The Second-Generation Biomethane from Mandarin Orange Peel under Cocultivation with Methanogens and the Armed Clostridium cellulovorans
Round 1
Reviewer 1 Report
Dear Authors,
I revised the manuscript “The second-generation biomethane from mandarin orange peel under cocultivation with methanogens and the armed Clostridium cellulovorans.” submitted to the Fermentation Journal. The paper is interesting. However, I have some concerns, which need to be addressed.
Line 1. Please state typ of the papier (Article, Review, Communication, etc.)
Introduction
Line 45. Explain the abbreviation IBE.
Line 72. More can be written about the pre-treatment of orange mandarin peel, e.g. mechanical, chemical and biological processing.
Materials and Methods
Line 88. Describe in more detail: Avicel and MN301. Not everyone knows these signs and names
Discussion
There is no discussion what the author writes in the discussion is rather a summary. In the discussion, the results of the research should be compared to other methods of pre-treatment of substrates containing lignocellulose fibres, e.g. mechanical or chemical pre-treatment.
Conclusions
No conclusions at work. Write down some of the most important conclusions.
Author Response
We appreciate your precious comments on my manuscript. We answered your questions and comments below. Could you please to read them. Since we corrected and rewrote some sentences in our manuscript, please review them again. If you have further questions or comments, please let us know.
Line 1. Please state type of the paper (Article, Review, Communication, etc.)
-->The type is Article.
Introduction
Line 45. Explain the abbreviation IBE.
--> I added a further explanation, “Isopropanol-Butanol-Ethanol (IBE) fermentation, which is a bacterial fermentation process producing isopropanol instead of acetone on acetone-ethanol-butanol (ABE) fermentation.”
Line 72. More can be written about the pre-treatment of orange mandarin peel, e.g. mechanical, chemical and biological processing.
-->We can not find any description at Line 72, but we wrote additional information in the Materials and Methods.
Materials and Methods
Line 88. Describe in more detail: Avicel and MN301. Not everyone knows these signs and names
--> Avicel is a crystalline cellulose powder that is industrially refined from natural cellulose, and the particle size of Avicel is less than 50 μm. MN301 is also industrially refined cellulose powder, and 80% of the particle size is less than 160 μm.
Discussion
There is no discussion what the author writes in the discussion is rather a summary. In the discussion, the results of the research should be compared to other methods of pretreatment of substrates containing lignocellulose fibres, e.g. mechanical or chemical pre-treatment.
--> We appreciate your kind comments to improve my manuscript. We modified and rewrote the Discussion.
Conclusions
No conclusions at work. Write down some of the most important conclusions.
--> Thank you again for your advice. We added and modified the Conclusions.
Reviewer 2 Report
Dear Authors,
The authors precisely described the types of microorganisms and culture conditio that they used for the fermentation process. Measuring equipment and parameters for conducting analytical research are described in detail. However, there are shortcomings in the descriptions of fermentation research - biogas production.
Please indicate clearly the novelty aspect of the research. The authors wrote: "Flavedo and albedo, hereafter called removed peel, were cut into strips with scissors." This information is not precise. Grain size should be provided. Substrate fragmentation is an important parameter in the methane fermentation process. The amount of produced methane and the fermentation time are closely related to the grain size of the substrate. The level of fragmentation should be correlated with the optimal efficiency of bacterial growth in the methane fermentation process. Pre-treatment of substrates (e.g. micronization) is one of the key procedures while conducting the fermentation process. Conducting the cycle of research on enzymatic treatment and further methane fermentation described in the paper, the Authors did not determine the effect of substrate grain size on the yield of combustible components in biogas. When conducting tests, all parameters and conditions of the process should be provided. The authors only provided the dry matter content of the substrate for the fermentation process, pH, types of microorganisms, temperature and duration of the process. However, there is no data on the ratio of nutrients C: N: P: S, C/N ratio, which are extremely important for the biogas production process. These data should be completed. The Authors did not provide information on the presence and concentration of inhibitors. These data should be completed. In the presented paper, the Authors completely omitted the aspect of scientific discussion. The results presented in graphical form were then described, but not commented or explained scientifically. Research results should be explained on the scientific level.
Author Response
We appreciate your precious comments on my manuscript. We answered your questions and comments below. Could you please to read them. Since we modified and rewrote some sentences in our manuscript, please review them again. If you have further questions or comments, please let us know.
Answers for your comments
Please indicate clearly the novelty aspect of the research.
-->The novel points are that it is clearly demonstrated C. cellulovorans can degrade orange wastes without any pretreatments by grinding and chemicals, and that methane is produced from orange wastes simultaneously. Moreover, although there have been many reports regarding the 2nd and 3rd generation biofuels, the cost of collecting raw materials from the agricultural fields was not discussed so much. For example, although seaweed is one of raw materials for the 3rd generation biofuel and a lot of seaweeds may be harvested, it takes huge energy to collect seaweeds from the sea. On the other hand, since orange wastes have already been in the orange juice factory, there is no additional energy to collect them. Therefore, we thought it would be the biggest advantage. This study clarified the expected amount of methane from orange wastes. Since the biorefinery plant in the orange juice factory is ready to go, we will expand this biorefinery system to other agricultural wastes.
The authors wrote: "Flavedo and albedo, hereafter called removed peel, were cut into strips with scissors." This information is not precise. Grain size should be provided. Substrate fragmentation is an important parameter in the methane fermentation process. The amount of produced methane and the fermentation time are closely related to the grain size of the substrate. The level of fragmentation should be correlated with the optimal efficiency of bacterial growth in the methane fermentation process.
-->As you mentioned, the grain size is very important for degradation and fermentation. We should describe the grain size. Peel just after removed from an orange was cut by scissors. The strip size was approximately 8 mm length and 2mm width. The peel was used as fresh, not dried and ground. Furthermore, the peel was not pretreated by any chemicals. Therefore, we would like to demonstrate in this study how C. cellulovorans degrades the orange peels. This is because ground powder is easy to degrade them enzymatically, however drying and grinding processes also need much energy, cost and time. Thus, our research is focusing on using agricultural waste as a raw material for biorefinery while remaining fresh.
Pre-treatment of substrates (e.g. micronization) is one of the key procedures while conducting the fermentation process. Conducting the cycle of research on enzymatic treatment and further methane fermentation described in the paper, the Authors did not determine the effect of substrate grain size on the yield of combustible components in biogas.
-->We explained the grain size at the above question.
When conducting tests, all parameters and conditions of the process should be provided. The authors only provided the dry matter content of the substrate for the fermentation process, pH, types of microorganisms, temperature and duration of the process. However, there is no data on the ratio of nutrients C: N: P: S, C/N ratio, which are extremely important for the biogas production process. These data should be completed.
-->We appreciate your important suggestion. N, P, S are initially included in the medium. C is supplied from a carbon source, such as cellulosic biomass by enzymatical degradation of C. cellulovorans. It is reported that the C/N ratio of orange wastes is approximately 40. We do not investigate the time courses of these compound concentrations. However, as you mentioned, the changes in the compound concentrations are important for sustainable degradation and fermentation. Thus, we will investigate these details, and we would like to mention this issue in the Discussion.
The Authors did not provide information on the presence and concentration of inhibitors. These data should be completed.
-->We have previously reported the effect of limonene concentration on microbial inhibition (Tomita et al. AMB Express (2019) 9:1). If the concentration of limonene is less than 0.05% (w/v), C. cellulovorans can degrade cellulose such as Avicel. Furthermore, we investigated C. cellulovorans can degrade orange wastes which include limonene. However, there are still many questions, for example, "When does limonene come out from the peel?", "How much is evaporating ratio of limonene?”, "Can biofilm protect microbiota from limonene?", etc. Although we are still working on these issues, this paper never described limonene much. Therefore, we only added the calculated limonene concentration in the Introduction.
In the presented paper, the Authors completely omitted the aspect of scientific discussion. The results presented in graphical form were then described, but not commented or explained scientifically. Research results should be explained on the scientific level.
-->We appreciate your kind comments to improve our manuscript. We modified and rewrote the Discussion part.
