Thermophilic Dark Fermentation for Simultaneous Mixed Volatile Fatty Acids and Biohydrogen Production from Food Waste
, Chonticha Mamimin 6,7 and Prawit Kongjan 1,2,*
Round 1
Reviewer 1 Report
The study is interesting, scientifically valid and the paper structure is not complex and adequate. However, the novelty of the study is not clear. What findings and information from this study fill the gaps in the literature on thermophilic dark fermentation of food waste?
Minor revision:
l.85: “H2”, 2 subscripted
l.136: “…….” ?
l. 208: extra space before “The 24”
l.279 Table 2: make the first line smaller because some words are not understood
Briefly comment on the results of the bacterial community analysis in the abstract and conclusion sections
some steps can be improved
Author Response
Responses to reviewers' comments
We are pleased to receive the valuable suggestions from the editor and reviewers, and have modified our manuscript to meet their concerns. Our specific response is provided below the comment of each referee. Grammatical errors are also corrected throughout the revised manuscript.
Reviewer 1
Comments and Suggestions for Authors:
1. The study is interesting, scientifically valid and the paper structure is not complex and adequate. However, the novelty of the study is not clear. What findings and information from this study fill the gaps in the literature on thermophilic dark fermentation of food waste?
Thank you for your question and suggestion. For about novelty, we’ve modified the last paragraph in “the Introduction” (Line 94-110) to demonstrate the challenges, including the inoculum pretreatment, which are required additional investigation prior full-scale implementation for food waste dark fermentation to recovery mixed VFA as the important building block chemicals with a greatly increasing market demand in industry for bioplastics, foods, composites, synthetic fibers, and coating.
“Inoculum pre-treatments, process optimization to achieve high and stable yields, the development of efficient techniques for selective recovery, and the cost-effectiveness of the whole process are the main challenges for full-scale implementation of food waste dark fermentation. Indeed, dark fermentation of food waste to efficiently recovering mixed-VFAs, which are currently the products of interest for further application in sustainable chemical industry is highly depending on sludge pretreatment to deactivated methanogens and physicochemical and operational parameters involved in the fermentative system. Applying thermal, acid, and alkali pretreatment was reported as efficient methods to select high activity microorganisms for dark fermentation [15,16]. Nevertheless, these pretreatment methods are still in doubt for economic viability. Therefore, in this investigation, original anaerobic digested sludge was pretreated by load shock means, which is considered as practical and economic pretreatment to subsequently establish mixed-cultures dark fermentation of food waste under thermophilic temperature (55 °C). Batch fermentation experiment was later investigated to estimate the first order kinetic constant (kh), which was further used to simply design suitable HRT for CSTR operation of mixed-cultures anaerobic fermentation of food waste to evaluating long term performance in continuous operation mode.”
Minor revision:
2. l.85: “H2”, 2 subscripted
It is subscripted throughout the revised manuscript “H2”
3. l.136: “…….” ?
It was deleted.
4. l. 208: extra space before “The 24”
It was edited.
5. l.279 Table 2: make the first line smaller because some words are not understood
It was revised. Abbreviation of ETOH for ethanol is added below Table 2.
6. Briefly comment on the results of the bacterial community analysis in the abstract and conclusion sections
The briefly comment is demonstrated in the in the abstract L 28-29.
“Clostridium thermobutyricum, Clostridium sporogenes, and Octadecobacter sp. were found dominant microbial community in the batch dark fermentation.”
Also in the conclusions, the dominantly bacteria were added in L 447-450.
“Without addition of nutrients and buffers, inoculum pretreated by load shock technique, which was detected dominantly with Clostridium thermobutyricum, Clostridium sporogenes, and Octadecobacter sp. could produce satisfactory mixed-VFA and hydrogen during batch and continuous food waste dark fermentation.”
7. Comments on the Quality of English Language some steps can be improved.
English language revised manuscript was carefully modified and corrected.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The novelty of this work is not very significant as many works in dark fermentation of food waste have been published in the last two years, namely between 2022 and the first months of 2023. In any case, the study shows interesting results.
The manuscript must be deeply corrected for the English language. Many sentences are very confusing jeopardizing the authors' ideas.
The title reflects the manuscript content.
The Abstract needs several explanations. Please, see the attached file containing the reviewer's comments.
Please, revise the keywords trying to select more specific ones.
The Introduction section needs several clarifications. The cited literature is old, being necessary for an update based on papers published in the last two years, namely in 2022 and 2023.
The Materials and Methods section needs a thorough revision. Many explanations are required. For specific comments, please, see the attached file containing the reviewer's comments.
Some Results must be better explained. Also, do not forget that Latin names of species and genera must be written in italic. For specific comments, please, see the attached file containing the reviewer's comments.
The Conclusions section is very short, giving the idea that the authors were already tired to write this part of the manuscript. In any case, some of the most important data is referred to in the Conclusions. Consequences of the work performed and future work and challenges are missing.
As cited above, the Bibliography must be updated as many works about dark fermentation of food wastes have been published recently.
Comments for author File: 
Comments.pdf
The manuscript must be deeply corrected for the English language. Many sentences are very confusing jeopardizing the authors' ideas.
Author Response
Responses to reviewers' comments
We are pleased to receive the valuable suggestions from the editor and reviewers, and have modified our manuscript to meet their concerns. Our specific response is provided below the comment of each referee. Grammatical errors are also corrected throughout the revised manuscript.
Reviewer 2
Comments and Suggestions for Authors:
1. The novelty of this work is not very significant as many works in dark fermentation of food waste have been published in the last two years, namely between 2022 and the first months of 2023. In any case, the study shows interesting results.
Thank you for your question and suggestion. For about novelty, we’ve modified the last paragraph in “the Introduction” (Line 94-110) to demonstrate the challenges, including the inoculum pretreatment, which are required additional investigation prior full-scale implementation for food waste dark fermentation to recovery mixed VFA as the important building block chemicals with a greatly increasing market demand in industry for bioplastics, foods, composites, synthetic fibers, and coating.
“Inoculum pre-treatments, process optimization to achieve high and stable yields, the development of efficient techniques for selective recovery, and the cost-effectiveness of the whole process are the main challenges for full-scale implementation of food waste dark fermentation. Indeed, dark fermentation of food waste to efficiently recovering mixed-VFAs, which are currently the products of interest for further application in sustainable chemical industry is highly depending on sludge pretreatment to deactivated methanogens and physicochemical and operational parameters involved in the fermentative system. Applying thermal, acid, and alkali pretreatment was reported as efficient methods to select high activity microorganisms for dark fermentation [15,16]. Nevertheless, these pretreatment methods are still in doubt for economic viability. Therefore, in this investigation, original anaerobic digested sludge was pretreated by load shock means, which is considered as practical and economic pretreatment to subsequently establish mixed-cultures dark fermentation of food waste under thermophilic temperature (55 °C). Batch fermentation experiment was later investigated to estimate the first order kinetic constant (kh), which was further used to simply design suitable HRT for CSTR operation of mixed-cultures anaerobic fermentation of food waste to evaluating long term performance in continuous operation mode.”
2. The manuscript must be deeply corrected for the English language. Many sentences are very confusing jeopardizing the authors' ideas.
Thank you very much for suggestion to improve English language. We did correct carefully English language throughout the revised manuscript.
3. The title reflects the manuscript content.
Thank you for this support.
4. The Abstract needs several explanations. Please, see the attached file containing the reviewer's comments.
Please see our response for the comment in the attached file.
5. Please, revise the keywords trying to select more specific ones.
The following keywords were modified in the revised manuscript.
“Keywords: Organic waste; Mixed culture fermentation; Volatile fatty acids; Bio-hydrogen”
6. The Introduction section needs several clarifications. The cited literature is old, being necessary for an update based on papers published in the last two years, namely in 2022 and 2023.
The introduction section was replaced with more new references as follow.
Villanueva-Galindo, E.; Vital-Jácome, M.; Moreno-Andrade, I. Dark fermentation for H2 production from food waste and novel strategies for its enhancement. International Journal of Hydrogen Energy 2023, 48 (27), 9957-9970.
Luo, L.; Sriram, S.; Johnravindar, D.; Martin, T. L. P.; Wong, J. W.; Pradhan, N. Effect of inoculum pretreatment on the microbial and metabolic dynamics of food waste dark fermentation. Bioresource Technology 2022, 358, 127404.
7. The Materials and Methods section needs a thorough revision. Many explanations are required. For specific comments, please, see the attached file containing the reviewer's comments.
Please see our response for the comment in the attached file.
8. Some Results must be better explained. Also, do not forget that Latin names of species and genera must be written in italic. For specific comments, please, see the attached file containing the reviewer's comments.
Please see our response for the comment in the attached file.
L228-242 “The main characteristics of food waste and original inoculum used in this investiga-tion were demonstrated in Table 1. The 24- C/N ratio of food waste collected from the uni-versity canteen indicates that amounts of carbon and nitrogen contained in food waste are suitable to be used as a major substrate for dark fermentation. …………………… Hydrogen yields of 135.2 ±8.6 mL H2/VSadded and 80.6±5.3 mL H2/VSadded, respectively, were consequently obtained in the stationary phase of batch fermentation of food waste and glucose at initial concentration of 10 g-VS/L (Fig.1).”
L271-287 “Presumably, further increasing food waste concentration higher than 13%W/V, mixed microbial cultures in the fermentation system could have declined hydrolysis activity. ……………….… Methanogens in original anaerobic sludge usually preferring pH range between pH 7 and pH 8 for their growth [27] could be eventually deactivated under acidic condition. The butyrate and acetate as the major metabolites in mixed-acid metabolic pathway were previously generated during dark fermentation [28]. The production of the mixed acids can be affected by various factors such as composition of the microbial consortium [15],”
L290-296 “Luo et al. [16] also reported that highest butyric acid concentration over other metabolites (acetic acid, propionic acid, lactic acid, and formic acid) ……….. Therefore, inoculum pretreatments could affect the economic viability for mixed-cultures dark fermentation process and requires careful consideration.”
L330-332 Formation of considerably high concentration of lactic acid in the dark fermentation process could be the indicator for having lower hydrogen yield and unstable or overloading conditions [23]. ”
L366-371 “. According to Chen et al. [38] research, VFAs, and hydrogen producer bacteria (Thermoanaerobacterium and Clostridium sensu stricto 1) were found as a domain bacteria more at the thermophilic temperature conditions than the mesophilic temperature in the dark fermentation process, due to this bacteria ability tolerance at high temperature conditions and acid-base range 5-6.”
L 373-387“. During the first 4-day start-up period, hydrogen composition in the fermented gas of 5, 11, 25, and 36 (%V/V) was respectively detected on day 1, day 2, day 3, and 4. Food waste with 26.4 g-VS/L was fed to the CSTR at designed HRT of 4 days, corresponding to organic loading rate (OLR) of 7.6 g-VS/L d under 55 ℃-thermophilic temperature. ………….. ………….. As demonstrated in Fig.4, catabolic metabolites including butyric acid, acetic acid, lactic acid, ethanol, propionic acid, and hydrogen were generated from food waste fermentation. Formation of such soluble metabolites must be the major cause for pH reduction in the fermentation system.”
L 393-396 “corresponding to hydrogen yield of 104.9± 11.0 mL-H2/g-VSadded. Villanueva-Galindo et al. [15] reviewed that hydrogen yields obtained from continuous dark fermentation of various types of food waste are in the range between 8.8±0.6 mL-H2/g-VS and 103.6±0.6 mL-H2/g-VS.”
L 401-406 “Additionally, cyclic hydrogen production (Fig.4a) with around 10% standard deviation is observed under steady state. This cyclic fluctuation could possibly due to pH always changed in the continuous fermentation system (without pH control). Hydrogen is generated from proton (H+) reduction by hydrogenase, which is highly inhibited by pH. Proton is poor electron acceptor. Therefore, electron is easily shifted to other reduced products of lactic acid and propionic acid [42].”
L 438-445 “Therefore, not only clean bio-ethanol fuel is produced but also greenhouse gases (CO and CO2) are sequestrated by applying syngas fermentation using suitable microorganisms. Furthermore, bio-based VFAs from organic wastes, as carbon source could be potentially utilized for biological nutrient removal in denitrification process and for microbial lipids for further producing bio-diesel [4]. Interestingly, Vu et al. [48] reported PHA content of 1.3 g/L was achieved successfully after 128 h of cultivation in the immersed membrane bioreactor (iMBR) fed with potato liquor and apple pomace-based VFAs with a total concentration of 8.8 g/L.”
Latin names of species and genera were edited in italic. The attached file containing the reviewer's comments were answered below.
9. The Conclusions section is very short, giving the idea that the authors were already tired to write this part of the manuscript. In any case, some of the most important data is referred to in the Conclusions. Consequences of the work performed and future work and challenges are missing.
The Conclusions section was modified as demonstrated in L 447-457.
“Without addition of nutrients and buffers, inoculum pretreated by load shock technique, which was detected dominantly with Clostridium thermobutyricum, Clostridium sporogenes, and Octadecobacter sp. could produce satisfactory mixed-VFAs and hydrogen during batch and continuous food waste dark fermentation. The appropriate HRT for CSTR operation can be simply designed by using kinetic data obtained from batch food waste dark fermentation. Thermophilic dark fermentation of food waste using mixed-cultures to simultaneously produce butyric rich VFAs, and hydrogen rich biogas was eventually established for continuous operation in the CSTR successfully. Therefore, experimental results obtained can be extrapolated to pilot scale implementation. To be more economically feasible, further investigation to replace glucose with food waste for inoculum load shock pretreatment.”
10. As cited above, the Bibliography must be updated as many works about dark fermentation of food wastes have been published recently.
The following recent publications were replaced.
Yahya, M.; Herrmann, C.; Ismaili, S.; Jost, C.; Truppel, I.; Ghorbal, A. Kinetic studies for hydrogen and methane co-production from food wastes using multiple models. Biomass and bioenergy 2022, 161, 106449.
Gottardo, M.; Dosta, J.; Cavinato, C.; Crognale, S.; Tonanzi, B.; Rossetti, S.; Bolzonella, D.; Pavan, P.; Valentino, F. Boosting butyrate and hydrogen production in acidogenic fermentation of food waste and sewage sludge mixture: a pilot scale demonstration. Journal of Cleaner Production 2023, 404, 136919.
Villanueva-Galindo, E.; Vital-Jácome, M.; Moreno-Andrade, I. Dark fermentation for H2 production from food waste and novel strategies for its enhancement. International Journal of Hydrogen Energy 2023, 48 (27), 9957-9970.
Luo, L.; Sriram, S.; Johnravindar, D.; Martin, T. L. P.; Wong, J. W.; Pradhan, N. Effect of inoculum pretreatment on the microbial and metabolic dynamics of food waste dark fermentation. Bioresource Technology 2022, 358, 127404.
Kim, S.-H.; Kumar, G.; Chen, W.-H.; Khanal, S. K. Renewable hydrogen production from biomass and wastes (ReBioH2-2020). Elsevier: 2021; Vol. 331, p 125024.
Moreno‐Andrade, I.; Berrocal‐Bravo, M. J.; Valdez‐Vazquez, I. Biohydrogen production from food waste and waste activated sludge in codigestion: influence of organic loading rate and changes in microbial community. Journal of Chemical Technology & Biotechnology 2023, 98 (1), 230-237
11. Comments on the Quality of English Language
The manuscript must be deeply corrected for the English language. Many sentences are very confusing jeopardizing the authors' ideas.
Thank you very much for suggestion to improve English language. We did correct carefully English language through the revised manuscript.
Reviewer 2
Comment from the attached file
1. L 20 “Food waste as abundant carbohydrates” Bad English! Re-phrase. Food waste is not only rich in carbohydrates, but also in proteins and lipids.
The modified sentence was stated in L 20.
“Food waste is organic solid waste having negative impact on sustainable environment.”
2. L 23 “The enriched inoculum” For what reason do you name the inoculum as an enriched inoculum?
We indeed mean inoculum pretreated by load shock pretreatment. Therefore “the pretreated inoculum” was used instead.
3. L 25 “(g-VS/L)” Please, insert the unit just after the last number and not in the beginning of a set of numbers.
It was change as stated in L 25. “….20.3, and 26.4 g-VS/L”
4. L 29 “The CSTR inoculated” For readers not used with this acronym, please, write it for the first time in the full mode and include the acronym between brackets.
The term of “The Continuous Stirred Tank Reactor (CSTR)” was stated in L 30-31.
5. L 29 “enriched mixed-cultures” "Enriched" in what?
The term of “enriched mixed-cultures” was changed to the term of “…mixed-cultures also prepared by load shock pretreatment” as stated in L 31.
6. L 30“was fed food waste” Bad English! Re-phrase it!
It was corrected to “…was operated by feeding 26.4 g-VS/L-food waste at kinetically designed HRT of 4 days,….” as stated in L 32-33.
7. L 33-34 “hydrogen production yield of 110.8 mL- H2/g-VSadded.” "Compared to the literature, what is the big finding of this research?
The hydrogen yield from dark fermentation is usually compared with 373.57 mL-H2/g-VS (Eq. 3 in the revised manuscript) as stated in L 34-35.
“….hydrogen yield of 110.8± 5.8 mL-H2/g-VSadded, which is higher than the reported yield in a range between 8.8±0.6 mL-H2/g-VS and 103.6±0.6 mL-H2/g-VS.”
8. L 35 “Bio-chemicals” This is too broad. Please, select a more specific keyword.
The key word of “Volatile fatty acids” was used instead of Bio-chemicals”
9. L 37“Introduction” References cited in the Introduction section are old. More recent references are needed. An example that authors have missed: Appl. Sci. 2022, 12(9), 4240; https://doi.org/10.3390/app12094240 But, many others can be cited: https://doi.org/10.1016/j.renene.2023.04.114
https://doi.org/10.1016/j.jclepro.2023.136919
https://doi.org/10.1016/j.fuel.2022.127376
https://doi.org/10.3390/en16073281
https://doi.org/10.1007/s13399-021-02056-x
Some old references were removed and current references were added.
Martins, I.; Surra, E.; Ventura, M.; Lapa, N. BioH2 from Dark Fermentation of OFMSW: Effect of the Hydraulic Retention Time and Organic Loading Rate. Applied Sciences 2022, 12 (9), 4240.
Banmairuroy, W.; Kritjaroen, T.; Homsombat, W. The effect of knowledge-oriented leadership and human resource development on sustainable competitive advantage through organizational innovation's component factors: Evidence from Thailand’s new S-curve industries. Asia Pacific Management Review 2022, 27 (3), 200-209.
Gottardo, M.; Dosta, J.; Cavinato, C.; Crognale, S.; Tonanzi, B.; Rossetti, S.; Bolzonella, D.; Pavan, P.; Valentino, F. Boosting butyrate and hydrogen production in acidogenic fermentation of food waste and sewage sludge mixture: a pilot scale demonstration. Journal of Cleaner Production 2023, 404, 136919.
Chen, H.; Yang, T.; Shen, Z.; Yang, E.; Liu, K.; Wang, H.; Chen, J.; Sanjaya, E. H.; Wu, S. Can digestate recirculation promote biohythane production from two-stage co-digestion of rice straw and pig manure? Journal of Environmental Management 2022, 319, 115655.
Villanueva-Galindo, E.; Vital-Jácome, M.; Moreno-Andrade, I. Dark fermentation for H2 production from food waste and novel strategies for its enhancement. International Journal of Hydrogen Energy 2023, 48 (27), 9957-9970.
Luo, L.; Sriram, S.; Johnravindar, D.; Martin, T. L. P.; Wong, J. W.; Pradhan, N. Effect of inoculum pretreatment on the microbial and metabolic dynamics of food waste dark fermentation. Bioresource Technology 2022, 358, 127404.
Nam, J.-Y. Optimum Conditions for Enhanced Biohydrogen Production from a Mixture of Food Waste and Sewage Sludge with Alkali Pretreatment. Energies 2023, 16 (7), 3281.
Moreno‐Andrade, I.; Berrocal‐Bravo, M. J.; Valdez‐Vazquez, I. Biohydrogen production from food waste and waste activated sludge in codigestion: influence of organic loading rate and changes in microbial community. Journal of Chemical Technology & Biotechnology 2023, 98 (1), 230-237
10. L 39 “total solid waste.” Do you mean "municipal solid wastes"?
Yes, it is municipal solid wastes food waste. It was replaced with “the municipal solid wastes” as shown in L 41.
11. L 42 “valorization” You should write "final destination/valorization" as landfilling is not a valorisation pathway, but a final disposal method.
It was changed to “final destination/valorization” as stated in L 45.
12. L 44“carbohydrates” Be careful, because food waste is not only rich in carbohydrates but also in proteins and lipids. The chemical properties are highly dependent on the food waste composition, such as the amounts of vegetables, fruits, meat, cooked meals, etc.
The modification for this comment is stated in L 45-47.
“Food waste is rich in carbohydrates, proteins, and lipids [2]. Nevertheless, food waste, which is majorly contained with carbohydrates…..”
13. L 49“butyric, acetic, and propionic are important” The term "acids" is missing in this part of the sentence.?
It is added as shown in L 51-52.
“Volatile fatty acids (VFA) such as butyric acid, acetic acid, and propionic acid..”
- L 55 "I'm sorry, but this is not an adequate way to define dark fermentation as within "mixed-cultures anaerobic fermentation" you may find several processes such as ethanolic fermentation, lactic fermentation, or even anaerobic digestion, and none of these fermentation pathways produces bioH2 as in the case of dark fermentation.
Thank you very much for this valuable comment. Mixed culture fermentation is then defined, regarding to the research work entitled Modeling Product Formation in Anaerobic
Mixed Culture Fermentations by Rodrı´guez et al, (2006) as stated in L 58-60.
“Mixed culture fermentation for potential production of VFA, lactic acid, and alchohols, which are the building blocks chemicals for others environmentally friendly industries is potentially feasible, according to the kinetic and thermodynamic modeling. Additionally, [6].”
Dark fermentation is typically called for applying mixed culture fermentation to produce hydrogen from carbohydrates rich substrate as stated in L 61-63.
“When the interested product generated from mixed culture fermentation of carbohydrates rich substrate is hydrogen, which is thermodynamically directed to only butyric acid and acetic acid, such fermentation process is typically called dark fermentation [2].”
15. L 73 “less risk of contamination by methanogenic archaea” I'm sorry, but I do not agree with this part of the sentence as methanogenic archaea also grow fast under thermophilic conditions.
Thank you very much for this comment. The term of “less risk of contamination by methanogenic archaea” was deleted.
16. L 76“enriched” "Enriched" in what?
Thank you for your concern. In this context, “the mixed cultures” (L 83) are better used.
17. L 91“enriched” "Enriched" in what?
To avoid confusing to use this “enriched”, The sentence stated for preparation original inoculum by using load shock pretreatment is written in L 103-104.
“Therefore, in this investigation, original anaerobic digested sludge was pretreated by load shock means”
18. L 100-101 “Food waste collected from the main canteen of Prince of Songkla University, Pattani campus, Thailand was separated bones out” Bad English! Please, re-phrase it!
We have re-phrased as demonstrated in L 108-110.
“Food waste was collected from the main canteen of Prince of Songkla University, Pattani campus, Thailand. The bones content in the food waste were picked out. Free bones food waste was then crushed with an electric blender to homogenize the food waste.”
19. L 102“temperature-4°C” Add a space!
We’ve corrected in L 116. “Temperature -4 °C”
20. L 104“fat” Do you mean "lipids"? and “carbohydrate” Do you mean "carbohydrates"?
The corrected words are added in L 118. “…lipids, carbohydrates, and…”
21. L 113 “-serum bottles” Do you think that serum bottles are adequate for these batch assays? Don't you think that the accumulation of H2 inside the reactors may have a negative impact on microbial populations?
Yes, hydrogen partial pressure could possibly cause negative impact. Nevertheless, having sufficient head space of around 52% of total volume and having gas release daily for measurement production gas volume left enough for gas production could potentially avoid the negative impact as reviewer’s concern.
22. L 117 “Closed bottle was purged” How is it possible to purge a "closed bottle"?
Closed bottle can be purged by inserting a pair of syringe needles through the butyl stopper as stated in L 132-133.
“Closed bottle was purged with N2 for 3-5 min to create anaerobic conditions by inserting a pair of syringe needles through the butyl stopper.”
23. L 117 “N2” How did you discount the N2 in the biogas collected and analysed from these batch
Purging N2 could be possible detected by GC-TCD in the first 2-3 days of the batch fermentation with slightly low concentration. As both total gas volume and gas composition measured, so we can easily subtract from the generated gas for further data synthesis.
24. L 121 “working” Do you mean "working volume"?
It is changed to “working volume” as shown in L 128.
25. L 123“(% Wet weight of food waste per working volume, %W/V)” Insert the unit after the last number of the set of numbers and not in the beginning.
It is edited as shown in L 139-140. “ …(% Wet weight of food waste per working volume, %W/V)”
26. L 125-126 “30 % of the working volume of the thermophilic inoculum and balanced volume of tap water was added” Bad English! Please, re-phrase it!
We’ve corrected as shown in L 141-143.
“The thermophilic inoculum around 30 % of the working volume was added to fermenter. Tap water was subsequently added to the fermenter until reaching 3.5 L-working volume.”
27. L 129 “-temperature by circulating” Bad English! Please, re-phrase it!
We’ve corrected as shown in L 145-146.
“The fermenter temperature was controlled at 55 °C by circulating hot water via the water jacket of the fermenter.”
28. L 131“Microbial communities were analyzed” Please, explain how microbial communities have been analysed.
We’ve explained as shown in L148-150
“Sludge samples were collected at the end of stationary phase for further analysis of microbial communities by using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method.”
29. L 133-134 “obtained from various initial food waste concentrations was subsequently evaluated the first order kinetic con” Bad English! Please, re-phrase it!
The modified sentence is given as shown in L151-153
“The cumulative results of butyric acid production obtained from batch food waste dark fermentation at different initial concentration were used to estimate the first order kinetic constant (kh, hr-1) according to Eq. (1) [17]].”
30. L 136“ equation (1)” For the first time that it is stated in research work, a first-order kinetic model must be shown in its original (exponential) form and not in its linear form. If you have used the linear form instead of the exponential form, then you must state it and show the linear equation that you have used.
The exponential form of the equation 1 is demonstrated in L 154-158.
(1)
B∞: maximum butyric acid concentration (g/L)
B: cumulative butyric acid concentration (g/L) at a given time
t: incubating time (hr.)”
31. L 143-145 “active volume” Do you mean "working volume"?
Yes, we mean “working volume”.
32. L 146 “(30.3 g-VS/L).” This concentration is a little bit higher than the one described above for 13% W/V. Can you add a short explanation on this, please?
We are sorry for this mistake. Indeed, the Food waste concentration of 13 % (W/V) is 26.4 g-VS/L.
33. L 148 “13% W/V-concentration” It is better to express this value in g VS/L.
The food waste concentration in g-VS/L is demonstrated in L 168.
“…with 26.4 g-VS/L-concentration was continuously…”
34. L 152 “ultimate hydrogen” How do you define the "ultimate hydrogen yield"? Please, add this explanation to the text of the manuscript.
The ultimate hydrogen yield is defined as demonstrated in L 171-172.
“Where ym is an ultimate/maximum hydrogen yield obtained from the stationary phase of the cumulative hydrogen yield of batch food waste dark fermentation as shown in Fig. 1”
The ym of batch food waste fermentation is reported in L 238-242.
“Hydrogen production yield of 135.2 ±8.6 mL H2/VSadded and 80.6±5.3 mL H2/VSadded, respectively, was obtained in the stationary phase of batch fermentation of food waste and glucose as shown in Fig.1.”
35. L 152 “BHP” What is the meaning of this acronym?
In this circumstance, the term of “batch food waste dark fermentation” (L 172) is used instead of BHP (Biological hydrogen potential).
36. L 153“which is around 80% of the ym” Why?
We did explanation for this concern as demonstrated in L 173-176.
“Kongjan et al. [19] reported that the HRT calculated by using the expected yield (y) around 80% of the ultimate yield (ym) is practically feasible to be operated the CSTR for dark fermentation at the maximum organic loading rate (OLR) of 11.3 g-VS/d L. The OLR of 7.6 g-VS/L·d was achieved for food waste fermentation by using calculated HRT of 4 days and initial food waste concentration of 26.4 g-VS/L.”
37. L 156 “linked to butyric acid and acetic acid pathway,” Also, PFOR may also participate in H2 production.
The pyruvate: ferredoxin oxidoreductase (PFOR) is added in L 179-180.
“catalysed by the pyruvate: ferredoxin oxidoreductase (PFOR) [20]”
38. L 157 “the product generation-time data for HRT estimation is better adopted from hydrogen production than the VFAs.” Using equation 2, what was the HRT calculated for continuous flow assay? Can you compare it with other HRT reported in the literature?
The HRT of 4 days was achieved as stated in L 375.
“…to the CSTR at designed HRT of 4 days…”
Other HRT reported in the literature are also stated in L 376-378
“The appropriate HRT between 2 days and 6 days is usually selected to evaluate optimum performance of continuous dark fermentation of an organic waste in the CSTR [39].”
39. L 162-164 "This method is controversial. It would be better that you have measured directly the total sugars
We are sorry for this concern. We had a technical problem to directly measure the total sugars during this experimental work. Nevertheless, Kaparaju et al. (2009) used the subtracting method to roughly estimate carbohydrates content. The reference was cited in L 186-188.
“The carbohydrates content was estimated by subtracting the protein lipid and total phenolic compounds from the volatile solids [22].”
40. L 170-171 “25oC/min” and “240oC.” Please, correct.
The term of “ °C ” was added as demonstrated in L 193-194.
41. L 205-207 “The COD/VS ratio of 1.30 is implied that food waste having high quantity of organic matters (carbohydrates, proteins, and lipids) in a form of VS is majorly consisted of carbohydrates,” This sentence is very confusing! It must be re-written.
We’ve stated the COD/VS ratio of 1.30 in the context of COD balance in L 412.
42. L 208-209 “24-C/N ratio of food waste” Such a high C/N ratio for food waste indicates that the used food waste must be composed mainly of vegetables and fruits. Food waste richer in meat or cooked food is currently richer in proteins giving rise to N-content and, consequently, decreasing the C/N ratio.
Thank you very much for this suggestion. Additional discussion is stated in L 231-236.
“Such a high C/N ratio for food waste indicates that the used food waste must be composed mainly of carbohydrate (rice, vegetables and fruits), which could be considerably degraded by fermentative bacteria to VFAs along with mixed hydrogen-carbon dioxide gas [24]. Food waste richer in meat or cooked food is currently richer in proteins giving rise to N-content and, consequently, decreasing the C/N ratio.”
43. L 213-214 “The mixed-cultures obtained by shock load pretreatment of anaerobic digested sludge to suppress methanogens was tested its performance” Bad English! Please, re-write.
The re-written sentences are stated in L 238-242.
“Mixed-cultures obtained by shock load pretreatment of anaerobic digested sludge were used as inoculum without addition of nutrients and buffer in batch fermentation. Hydrogen yields of 135.2 ±8.6 mL H2/VSadded and 80.6±5.3 mL H2/VSadded, respectively, were consequently obtained in the stationary phase of batch fermentation of food waste and glucose at initial concentration of 10 g-VS/L (Fig.1).”
44. L 228-230 “Therefore, inoculum pretreatments could affect the economic viability for mixed-cultures dark fermentation process and requires careful consideration.” I don't see the relation of this idea with the previous discussion on the fact that hydrogen yield does not reach the maximum theoretical value. Can you explain better, please?
We’ve stated the concerned sentence with another relevance as shown in L 290-296.
“Luo et al. [16] also reported that highest butyric acid concentration over other metabolites (acetic acid, propionic acid, lactic acid, and formic acid) was generated from food waste fermentation using mixed microbial cultures, previously pretreated by acid, alkali, and thermal pretreatment. These pretreatment techniques seem to be expensive for implementation in the industrial scale because chemicals and energy are required for inoculum pretreatment. Therefore, inoculum pretreatments could affect the economic viability for mixed-cultures dark fermentation process and requires careful consideration.”
45. L 246-248 “indicating that mixed microbial cultures were reaching fully capable of converting food waste to metabolite products.” This part of the sentence is confusing. What do authors mean?
We actually mean substrate degradation activity of mixed-cultures is lowered by increasing food waste concentration higher than 13%W/V. The explanation is stated in L 271-272.
“Presumably, further increasing food waste concentration higher than 13%W/V, mixed microbial cultures in the fermentation system could have declined hydrolysis activity.”
46. L 250-251 “The production of the mixed acids can be affected by various factors such as the concentration and characteristics of the substrates, pH, redox potential, etc. [20].” Also, the composition of the microbial consortium is very important. If lactic fermentative bacteria are predominant (effectively, they are present in food wastes), then high concentrations of lactate will be produced without H2 production.
Thank you very much
47. L 254-257 “However, the detected ethanol confirms that acetate-ethanol type metabolic pathway also accounting in this fermentation. Luo et al. [21] also reported that highest butyric acid concentration over other metabolites (acetic acid, propionic acid, lactic acid, and formic acid) was generated from food waste fermentation using mixed microbial cultures.” "What is the relation between these two sentences in this same paragraph? For what reason are you relating acetate-ethanol metabolic pathway with butyric acid pathway?
We are sorry for this mistaken. As no relation be
48. L 278“Table 2.” This table is very difficult to read as the columns are very narrow. Please, re-arrange the table for better reading.
We’ve re-arranged Table 2 in the revised manuscript.
49. L 288-290 “Clostridium thermobutyricum, butyric acid producer [27] and genus Lactobacillus (Lactobacillus fermentum, Lactobacillus delbrueckii, and Lactobacillus crispatus), lactic acid producing bacteria” The names in the Latin language must be written in italic. Therefore, the species and genus names must be written in italic. This comment applies throughout the manuscript whenever species and genera are cited.
We’ve checked and re-written in italic throughout the revised manuscript.
50. L 291 “lactic acid as one of major metabolites” What is the consequence of having the lactic fermentation as one of the most relevant metabolic pathways in the dark fermentation process?
We have stated the consequence of having the lactic fermentation as shown in L 330-332.
“Formation of considerably high concentration of lactic acid in the dark fermentation process could be the indicator for having lower hydrogen yield and unstable or overloading conditions [23].”
51. L 304-305 “Lactobacillus such as Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus delbrueckii”The names in the Latin language must be written in italic. Therefore, the species and genera names must be written in italic.
We have re-written the species and genera names in italic throughout the revised manuscript.
- L 318 “hydrogen” For what reason do you think the H2 production is cyclic (Figure 4a)?
We’ve tried to explain this phenomenon as stated in L 401-406.
“Additionally, cyclic hydrogen production (Fig.4a) with around 10% standard deviation is observed under steady state. This cyclic fluctuation could possibly due to pH always changed in the continuous fermentation system (without pH control). Hydrogen is generated from proton (H+) reduction by hydrogenase, which is highly inhibited by pH. Proton is poor electron acceptor. Therefore, electron is easily shifted to other reduced products of lactic acid, propionic acid [42].”
53. L 318 “(%V/V)” Write the unit after the values and not before. Otherwise, the sentence becomes difficult to read.
The unit of “(%V/V) is written after the values in L 374.
54. L 326 “Formation of mixed soluble metabolites, accompanied by pH reduction in the fermentation system.” Bad English! Please, re-phrase it.
We’ve re-phrased this sentence as stated in L 386-387.
“Formation of these soluble metabolites must be the major cause for pH reduction in the fermentation system.”
55. L 342-346 “Operating dark fermentation of food waste by using modified CSTR equipped with radial impellers at OLR of 6 g-VS/L d (HRT 5 days) and temperature 37ฐC), total VFA concentration of 16.04 g-VS/L (0.52 g/g-VS), containing acetic acid 39.5%, butyric acid, 23.6%, caproic acid: 24.9%, propionic 5.7%, and valeric acid 2.7% was previously reported by Wainaina et al., [36].” Too complex sentence! It must be spared in at least two sentences.
This complex sentence is separated in two sentences as demonstrated in L 231-236.
“The modified CSTR equipped with radial impellers was fed with food waste OLR of 6 g-VS/L d (HRT 5 days) and temperature 37 °C). Consequently, total VFAs concentration of 16.04 g-VS/L (0.52 g/g-VS), containing acetic acid 39.5%, butyric acid, 23.6%, caproic acid: 24.9%, propionic 5.7%, and valeric acid 2.7% was achieved[24].”
56. L 368-370“hydrogen rich bio-syngas after being used as sparging gas to recover butanol from ABE fermentation broth by as previously stated, could be possibly converted to ethanol” From an energy point of view, what is the interest in converting H2 into ethanol?
We’ve stated the interest in converting hydrogen rich syngas, which is not pure hydrogen in L 438-440.
“Therefore, not only clean bio-ethanol fuel is produced but also greenhouse gases (CO and CO2) are sequestrated by applying syngas fermentation using suitable microorganisms.”
57. L 374-377“Bio-based VFA from organic wastes, as carbon source could be potentially utilized for biological nutrient removal in denitrification process and for microbial lipids for further producing bio-diesel” Don't you think that VFAs may be used in more interesting processes such as the feeding of microbial populations able to produce biopolymers (such as PHA or PHB)?
58. L 378“Conclusions” Even very short, the conclusions fit the most interesting data from your work.
We’ve revised the conclusions with additional composition of the microbial community in L447-457.
“Without addition of nutrients and buffers, inoculum pretreated by load shock technique, which was detected dominantly with Clostridium thermobutyricum, Clostridium sporogenes, and Octadecobacter sp. could produce satisfactory mixed-VFAs and hydrogen during batch and continuous food waste dark fermentation. The appropriate HRT for CSTR operation can be simply designed by using kinetic data obtained from batch food waste dark fermentation. Thermophilic dark fermentation of food waste using mixed-cultures to simultaneously produce butyric rich VFAs, and hydrogen rich biogas was eventually established for continuous operation in the CSTR successfully. Therefore, experimental results obtained can be extrapolated to pilot scale implementation. To be more economically feasible, further investigation to replace glucose with food waste for inoculum load shock pretreatment.”
59. L 402“References” Bibliographic references must be updated with more recent bibliographic citations. Several studies have been published in the last three years (2021-2023) on the dark fermentation of food waste.
The following recent publications have been cited in the References
Yahya, M.; Herrmann, C.; Ismaili, S.; Jost, C.; Truppel, I.; Ghorbal, A. Kinetic studies for hydrogen and methane co-production from food wastes using multiple models. Biomass and bioenergy 2022, 161, 106449.
Gottardo, M.; Dosta, J.; Cavinato, C.; Crognale, S.; Tonanzi, B.; Rossetti, S.; Bolzonella, D.; Pavan, P.; Valentino, F. Boosting butyrate and hydrogen production in acidogenic fermentation of food waste and sewage sludge mixture: a pilot scale demonstration. Journal of Cleaner Production 2023, 404, 136919.
Villanueva-Galindo, E.; Vital-Jácome, M.; Moreno-Andrade, I. Dark fermentation for H2 production from food waste and novel strategies for its enhancement. International Journal of Hydrogen Energy 2023, 48 (27), 9957-9970.
Luo, L.; Sriram, S.; Johnravindar, D.; Martin, T. L. P.; Wong, J. W.; Pradhan, N. Effect of inoculum pretreatment on the microbial and metabolic dynamics of food waste dark fermentation. Bioresource Technology 2022, 358, 127404.
Kim, S.-H.; Kumar, G.; Chen, W.-H.; Khanal, S. K. Renewable hydrogen production from biomass and wastes (ReBioH2-2020). Elsevier: 2021; Vol. 331, p 125024.
Moreno‐Andrade, I.; Berrocal‐Bravo, M. J.; Valdez‐Vazquez, I. Biohydrogen production from food waste and waste activated sludge in codigestion: influence of organic loading rate and changes in microbial community. Journal of Chemical Technology & Biotechnology 2023, 98 (1), 230-237
Martins, I.; Surra, E.; Ventura, M.; Lapa, N. BioH2 from Dark Fermentation of OFMSW: Effect of the Hydraulic Retention Time and Organic Loading Rate. Applied Sciences 2022, 12 (9), 4240.
Nam, J.-Y. Optimum Conditions for Enhanced Biohydrogen Production from a Mixture of Food Waste and Sewage Sludge with Alkali Pretreatment. Energies 2023, 16 (7), 3281.
Author Response File: Author Response.docx
Reviewer 3 Report
In the manuscript food waste was used as abundant carbohydrates for simultaneously feasible generation of mixed volatile fatty acids (VFA) and bio-hydrogen by deploying dark fermentation. The effect of different concentrations of food waste (g-VS/L), organic loading rate (OLR) and the microbial characteristics were investigated. The results support that thermophilic dark fermentation of food waste using enriched mixed-cultures to produce butyric rich VFA and hydrogen rich bio-syngas was established successfully. However, necessary modifications still need to be made before its final publication.
Specific comments：
1. Abstract can be improved. Stand out the innovation and focus on the topic.
2. Keywords can be supplemented, i.e. "anaerobic digestion, volatile fatty acids (VFA), hydrogen production yield..." can be added.
3. Insufficient innovation was exhibited in the text.
4. Table 1 can be improved.
5. Figure 1 and Figure 3 can be improved.
6. Some recent results can be cited, such as Journal of Environmental Management, 2022, 319, 115655; Chemosphere, 2022, 286(1), 131655; Bioresource Technology, 2021, 320(A): 124303.
7. Further discussions on the dark fermentation of the food waste can be supplemented with in-depth comparison analyses.
8. Check the consistency of the manuscript. Fix grammar and formatting mistakes.
Author Response
Responses to reviewers' comments
We are pleased to receive the valuable suggestions from the editor and reviewers, and have modified our manuscript to meet their concerns. Our specific response is provided below the comment of each referee. Grammatical errors are also corrected throughout the revised manuscript.
Reviewer3
Comments and Suggestions for Authors:
1. In the manuscript food waste was used as abundant carbohydrates for simultaneously feasible generation of mixed volatile fatty acids (VFA) and bio-hydrogen by deploying dark fermentation. The effect of different concentrations of food waste (g-VS/L), organic loading rate (OLR) and the microbial characteristics were investigated. The results support that thermophilic dark fermentation of food waste using enriched mixed-cultures to produce butyric rich VFA and hydrogen rich bio-syngas was established successfully. However, necessary modifications still need to be made before its final publication.
Thank you for your valuable comments. This manuscript was corrected and modified, accordingly.
2. Abstract can be improved. Stand out the innovation and focus on the topic.
The abstract was modified to focusing on the innovation for inoculum pretreatment by using load shock pretreatment as demonstrated in L 20-36.
“Food waste is organic solid waste having negative impact on sustainable environment. It was used for simultaneously feasible generation of mixed volatile fatty acids (VFAS) and bio-hydrogen by deploying dark fermentation. Original anaerobic digested sludge was prepared by shock technique with glucose 50 g/L under thermophilic temperature (55 °C). The pretreated inoculum was capable of converting 10 g-VS/L food waste to hydrogen with rather high yield of 135.2±7 mL H2/VSadded. The effect of various food waste concentrations of 10.2, 16.3, 20.3, and 26.4 g-VS/L on mixed- VFASs production was subsequently carried out in batch dark fermentation. Highest butyric acid concentration (5.26±0.22 g/L) in soluble metabolites was obtained from batch dark fermentation with 26.4 g-VS/L of food waste. Dominant Clostridium thermobutyricum, Clostridium sporogenes, and Octadecobacter sp. found in the batch dark fermentation of food waste could confirm the effectiveness of the load shock pretreatment for inoculum preparation. The Continuous stirred tank reactor (CSTR) inoculated with mixed-cultures also prepared by load shock pretreatment and without external nutrients addition was operated by feeding 26.4 g-VS/L-food waste at kinetically designed HRT of 4 days, corresponding to organic loading rate (OLR) of 7.6 g-VS/L d. Under steady-state conditions, butyric acid (5.65±0.51 g/L) rich mixed VFAS was achieved promisingly along with hydrogen yield of 110.8± 5.8 mL-H2/g-VSadded, which is higher than the reported yield in a range between 8.8±0.6 mL-H2/g-VS and 103.6±0.6 mL-H2/g-VS.”
- Keywords can be supplemented, i.e. "anaerobic digestion, volatile fatty acids (VFA), hydrogen production yield" can be added.
The following keywords were modified in the revised manuscript.
“Keywords: Organic waste; Mixed culture fermentation; Volatile fatty acids; Bio-hydrogen”
4. Insufficient innovation was exhibited in the text.
Thank you for your question and suggestion. For about novelty, we’ve modified the last paragraph in “the Introduction” (Line 89-105) to demonstrate the challenges, including the inoculum pretreatment, which are required additional investigation prior full-scale implementation for food waste dark fermentation to recovery mixed VFA as the important building block chemicals with a greatly increasing market demand in industry for bioplastics, foods, composites, synthetic fibers, and coating.
“Inoculum pre-treatments, process optimization to achieve high and stable yields, the development of efficient techniques for selective recovery, and the cost-effectiveness of the whole process are the main challenges for full-scale implementation of food waste dark fermentation. Indeed, dark fermentation of food waste to efficiently recovering mixed-VFAs, which are currently the products of interest for further application in sustainable chemical industry is highly depending on sludge pretreatment to deactivated methanogens and physicochemical and operational parameters involved in the fermentative system. Applying thermal, acid, and alkali pretreatment was reported as efficient methods to select high activity microorganisms for dark fermentation [15,16]. Nevertheless, these pretreatment methods are still in doubt for economic viability. Therefore, in this investigation, original anaerobic digested sludge was pretreated by load shock means, which is considered as practical and economic pretreatment to subsequently establish mixed-cultures dark fermentation of food waste under thermophilic temperature (55 °C). Batch fermentation experiment was later investigated to estimate the first order kinetic constant (kh), which was further used to simply design suitable HRT for CSTR operation of mixed-cultures anaerobic fermentation of food waste to evaluating long term performance in continuous operation mode.”
5. Table 1 can be improved.
Table 1 was improved by adding the standard deviation data of carbohydrates proteins and lipids.
6. Figure 1 and Figure 3 can be improved.
Figure 1 was improved by adding the standard deviation data on a graph. Meanwhile, Figure 3 was improved visualization by linking DGGE bands and bacterial species.
7. Some recent results can be cited, such as Journal of Environmental Management, 2022, 319, 115655; Chemosphere, 2022, 286(1), 131655; Bioresource Technology, 2021, 320(A): 124303.
The following suggested papers are cited in the revised manuscript.
“Chen, H.; Huang, R.; Wu, J.; Zhang, W.; Han, Y.; Xiao, B.; Wang, D.; Zhou, Y.; Liu, B.; Yu, G. Biohythane production and microbial characteristics of two alternating mesophilic and thermophilic two-stage anaerobic co-digesters fed with rice straw and pig manure. Bioresource technology 2021, 320, 124303..”
“Chen, H.; Yang, T.; Shen, Z.; Yang, E.; Liu, K.; Wang, H.; Chen, J.; Sanjaya, E. H.; Wu, S. Can digestate recirculation promote biohythane production from two-stage co-digestion of rice straw and pig manure? Journal of Environmental Management 2022, 319, 115655.”
8. Further discussions on the dark fermentation of the food waste can be supplemented with in-depth comparison analyses.
Comparison discussion on inoculum pretreatment and the kh was added in L 272-283.
“For batch dark fermentation of food waste, Yahya et al. [26] reported previously, an inoculum obtained by applying autoclave heat pretreatment (100 °C and 20 min) of anaerobic digestate sludge provided the kh in a range between 0.021 hr-1 and 0.041 hr-1, which is similar range of kh obtained from batch food waste dark fermentation in this current investigation. This could be evidently proven that shock load pretreatment could be one of effective method for inoculum preparation for dark fermentation. During the load shock operation, mixed organic acids are rapidly generated in rather high concentration, consequently the pH in fermentative system was rapidly decreased to acidic range between pH 5 and pH 6 by self-adjustment via high concentration of generated organic acid based acidogenesis. Methanogens in original anaerobic sludge usually preferring pH range between pH 7 and pH 8 for their growth [27] could be eventually deactivated under acidic condition.”
Metabolites obtained from the previous research using other inoculum pretreatments were additionally stated in L 290-296.
“Luo et al. [16] also reported that highest butyric acid concentration over other metabolites (acetic acid, propionic acid, lactic acid, and formic acid) was generated from food waste fermentation using mixed microbial cultures, previously pretreated by acid, alkali, and thermal pretreatment.”
Additional discussion on the effect of HRT used for CSTR operation was stated in L 374-384.
“ Food waste with 26.4 g-VS/L was fed to the CSTR at designed HRT of 4 days, corresponding to organic loading rate (OLR) of 7.6 g-VS/L d under 55 ℃-thermophilic temperature. The appropriate HRT between 2 days and 6 days is usually selected to evaluate optimum performance of continuous dark fermentation of an organic waste in the CSTR [39]. Since having slow growth rate, methanogens normally require HRT longer than 10 days for traditional anaerobic digestion of organic wastes under 55 °C -thermophilic temperature in the CSTR [18]. Methane was not detected during the continuous operation for 51 days, indicating that methanogenic archaea were completely deactivated by operating HRT below 6 days and using initial inoculum obtained by load shock pretreatment of anaerobic sludge, leading to have high VFA concentration [14].”
9. Check the consistency of the manuscript. Fix grammar and formatting mistakes.
English language revised manuscript was thoroughly modified and corrected.
Round 2
Reviewer 1 Report
Thanks to the authors for following my suggestions.
Reviewer 3 Report
The revised manuscript can be accepted for publication. Personally, Lines 155-157, it can be revised as lines 171-173.
