Production of Biofuels from Glycerol from the Biodiesel Production Process—A Brief Review

Round 1

Reviewer 1 Report

 

In their manuscript, the Authors describe the use of glycerol in biotechnological processes. Some comments are below.

Why the authors mainly used older literature data on the process of anaerobic fermentation of glycerol (2009-2013). A cursory search of the ScienceDirect database returns several thousand records from 2020 -2024. In the opinion of the Reviewer, it would be possible to find several newer articles than those used by the Authors.

The process of producing hydrogen by anaerobic digestion is called dark fermentation. In the opinion of the reviewer, it would be worth adding an explanation in the text. The authors indirectly describe the process itself, e.g. in lines 227-234. However, this may not be known to all readers.

In the opinion of the Reviewer, Latin names should be written in italics.

In the opinion of the Reviewer, when describing the yield of ethanol from pure glycerol, it would be worth specifying the theoretical yield of ethanol from the substrate. This will allow you to better visualize the efficiency of the process.

The Authors state that the use of glycerol is economically and energetically profitable, but they do not provide data to confirm it. In the opinion of the reviewer, it was worth providing data and calculations regarding the economic and energy profitability of using glycerol as a substrate for biotechnological processes.

Minor stylistic and grammatical errors.

Author Response

Dear Reviewer:

 

Thank you for reviewing our manuscript and for your valuable comments and suggestions to improve the manuscript quality. Based on your good suggestions, the manuscript has been carefully corrected. We are looking forward to receiving your positive responses.

 

 

With best wishes,

Cid Marcos G Andrade.

 

“In their manuscript, the Authors describe the use of glycerol in biotechnological processes. Some comments are below.

Why the authors mainly used older literature data on the process of anaerobic fermentation of glycerol (2009-2013). A cursory search of the ScienceDirect database returns several thousand records from 2020 -2024. In the opinion of the Reviewer, it would be possible to find several newer articles than those used by the Authors.”

The authors agree with the reviewer and we have added the following references in the text.

Alves, I.R.F.S.; Mahler, C.F.; Oliveira, L.B.; Reis, M.M.; Bassin, J.P. Assessing the Use of Crude Glycerol from Biodiesel Production as an Alternative to Boost Methane Generation by Anaerobic Co-Digestion of Sewage Sludge. Biomass Bioenergy 2020, 143, doi:10.1016/j.biombioe.2020.105831.

Prasertsan, P.; Leamdum, C.; Chantong, S.; Mamimin, C.; Kongjan, P.; O-Thong, S. Enhanced Biogas Production by Co-Digestion of Crude Glycerol and Ethanol with Palm Oil Mill Effluent and Microbial Community Analysis. Biomass Bioenergy 2021, 148, doi:10.1016/j.biombioe.2021.106037.

Takeda, P.Y.; Gotardo, J.T.; Gomes, S.D. Anaerobic Co-Digestion of Leachate and Glycerol for Renewable Energy Generation. Environmental Technology (United Kingdom) 2022, 43, 1118–1128, doi:10.1080/09593330.2020.1818832.

Bułkowska, K.; Mikucka, W.; Pokój, T. Enhancement of Biogas Production from Cattle Manure Using Glycerine Phase as a Co-Substrate in Anaerobic Digestion. Fuel 2022, 317, doi:10.1016/j.fuel.2022.123456.

Alves, I.R.F.S.; Mahler, C.F.; Oliveira, L.B.; Reis, M.M.; Bassin, J.P. Investigating the Effect of Crude Glycerol from Biodiesel Industry on the Anaerobic Co-Digestion of Sewage Sludge and Food Waste in Ternary Mixtures. Energy 2022, 241, doi:10.1016/j.energy.2021.122818.

 

“The process of producing hydrogen by anaerobic digestion is called dark fermentation. In the opinion of the reviewer, it would be worth adding an explanation in the text. The authors indirectly describe the process itself, e.g. in lines 227-234. However, this may not be known to all readers.”

 

The authors agree with the reviewer and are grateful for the suggestion. The following paragraph has been added to the text.”

 

The hydrogen production process via the fermentation process is mostly carried out by anaerobic digestion, called dark fermentation [1,2] . In this process, organic substrates, such as glycerol, are decomposed by hydrogen-producing microorganisms in the absence of photosynthetic reactions [3]. Under anaerobic environments, H+ protons can operate as electron acceptors neutralizing the electrons generated by the oxidation of organic substrates, consequently producing hydrogen [4].

“In the opinion of the Reviewer, Latin names should be written in italics.”

Thanks for the comments, the Latin names have been revised.

“In the opinion of the Reviewer, when describing the yield of ethanol from pure glycerol, it would be worth specifying the theoretical yield of ethanol from the substrate. This will allow you to better visualize the efficiency of the process.”

 

The authors agree with the reviewer and are grateful for the suggestion, so the following paragraph has been added to the text.

 

During the review process, processes that used pure glycerol and crude glycerol as raw material were mentioned, such as the work carried out by Lee et al. (2017) [5]. However, it sought to present mostly works that used crude glycerol, since the glycerol purification process is neither energetically nor economically viable, as presented by Zhang et al. (2016) [6].  Another question is: “Currently, the ethanol production process using glycerol as a raw material is not considered economically viable when compared to other conventional processes [7,8]. However, the use of glycerol from the biodiesel production process is interesting because of some factors, such as: making glycerol a source of biomass, and from it producing ethanol, which is one of the alcohols that can be used in the process. of biodiesel production, encouraging the development of integrated biorefineries (industrial symbiosis) [9,10], as well as the development of new methods and technologies that may be technically and economically viable in the future.” – This excerpt was added to the work with the purpose of clarifying the question of the viability of the ethanol production process using glycerol as a raw material.

“The Authors state that the use of glycerol is economically and energetically profitable, but they do not provide data to confirm it. In the opinion of the reviewer, it was worth providing data and calculations regarding the economic and energy profitability of using glycerol as a substrate for biotechnological processes.”

The authors agree with the reviewer and are grateful for the observation. We believe that we have not made this matter clear in the text. Since we do not claim that glycerol used as a substrate is an economically and energetically more viable alternative, but rather that glycerol can be a promising renewable alternative for the production of other biofuels; and consequently, make glycerol a by-product economically and energetically more viable in the future. This excerpt was added to the text, with the aim of clarifying the subject mentioned by the reviewer:

As already mentioned, biodiesel is a potential successor to diesel, which is a reality in many countries. The biodiesel production process via transesterification reaction is the most used, and during this process there is production of glycerol, currently considered a by-product [11,12]. The considerable increase of glycerol in the market, according to studies, is directly associated with the production of biodiesel [13,14]. This increase in glycerol on the market caused a devaluation of the commercial value of glycerol, making separation and purification processes energetically and economically unfeasible [6]. Therefore, proposing the use of glycerol as a renewable raw material for the production of other biofuels is interesting from an economic, energy and technological development point of view. It is an attempt to value this by-product that is saturated in the market. In this way, considering the current scenario, seeking the development of new forms of biofuel production, and the whole problem presented in relation to the biodiesel production process and glycerol, this work aims to present works that had as objective the production of other biofuels, including methane, hydrogen and ethanol, via fermentation processes, using glycerol from the biodiesel production process as raw material. It is important to highlight that this work aimed to carry out a review with a focus on fermentation processes because it is a more economically viable route, since it makes it possible to use crude glycerol as a source of raw material. We did not find a review in the consulted literature that performs this task, which is our main contribution.

References

1. Wong, Y.M.; Wu, T.Y.; Juan, J.C. A Review of Sustainable Hydrogen Production Using Seed Sludge via Dark Fermentation. Renewable and Sustainable Energy Reviews 2014, 34, 471–482.
2. De Gioannis, G.; Muntoni, A.; Polettini, A.; Pomi, R. A Review of Dark Fermentative Hydrogen Production from Biodegradable Municipal Waste Fractions. Waste Management 2013, 33, 1345–1361, doi:10.1016/j.wasman.2013.02.019.
3. Chookaew, T.; Prasertsan, P.; Ren, Z.J. Two-Stage Conversion of Crude Glycerol to Energy Using Dark Fermentation Linked with Microbial Fuel Cell or Microbial Electrolysis Cell. N Biotechnol 2014, 31, 179–184, doi:10.1016/j.nbt.2013.12.004.
4. Ghimire, A.; Frunzo, L.; Pirozzi, F.; Trably, E.; Escudie, R.; Lens, P.N.L.; Esposito, G. A Review on Dark Fermentative Biohydrogen Production from Organic Biomass: Process Parameters and Use of by-Products. Appl Energy 2015, 144, 73–95.
5. Lee, S.J.; Lee, J.H.; Yang, X.; Yoo, H.Y.; Han, S.O.; Park, C.; Kim, S.W. Re-Utilization of Waste Glycerol for Continuous Production of Bioethanol by Immobilized Enterobacter Aerogenes. J Clean Prod 2017, 161, 757–764, doi:10.1016/j.jclepro.2017.05.170.
6. Zhang, X.; Yan, S.; Tyagi, R.D.; Surampalli, R.Y.; Valéro, J.R. Energy Balance of Biofuel Production from Biological Conversion of Crude Glycerol. J Environ Manage 2016, 170, 169–176, doi:10.1016/j.jenvman.2015.09.031.
7. Posada, J.A.; Cardona, C.A. Design and Analysis of Fuel Ethanol Production from Raw Glycerol. Energy 2010, 35, 5286–5293, doi:10.1016/j.energy.2010.07.036.
8. Sunarno, J.N.; Prasertsan, P.; Duangsuwan, W.; Kongjan, P.; Cheirsilp, B. Mathematical Modeling of Ethanol Production from Glycerol by Enterobacter Aerogenes Concerning the Influence of Impurities, Substrate, and Product Concentration. Biochem Eng J 2020, 155, doi:10.1016/j.bej.2019.107471.
9. Chozhavendhan, S.; Karthiga Devi, G.; Bharathiraja, B.; Praveen Kumar, R.; Elavazhagan, S. Assessment of Crude Glycerol Utilization for Sustainable Development of Biorefineries. In Refining Biomass Residues for Sustainable Energy and Bioproducts: Technology, Advances, Life Cycle Assessment, and Economics; Elsevier, 2019; pp. 195–212 ISBN 9780128189962.
10. Gonela, V.; Zhang, J. Design of the Optimal Industrial Symbiosis System to Improve Bioethanol Production. J Clean Prod 2014, 64, 513–534, doi:10.1016/j.jclepro.2013.07.059.
11. Elgharbawy, A.S.; Sadik, W.A.; Sadek, O.M.; Kasaby, M.A. Maximizing Biodiesel Production from High Free Fatty Acids Feedstocks through Glycerolysis Treatment. Biomass Bioenergy 2021, 146, doi:10.1016/j.biombioe.2021.105997.
12. Almeida, E.L.; Andrade, C.M.G.; Andreo Dos Santos, O. Production of Biodiesel Via Catalytic Processes: A Brief Review. International Journal of Chemical Reactor Engineering 2018, 16.
13. Bournay, L.; Casanave, D.; Delfort, B.; Hillion, G.; Chodorge, J.A. New Heterogeneous Process for Biodiesel Production: A Way to Improve the Quality and the Value of the Crude Glycerin Produced by Biodiesel Plants. In Proceedings of the Catalysis Today; October 15 2005; Vol. 106, pp. 190–192.
14. Ramadhas, A.S.; Jayaraj, S.; Muraleedharan, C. Biodiesel Production from High FFA Rubber Seed Oil. Fuel 2005, 84, 335–340, doi:10.1016/j.fuel.2004.09.016. 

Reviewer 2 Report

A suggestion of “major revision” is put forward for this work and the review comments are as follows:

1. The degree of novelty of this manuscript must be clearly stated.

2. It is best to describe other sources of glycerol. Compare the physicochemical properties of glycerol, a byproduct of biodiesel, with glycerol from other sources.

3. Lines 30-33: Please briefly introduce the situation of acidic catalysts.

4. I suggest the author to add reaction conditions such as reaction time, temperature, and pH in Table 2.

5. Please provide a detailed explanation of the advantages of using glycerol as raw material to produce hydrogen in section 3.2.

6. Please provide an economic analysis of using glycerol as raw material to produce ethanol in section 3.3.

The quality of this English language is good.

Author Response

Dear Reviewer:

 

Thank you for reviewing our manuscript and for your valuable comments and suggestions to improve the manuscript quality. Based on your good suggestions, the manuscript has been carefully corrected. We are looking forward to receiving your positive responses.

 

 

With best wishes,

Cid Marcos G Andrade.

“A suggestion of “major revision” is put forward for this work and the review comments are as follows:

1. The degree of novelty of this manuscript must be clearly stated.”

We agree and thank the reviewer. We add the following paragraph to the text.

As already mentioned, biodiesel is a potential successor to diesel, which is a reality in many countries. The biodiesel production process via transesterification reaction is the most used, and during this process there is production of glycerol, currently considered a by-product (Almeida et al., 2018; Elgharbawy et al., 2021). The considerable increase of glycerol in the market, according to studies, is directly associated with the production of biodiesel (Bournay et al., 2005; Ramadhas et al., 2005). This increase in glycerol on the market caused a devaluation of the commercial value of glycerol, making separation and purification processes energetically and economically unfeasible (Zhang et al., 2016). Therefore, proposing the use of glycerol as a renewable raw material for the production of other biofuels is interesting from an economic, energy and technological development point of view. It is an attempt to value this by-product that is saturated in the market. In this way, considering the current scenario, seeking the development of new forms of biofuel production, and the whole problem presented in relation to the biodiesel production process and glycerol, this work aims to present works that had as objective the production of other biofuels, including methane, hydrogen and ethanol, via fermentation processes, using glycerol from the biodiesel production process as raw material. It is important to highlight that this work aimed to carry out a review with a focus on fermentation processes because it is a more economically viable route, since it makes it possible to use crude glycerol as a source of raw material. We did not find a review in the consulted literature that performs this task, which is our main contribution.

“2. It is best to describe other sources of glycerol. Compare the physicochemical properties of glycerol, a byproduct of biodiesel, with glycerol from other sources.”

We appreciate the reviewer's observations and suggestions, so we have added the following paragraph to the text.

Glycerol can be obtained from sources considered renewable, including vegetable oils and animal fats, and also from sources considered non-renewable, such as petroleum. Glycerol can be obtained from fermentation processes, chemical processes such as the hydrogenation of carbohydrates of petrochemical origin, through the saponification process, and through the biodiesel production process. The physicochemical properties of glycerol may vary depending on the raw material used. The work carried out by Quispe et al. (2013) shows that when using different vegetable oils as raw materials, the formed glycerol had different viscosities (Quispe et al., 2013).

“3. Lines 30-33: Please briefly introduce the situation of acidic catalysts.”

The actors thank the reviewer and therefore we have added the following paragraph to the text.

According to Alcañiz-Monge et al. (2013) heterogeneous acid catalysts have the advantage of obtaining biodiesel both through the esterification reaction of fatty acids and by transesterification of triglycerides, formation of purer products, and the possible reuse in a new process (Alaba et al., 2016; Alcañiz-Monge et al., 2013). However, when compared to conventional catalysis, both heterogeneous and homogeneous acid catalysis have some disadvantages, such as requiring longer reaction time and higher operating costs (Lee & Wilson, 2015; Vieira et al., 2013).

“4. I suggest the author to add reaction conditions such as reaction time, temperature, and pH in Table 2.”

Thanks, as suggested, operating conditions have been added

“5. Please provide a detailed explanation of the advantages of using glycerol as raw material to produce hydrogen in section 3.2.”

The authors are grateful for the suggestion and so the following paragraph has been added to the text.

Currently, hydrogen is seen as a form of clean energy, since the only product of the combustion process is water (Wang et al., 2020). Conventional forms of hydrogen production are from the process of catalytic reforming of petroleum, the steam reforming of methane present in natural gas, however, these methods cannot be considered renewable, since both use raw materials of fossil origin (Cortright et al., 2002; Kondratenko et al., 2013). Another conventional method also used for the production of hydrogen is through the electrolysis of water, however, this method demands a high energy cost (Wang et al., 2020). Due to the problems involving conventional methods, the use of glycerol as biomass for the production of hydrogen can be an interesting alternative method, since it causes less environmental impact and requires less energy expenditure (Pott et al., 2014; Sarma et al., 2019; Varella Rodrigues et al., 2020).

“6. Please provide an economic analysis of using glycerol as raw material to produce ethanol in section 3.3.”

The authors agree and thank the reviewer for comments and suggestions, so we have added the following paragraph to the text.

Currently, the ethanol production process using glycerol as a raw material is not considered economically viable when compared to other conventional processes (Posada & Cardona, 2010; Sunarno et al., 2020). However, the use of glycerol from the biodiesel production process is interesting because of some factors, such as: making glycerol a source of biomass, and from it producing ethanol, which is one of the alcohols that can be used in the process. of biodiesel production, encouraging the development of integrated biorefineries (industrial symbiosis) (Chozhavendhan et al., 2019; Gonela & Zhang, 2014), as well as the development of new methods and technologies that may be technically and economically viable in the future.

 

References

Alaba, P. A., Sani, Y. M., & Ashri Wan Daud, W. M. (2016). Efficient biodiesel production: Via solid superacid catalysis: A critical review on recent breakthrough. In RSC Advances (Vol. 6, Issue 82, pp. 78351–78368). Royal Society of Chemistry. https://doi.org/10.1039/c6ra08399d

Alcañiz-Monge, J., Trautwein, G., & Marco-Lozar, J. P. (2013). Biodiesel production by acid catalysis with heteropolyacids supported on activated carbon fibers. Applied Catalysis A: General, 468, 432–441. https://doi.org/10.1016/j.apcata.2013.09.006

Almeida, E. L., Andrade, C. M. G., & Andreo Dos Santos, O. (2018). Production of Biodiesel Via Catalytic Processes: A Brief Review. In International Journal of Chemical Reactor Engineering (Vol. 16, Issue 5). Walter de Gruyter GmbH. https://doi.org/10.1515/ijcre-2017-0130

Bournay, L., Casanave, D., Delfort, B., Hillion, G., & Chodorge, J. A. (2005). New heterogeneous process for biodiesel production: A way to improve the quality and the value of the crude glycerin produced by biodiesel plants. Catalysis Today, 106(1–4), 190–192. https://doi.org/10.1016/j.cattod.2005.07.181

Chozhavendhan, S., Karthiga Devi, G., Bharathiraja, B., Praveen Kumar, R., & Elavazhagan, S. (2019). Assessment of crude glycerol utilization for sustainable development of biorefineries. In Refining Biomass Residues for Sustainable Energy and Bioproducts: Technology, Advances, Life Cycle Assessment, and Economics (pp. 195–212). Elsevier. https://doi.org/10.1016/B978-0-12-818996-2.00009-0

Cortright, R. D., Davda, R. R., & Dumesic, J. A. (2002). Hydrogen from catalytic reforming of biomass-derived hydrocarbons in liquid water. Nature, 418(6901), 964–967. https://doi.org/10.1038/nature01009

Elgharbawy, A. S., Sadik, W. A., Sadek, O. M., & Kasaby, M. A. (2021). Maximizing biodiesel production from high free fatty acids feedstocks through glycerolysis treatment. Biomass and Bioenergy, 146. https://doi.org/10.1016/j.biombioe.2021.105997

Gonela, V., & Zhang, J. (2014). Design of the optimal industrial symbiosis system to improve bioethanol production. Journal of Cleaner Production, 64, 513–534. https://doi.org/10.1016/j.jclepro.2013.07.059

Kondratenko, E. V., Mul, G., Baltrusaitis, J., Larrazábal, G. O., & Pérez-Ramírez, J. (2013). Status and perspectives of CO2 conversion into fuels and chemicals by catalytic, photocatalytic and electrocatalytic processes. In Energy and Environmental Science (Vol. 6, Issue 11, pp. 3112–3135). https://doi.org/10.1039/c3ee41272e

Lee, A. F., & Wilson, K. (2015). Recent developments in heterogeneous catalysis for the sustainable production of biodiesel. Catalysis Today, 242(Part A), 3–18. https://doi.org/10.1016/j.cattod.2014.03.072

Posada, J. A., & Cardona, C. A. (2010). Design and analysis of fuel ethanol production from raw glycerol. Energy, 35(12), 5286–5293. https://doi.org/10.1016/j.energy.2010.07.036

Pott, R. W. M., Howe, C. J., & Dennis, J. S. (2014). The purification of crude glycerol derived from biodiesel manufacture and its use as a substrate by Rhodopseudomonas palustris to produce hydrogen. Bioresource Technology, 152, 464–470. https://doi.org/10.1016/j.biortech.2013.10.094

Quispe, C. A. G., Coronado, C. J. R., & Carvalho, J. A. (2013). Glycerol: Production, consumption, prices, characterization and new trends in combustion. In Renewable and Sustainable Energy Reviews (Vol. 27, pp. 475–493). Elsevier Ltd. https://doi.org/10.1016/j.rser.2013.06.017

Ramadhas, A. S., Jayaraj, S., & Muraleedharan, C. (2005). Biodiesel production from high FFA rubber seed oil. Fuel, 84(4), 335–340. https://doi.org/10.1016/j.fuel.2004.09.016

Sarma, S., Ortega, D., Minton, N. P., Dubey, V. K., & Moholkar, V. S. (2019). Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol. Bioresource Technology, 284, 168–177. https://doi.org/10.1016/j.biortech.2019.03.074

Sunarno, J. N., Prasertsan, P., Duangsuwan, W., Kongjan, P., & Cheirsilp, B. (2020). Mathematical modeling of ethanol production from glycerol by Enterobacter aerogenes concerning the influence of impurities, substrate, and product concentration. Biochemical Engineering Journal, 155. https://doi.org/10.1016/j.bej.2019.107471

Varella Rodrigues, C., Oliveira Santana, K., Nespeca, M. G., Varella Rodrigues, A., Oliveira Pires, L., & Maintinguer, S. I. (2020). Energy valorization of crude glycerol and sanitary sewage in hydrogen generation by biological processes. International Journal of Hydrogen Energy, 45(21), 11943–11953. https://doi.org/10.1016/j.ijhydene.2020.02.168

Vieira, S. S., Magriotis, Z. M., Santos, N. A. V., Saczk, A. A., Hori, C. E., & Arroyo, P. A. (2013). Biodiesel production by free fatty acid esterification using lanthanum (La3+) and HZSM-5 based catalysts. Bioresource Technology, 133, 248–255. https://doi.org/10.1016/j.biortech.2013.01.107

Wang, R., Liu, S., Liu, S., Li, X., Zhang, Y., Xie, C., Zhou, S., Qiu, Y., Luo, S., Jing, F., & Chu, W. (2020). Glycerol steam reforming for hydrogen production over bimetallic MNi/CNTs (M[dbnd]Co, Cu and Fe) catalysts. Catalysis Today, 355, 128–138. https://doi.org/10.1016/j.cattod.2019.07.040

Zhang, X., Yan, S., Tyagi, R. D., Surampalli, R. Y., & Valéro, J. R. (2016). Energy balance of biofuel production from biological conversion of crude glycerol. Journal of Environmental Management, 170, 169–176. https://doi.org/10.1016/j.jenvman.2015.09.031

Round 2

Reviewer 1 Report

The Authors have made the suggested corrections. However, one passage in the manuscript would require minor clarification.
Line 283 - What are they photosynthetic reactions?
Did the Authors mean the processes that take place during photofermentation? The process of photofermetnation and dark fermentation are not the same. They can be combined to increase hydrogen yield and substrate reduction.
In the reviewer's opinion, when describing dark fermentation, it is not necessary to go into details about electron transport (especially since it probably concerned photofermentation) It is enough to say that methanogens must be eliminated from the process so that they do not produce methane. In this situation, hydrogen is not used in the 3rd and 4th phases of the anaerobic digestion process.

Author Response

Dear Reviewer:

Thank you for reviewing our manuscript and for your valuable comments and suggestions to improve the manuscript quality. Based on your good suggestions, the manuscript has been carefully corrected. We are looking forward to receiving your positive responses.

With best wishes,

Cid Marcos G Andrade.

Comments and Suggestions for Authors

The Authors have made the suggested corrections. However, one passage in the manuscript would require minor clarification.

Line 283 - What are they photosynthetic reactions? Did the Authors mean the processes that take place during photofermentation? The process of photofermetnation and dark fermentation are not the same. They can be combined to increase hydrogen yield and substrate reduction.
In the reviewer's opinion, when describing dark fermentation, it is not necessary to go into details about electron transport (especially since it probably concerned photofermentation) It is enough to say that methanogens must be eliminated from the process so that they do not produce methane. In this situation, hydrogen is not used in the 3rd and 4th phases of the anaerobic digestion process.

Line 283 - What are they photosynthetic reactions? Did the Authors mean the processes that take place during photofermentation?

Yes, there was a typing error, instead of saying “photofermentation” we said “photosynthetic reactions”. Thank you for your observation.

The process of photofermetnation and dark fermentation are not the same.

We appreciate the information. In fact they are not the same, the text was rewritten.

They can be combined to increase hydrogen yield and substrate reduction.

Corroborating what the reviewer mentioned: “according to Maru et. al (2013) dark fermentation is different from photofermentation, and particularly more promising because it is simpler and cheaper. Maru et. al (2013) also mentions that photofermentation can be coupled to the dark fermentation process in order to make the process more profitable and sustainable – being a way to ensure that the acetate is completely oxidized [1].”

In the reviewer's opinion, when describing dark fermentation, it is not necessary to go into details about electron transport (especially since it probably concerned photofermentation) It is enough to say that methanogens must be eliminated from the process so that they do not produce methane. In this situation, hydrogen is not used in the 3rd and 4th phases of the anaerobic digestion process.

We appreciate the suggestions; the text has been rewritten.

The process of producing hydrogen through the fermentation process is mainly carried out by anaerobic digestion, called dark fermentation [2,3]. According to Maru (2016), dark fermentation offers significant advantages when compared to other hydrogen production processes, as it requires less investment, the operating conditions are simpler, and it is environmentally more advantageous [4]. In this process, organic substrates, such as glycerol, are decomposed by hydrogen-producing microorganisms, and methanogenic microorganisms must be eliminated from the process so that they do not produce methane [5,6].

1. Maru, B.T.; Constanti, M.; Stchigel, A.M.; Medina, F.; Sueiras, J.E. Biohydrogen Production by Dark Fermentation of Glycerol Using Enterobacter and Citrobacter Sp. Biotechnol Prog 2013, 29, 31–38, doi:10.1002/btpr.1644.
2. Wong, Y.M.; Wu, T.Y.; Juan, J.C. A Review of Sustainable Hydrogen Production Using Seed Sludge via Dark Fermentation. Renewable and Sustainable Energy Reviews 2014, 34, 471–482.
3. De Gioannis, G.; Muntoni, A.; Polettini, A.; Pomi, R. A Review of Dark Fermentative Hydrogen Production from Biodegradable Municipal Waste Fractions. Waste Management 2013, 33, 1345–1361, doi:10.1016/j.wasman.2013.02.019.
4. Maru, B.T.; López, F.; Kengen, S.W.M.; Constantí, M.; Medina, F. Dark Fermentative Hydrogen and Ethanol Production from Biodiesel Waste Glycerol Using a Co-Culture of Escherichia Coli and Enterobacter Sp. Fuel 2016, 186, 375–384, doi:10.1016/j.fuel.2016.08.043.
5. Chookaew, T.; Prasertsan, P.; Ren, Z.J. Two-Stage Conversion of Crude Glycerol to Energy Using Dark Fermentation Linked with Microbial Fuel Cell or Microbial Electrolysis Cell. N Biotechnol 2014, 31, 179–184, doi:10.1016/j.nbt.2013.12.004.
6. Ghimire, A.; Frunzo, L.; Pirozzi, F.; Trably, E.; Escudie, R.; Lens, P.N.L.; Esposito, G. A Review on Dark Fermentative Biohydrogen Production from Organic Biomass: Process Parameters and Use of by-Products. Appl Energy 2015, 144, 73–95.