Bioethanol: A New Synergy between Marine Chitinases from Bacillus haynesii and Ethanol Production by Mucor circinelloides
and Keyur Raval 1,*
Round 1
Reviewer 1 Report
This paper entitled "Bioethanol: A new synergy between marine chitinases from 2 Bacillus haynesii and ethanol production by Mucor circinelloides" is a good addition to the literature in the search for alternative routes towards the production of bioethanol, in this case using chitin as starting material and chito-oligosaccharides as the carbon source for ethanol production. The titers are still low from an economical perspective but this is a proof of concept for a totally biological processing of chitin towards bioethanol. In particular, it is to expect that only a very reduce number of microbial strains are able to life and to produce on chito-oligosaccharides, turning the process more controlable. The work is very well performed and described and, in my opinion, there is only a minor detail: other uses of chito-oligosaccharides, for example, as biostimulants are well described in the literature, but they are not mentioned in the introduction section. Maybe the authors can extend this part of the manuscript to comment on this subject, due to its importance, as chito-oligosaccharides are also important biorefinery products.
Author Response
Reviewer 1
Query 1: This paper entitled "Bioethanol: A new synergy between marine chitinases from 2 Bacillus haynesii and ethanol production by Mucor circinelloides" is a good addition to the literature in the search for alternative routes towards the production of bioethanol, in this case using chitin as starting material and chito-oligosaccharides as the carbon source for ethanol production. The titers are still low from an economical perspective but this is a proof of concept for a totally biological processing of chitin towards bioethanol. In particular, it is to expect that only a very reduce number of microbial strains are able to life and to produce on chito-oligosaccharides, turning the process more controlable. The work is very well performed and described and, in my opinion, there is only a minor detail: other uses of chito-oligosaccharides, for example, as biostimulants are well described in the literature, but they are not mentioned in the introduction section. Maybe the authors can extend this part of the manuscript to comment on this subject, due to its importance, as chito-oligosaccharides are also important biorefinery products.
Reply to Query1: As suggested by the reviewer, the other uses of the chito-oligosaccharides have been included in the introduction.
Biostimulants:
Chitin derived polymers are biostimulators in vegetative crops prone to stressors. Employing biostimulants in agriculture are considered as an eco-friendly practice as it can reduce the usage of mineral inorganic fertilizers without affecting yield and quality of vegetables. Also, it stimulates the root growth, improves the nutrient uptake and enhances anti-oxidant potential in plants. Chitin is one of the most common component in biostimulants 1,2. Chitin oligosaccharides can act as Pathogen associated molecular patterns (PAMPs) in plants and elicits its immune response. Hence, the pathogens can be eliminated without application of fungicides 3.
(1) Bulgari, R.; Cocetta, G.; Trivellini, A.; Vernieri, P.; Ferrante, A. Biostimulants and Crop Responses: A Review. Biological Agriculture and Horticulture. Taylor and Francis Ltd. January 2, 2015, pp 1–17.
(2) Shahrajabian, M. H.; Chaski, C.; Polyzos, N.; Tzortzakis, N.; Petropoulos, S. A. Sustainable Agriculture Systems in Vegetable Production Using Chitin and Chitosan as Plant Biostimulants. Biomolecules. MDPI AG June 1, 2021.
(3) Kobayashi, H.; Suzuki, Y.; Sagawa, T.; Saito, M.; Fukuoka, A. Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst. Angewandte Chemie International Edition 2022.
Reviewer 2 Report
Herein, I submit my comments for initial submission of the manuscript entitled: “Bioethanol: A new synergy between marine chitinases from Bacillus haynesii and ethanol production by Mucor circinelloides”.
New renewable energy sources are an interesting topic, considering the treat that fossil fuels represent for climatic change and species on Earth. Therefore, efforts are being done in order to reduce the use of petroleum fuels for reducing greenhouse gases emissions by means of renewable resources. Nowadays, low-cost biomasses are used in the production of biofuels like bioethanol. Nevertheless, this production requires large amounts utilities associated with the obtention of the raw biomass. In order to present an alternative to the production of bioethanol from lignocellulosic biomass, this manuscript presents a method for its obtention from chitin (one of the most abundant polymers in the environment) under biorefinery concept.
I consider this manuscript suitable for publication. However, some ambiguities arose during the reading. Therefore, I came to a conclusion to encourage you to review it again. Please, use my comments listed below:
Comment 1: Supplementary Figure 1 should be in the supplementary file attached to the article and not in the manuscript.
Comment 2: Please, delete the methodological text included in the section 3. Results and discussion, considering that information is reported in the section 2. Materials and methods.
Comment 3: Further discussion of results with the literature is needed. Specially, regarding with the effect of the evaluated parameters. There is a lack of comparison of the results obtained in the present study; the discussion should give an interpretation of the significance of the results obtained with reference to similar works done by other authors. In terms of efficiency and reactive, is the production from chitin used in the present study better than the second-gen methods from waste biomass?
Comment 4: Lines 320-321: why do the higher amounts of substrate produce production of inhibitory compounds or end product inhibition? Please, explain that affirmation.
Comment 8: In Figure 8, there is no clarity regarding the correspondence of the line in the plot with the substrates named in the legend of the figure.
Comment 9: Rewrite the abstract, (the Abstract should give brief information about the aims, results, methods, and results of the research).
Comment 10: The reference section does not match with the style of the manuscripts published in Fermentation.
Author Response
Reviewer 2
Query 1: Supplementary Figure 1 should be in the supplementary file attached to the article and not in the manuscript.
Reply to Query 1: As suggested by the reviewer, the supplementary figures have been removed and attached as a separate file.
Query 2: Please, delete the methodological text included in the section 3. Results and discussion, considering that information is reported in the section 2. Materials and methods.
Reply to Query 2: As suggested by the reviewer, the modifications have been made. The changes are as under
Deleted mass spec range 50 -2000 in results and discussion
Deleted centrifugation rpm time conditions and filtration of broth prior to enzyme reaction in production of COS in results.
Query 3: Further discussion of results with the literature is needed. Specially, regarding with the effect of the evaluated parameters. There is a lack of comparison of the results obtained in the present study; the discussion should give an interpretation of the significance of the results obtained with reference to similar works done by other authors.
In terms of efficiency and reactive, is the production from chitin used in the present study better than the second-gen methods from waste biomass?
Reply to Query 3: As suggested by the reviewer, the modifications have been made in the text and highlighted. The modifications are as under.
Second generation feedstocks include nonedible feedstocks such as woody biomass and forest residues. Second generation biofuels possess several advantages like there is no competition between food and fuel and also there is no need to cultivate feedstock as it is utilised from waste.
Annually, 5-8 Mt of lignocellulosic biomass is generated from agriculture and deforestation but the generated biomass is lower than the consumption of crude oil per annum (aron). Also, second generation biomass requires pre treatment process inorder to convert the rigid recalcitrance structure of lignocellulosic biomass to fermentable sugar (Sankaran). In Brazil, for a conventional ethanol production plant, the electricity consumption is about 28 kWh/t of canes when steam consumption (2.5 barr) was 372 kg/t of canes (walter). Hence, second generation biofuels include high capital cost and involves complicated processing equipment.
Bioethanol production from feedstocks such as corn stover, barley straw, corn meal were 5.85 L/Kg (Da Silva techno economical), 0.054 L/ Kg (Rooni 2017) and 9.67±0.11 L/kg (Nikolic) of dried biomass respectively. Mithra et al., reported bioethanol yield of 34-44 g/L was obtained from lignocellulos-starch biomass by fed batch separate hydrolysis and fermentation (Mithra).
Query 4: Lines 320-321: why do the higher amounts of substrate produce production of inhibitory compounds or end product inhibition? Please, explain that affirmation.
Reply to Query 4: The authors meant that an increased amount of COS incorporated resulted in increased amount of ethanol production and beyond 8g/L , the organism was not able to tolerate the ethanol produced, hence we could not achieve higher production of the bioethanol in spite of increasing the substrate concentration.
Query 5: In Figure 8, there is no clarity regarding the correspondence of the line in the plot with the substrates named in the legend of the figure.
Reply to Query 5: As suggested by the reviewer, the changes have been incorporated and highlighted. The symbols have also been added in the legends.
Query 6: Rewrite the abstract, (the Abstract should give brief information about the aims, results, methods, and results of the research).
Reply to Query 6: As suggested by the reviewer, the abstract has been modified and highlighted. The changes are as under
The fourth generation of bioethanol production is on a lookout for non-lignocellulosic biomass waste. One such candidate is chitin, the second most abundant biopolymer on earth. However, the crystalline nature of chitin hinders its application potential for bioethanol production. This limitation can be circumvented by hydrolysing this polymer into oligomers using chitinases. We used this hypothesis and isolated a Bacillus haynesii, a marine bacterium capable of utilizing colloidal chitin as a substrate and producing chitin oligomers. The bacterium demonstrated maximum chitinase activity of 3.08 U/mL with specific activity of 96 U/mg at the 90th hour. Chitin oligosaccharides produced by Bacillus haynesii were utilized for ethanol production using Mucor circinelloides and the process parameters for ethanol production were optimized. It resulted in 7.4 g/L of ethanol from 30 g/L of chitin oligomers with a maximum ethanol yield of 0.25 g of ethanol / g of colloidal chitin at 55th h with 48 h inoculum in 80 mL of fermentation medium.
Query 7: The reference section does not match with the style of the manuscripts published in Fermentation.
Reply to Query 7: As suggested the changes were incorporated. The DOI link for the references were removed.
Reviewer 3 Report
The authors worked on isolated a Bacillus haynesii, a marine bacterium capable of utilizing colloidal 19 chitin as a substrate and producing chitin oligomers.
Gas Chromatography and Mass spectroscopy was used to analyse.
HPLC can be also included to understand more
English can be improved
Typographical mistakes are there
Introduction can be elaborated can cite certain literature
Author Response
Reviewer 3
Query 1: The authors worked on isolated a Bacillus haynesii, a marine bacterium capable of utilizing colloidal chitin as a substrate and producing chitin oligomers.
Gas Chromatography and Mass spectroscopy was used to analyse.
HPLC can be also included to understand more
English can be improved
Typographical mistakes are there
Introduction can be elaborated can cite certain literature
Reply to Query 1:
We would like to thank the reviewer for the constructive comments. The detection of ethanol was performed using gas chromatography as it is volatile compound and this technique is the preferred for analysis. As suggested, the introduction has been improvised and the typological and English errors corrected.
Round 2
Reviewer 2 Report
Herein, I submit my comments for the second submission of the manuscript entitled: “Bioethanol: A new synergy between marine chitinases from Bacillus haynesii and ethanol production by Mucor circinelloides”.
I consider this manuscript suitable for publication. However, there are still some persisting mistakes in the manuscript. Please, use my comments listed below:
Comment 1: The Abstract still does not give brief information about the aims, results, methods, and results of the research, especially the methods used during the study.
Comment 2: The reference section still does not match with the style of the manuscripts published in Fermentation, as follow: References should be numbered in order of appearance and indicated by a numeral or numerals in square brackets—e.g., [1] or [2,3], or [4–6]. References must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript. The references must have the subsequent format:
1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
2. Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, 2007; Volume 3, pp. 154–196.
Comment 3: I consider that still there is a lack respect the discussion, considering it does not provide an interpretation of the significance of the results obtained with reference to similar works done by other authors. The above, considering the changes were included in the Introduction chapter and were not related to the obtained results.
Author Response
Dear Esteemed Colleague,
Herein, I submit my comments for the second submission of the manuscript entitled: “Bioethanol: A new synergy between marine chitinases from Bacillus haynesii and ethanol production by Mucor circinelloides”.
Comment: I consider this manuscript suitable for publication. However, there are still some persisting mistakes in the manuscript. Please, use my comments listed below:
Comment 1: The Abstract still does not give brief information about the aims, results, methods, and results of the research, especially the methods used during the study.
Reply: As suggested by the reviewer, the abstract is modified and aims, results, methods have been included.
Comment 2: The reference section still does not match with the style of the manuscripts published in Fermentation, as follow: References should be numbered in order of appearance and indicated by a numeral or numerals in square brackets—e.g., [1] or [2,3], or [4–6]. References must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript. The references must have the subsequent format:
- Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal NameYear, Volume, page range.
- Author 1, A.; Author 2, B. Title of the chapter. In BookTitle, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, 2007; Volume 3, pp. 154–196.
Reply: As suggested by the reviewer, the reference have been modified.
Comment 3: I consider that still there is a lack respect the discussion, considering it does not provide an interpretation of the significance of the results obtained with reference to similar works done by other authors. The above, considering the changes, were included in the Introduction chapter and were not related to the obtained results.
Reply: As suggested by the reviewer, the modification has been made in text and highlighted in red colour font.
Abstract: Lines: 19-23, 26-27, 29-31
Introduction: Lines: 52-62, 96-99, 100-102, 104-106, 109-110
Results and discussion: Lines: 273-279, 322-324, 327-333, 352-359, 380-381, 389-397, 411-413, 425-428.
Hope our reply would satisfy the comments raised by the esteemed reviewers.
Thank you very much to the Reviewers and the Journal team
Author Response File: 
Author Response.pdf
