

 

- L161-163 Please add the reference in the correct format and report it in the reference list.

Response: References were added the right way into the text (L163-192) and also reported at the References List (L498-505) – highlighted in yellow.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is of interest.

The study assessed the biogas yield and quality, and the stability of the AcoD process involving cattle manure and corn grain residues derived from a grain processing agroindustry, by exploring the enzyme effect.

 

Here are some suggestions:

1. Abstract: I suggest enhancing the take-home message and focusing on the methane more than the biogas. I suggest stating the scale of the fermentation
2. Introduction: I suggest explaining why you focused attention on these substrates (a part of the capability of producing methane, since there are a lot of feedstocks with this capability), the gap with the literature, and the novelty of your study
3. Materials and methods: Why did you decide to proceed like that? Industrial CG waste was first dried naturally to remove excess moisture. Next, it was ground using a knife mill (Marconi, MA580) and passed through a 4-mm sieve. The material was put into LDPE (low-density polyethene) bags and kept in the freezer during the entire experiment.
4. Material and methods: section 2.3: Which is the volume/scale of the reactor? How did you set the OLR?
5. Material and methods: section 2.4: How did you select the enzyme and operative conditions? I suggest you read these publications about enzyme application in AD:
   10. https://doi.org/10.1016/j.biortech.2011.07.048
   11. https://doi.org/10.1016/j.jclepro.2024.143760
   12. https://doi.org/10.1016/j.rser.2018.03.103
   13. https://doi.org/10.1016/j.foodres.2015.03.027
6. Materials: Did you perform the tests in triplicate?
7. Table 4: Did you measure the FOS/TAC?
8. Conclusions: Could you be more quantitative in reporting the findings and providing the potential applicability of this work?

Author Response

Manuscript ID: fermentation-3991477

Type of manuscript: Article

Title: Adding digestive enzymes to anaerobic co-digestion of cattle manure and industrial corn grain waste

 

COMMENTS – REVIEWER 2

 

Comments and Suggestions for Authors:

Comments 1 - Abstract: I suggest enhancing the take-home message and focusing on the methane more than the biogas. I suggest stating the scale of the fermentation

Response 1: Suggestions were made into the Abstract and are highlighted in yellow.

 

Comments 2 - Introduction: I suggest explaining why you focused attention on these substrates (a part of the capability of producing methane, since there are a lot of feedstocks with this capability), the gap with the literature, and the novelty of your study

Response 2:

Brazil has the largest commercial bovine herd in the world, and occupies the second global place in meat production, with an estimated production of 11 million tons. The country is considered the largest global exporter of bovine meat – around 3 million tons in 2024. Such expressive numbers also represent and environmental challenge [1, 2]

As far as corn production, Brazil is currently the third largest global producer, after United States and China. During 2023, the country produced around 132 million tons of corn. Most recent estimates (2024/25 harvest) point to a corn production in Brazil of approximately 126 to 131 million tons, which reinforces the third place as largest corn producer in the world [3].

On the other hand, animal dejects are one of the main contributors for environmental pollution and greenhouse gas (GHG) emissions, representing 14,5% of total anthropogenic emissions, including CH₄, CO₂ and N₂O. More than 1,4 billion of tons of CO₂eq are attributed to animal dejects, with the bovine dejects being the major contributor and the one with the largest participation in the general dejects generation [4].

Thinking about processing Animal Dejects (AD), the bovine deject can cause a stabilizer effect on the biogas process when there is some kind of disruption in the process, such as, for example, the presence of residues which acidify the medium, like corn (biogas manual). Therefore, the treatment with co-digestion of such substrates are extremely interesting to the country and to the world, in compliance to the UN ODS, and providing renewable energy generation, as well as promoting the environmental impact mitigation, in addition to its potential of biofertilizers production and soil enrichment, replacing conventional chemical fertilizations.

References:

1. Available at: https://www.brasilagro.com.br/conteudo/pais-e-o-4-produtor-de-graos-e-o-maior-exportador-de-carne-bovina-do-mundo.html?utm_source=chatgpt.com. access: 26 Nov. 2025.
2. Available at: https://www.fao.org/faostat/en/#data/QCL. access 26 Nov. 2025.
3. Available at: https://www.embrapa.br/en/agropensa/agro-em-dados/agricultura/milho?utm_source=chatgpt.com. access 26 Nov 2025.
4. Available at: https://doi.org/10.3390/en17030546. access em 26 Nov 2025.

 

Comments 3 - Materials and methods: Why did you decide to proceed like that? Industrial CG waste was first dried naturally to remove excess moisture. Next, it was ground using a knife mill (Marconi, MA580) and passed through a 4-mm sieve. The material was put into LDPE (low-density polyethene) bags and kept in the freezer during the entire experiment.

Response 3:

The industrial residues of corn grain went through a pre-drying during 4 (four) days, exposed to the sun and with room temperature varying from 25 to 28^oC, with the aim to reduce the excessive humidity and make it possible to grind the material without clogging the mill’s sieves and knifes. The fibrous mass of the corn grains, with its original humidity, promoted clogging of the mill’s knifes.

Grinding of the substrate increases the availability of the organic material, and, consequently, the solubility of the material. The grinding promotes the increase of the total area of the material available for the digestion process. Many microorganisms, especially those active at the initial steps of hydrolysis, are favored by grinding. The smaller the particles, the larger the total area available for the process of hydrolysis by microorganisms.  The smaller the granulometry of organic material at the beginning of the anaerobic digestion (hydrolysis), the higher the efficiency of the microorganisms in degrading such material. The united microorganisms’ secret digestive enzymes almost simultaneously, promoting the breach of the cells and increasing degradation rate. Literature describes a clear correlation between the size of particles and methane production.

The grinding process was performed to reduce the particles’ size and optimization of the degradation process. Later, when the substrates were diluted in the inoculum and water, they were sifted once more for the separation of the solid fraction.

The procedures described were necessary because the semi-continuous experimental biodigesters, with a useful volume of 28 liters, did not have agitation of the content (interior movements of the biodigesters), nor did they have heating.

The material was placed in plastic bags and kept in freezer (-18^oC throughout the experiment, being defrosted the day before supply). Such temperature is adequate to guarantee that the materials are frozen quickly and safely, preventing bacterial proliferation and excessive formation of ice crystals.

The procedures described have the finality of preserving the corn grains residues’ characteristics, preventing further fermentation during storage throughout the experimental period, as well as preventing alterations in its chemical composition and levels of total solids and volatile solids already analyzed.  The process guaranteed that the daily supplies were  performed with a standard material, with its main characteristics preserved throughout the 56 days of experimental trial. When the residues are in storage inadequately, there is a high risk of unwanted fermentation, decreasing the biogas potential production of the material.

 

Comments 4 - Material and methods: section 2.3: Which is the volume/scale of the reactor? How did you set the OLR?

Resposta 4:

The biodigesters useful volume is 28 liters. The organic load was defined considering the operability conditions of the biodigesters, which did not have agitation nor heating. It was considered a level of TS of 3%, to prevent excessive decantation in the interior of the biodigesters, as preconized by literature.

In the continuous process reactor, the material is bombed continuously to its interior, which allows a continuous flow of raw material throughout the day, and, therefore, constant biogas production. Such a process is possible if the substrate is liquid with levels of TS inferior of 5% (KARLSSON et al., 2014).

According to Silva (2001), when searching for a maximal production of biogas for the biodigester volume and time unit, the maximum concentration of TS should be inferior of 8%, range in which it is also possible to facilitate the movement of the material inside the biodigester, in addition to preventing clogging of entrance and exit pipes of the reactor.

References:

KARLSSON, T.; KONRAD, O.; LUMI, M.; SCHMEIER, N.P.; MARDER, M.; CASARIL, C.E.; KOCH, F.F.; PEDROSO, A.G. Manual Básico de Biogás. Lajeado: Ed.Univates, 2014. 69p.

SILVA, M.S. Biodigestão anaeróbia no saneamento rural. Lavras: UFLA/FAEPE, 71p. (Textos Acadêmicos). 2001.

 

Comments 5 - Material and methods: section 2.4: How did you select the enzyme and operative conditions? I suggest you read these publications about enzyme application in AD.

Response 5:

Enzymes were donated to the study by a private company, and dosages were the ones recomemnded for the use with domestic efluents.

 

Comments 6 - Materials: Did you perform the tests in triplicate?

Response 6: All analysis were performed in triplicates.

 

Comments 7- Table 4: Did you measure the FOS/TAC?

Response 7: No, FOS/TAC was not measured.

 

Comments 8- Conclusions: Could you be more quantitative in reporting the findings and providing the potential applicability of this work?

Response 8: Significant values were added directly into the conclusion text, highlighted in yellow.

“The combining cattle manure with corn-grain waste in anaerobic co-digestion leads to higher daily biogas production (52,12%) and improved specific yields of methane (41,35%) and biogas (48,55%) compared to just using cattle manure (mono-digestion).”

As for the potential applicability of the study, it is recommended more research, especially with doses of enzymes studied, as stated into the text.

“The findings provide a basis for guiding further discussions on the application of enzymatic compounds at higher dosages and the effectiveness of enzymes in carbohydrate-rich substrates.”

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, 

thank you for your work. I suggest explicitly stating in the text that the test was performed in triplicate.

Thank you