Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production
Round 1
Reviewer 1 Report
The experimental methodology is adequate, and at the end, butanol was obtained with high productivity and high product concentration.
Authors may provide some information about the cassava source they employed. Which is the amount of cassava, as such, remaining after batch fermentation at different cassava concentrations?
The TSH06 system performs better than other biological agents? (Authors mention that batch results are similar to those obtained by clostridium species: ~12 g/L).
The manuscript can be read without difficulty, however, there are a few grammar mistakes along the text that should be corrected.
Author Response
Comments and Suggestions for Authors
The experimental methodology is adequate, and at the end, butanol was obtained with high productivity and high product concentration.
Authors may provide some information about the cassava source they employed. Which is the amount of cassava, as such, remaining after batch fermentation at different cassava concentrations?
Thank you for your suggestion! Cassava was purchased from Guangdong province of China. The information has been added in “Materials and Methods”. The starch concentration remained in the fermentation broth after batch fermentation was shown in Table 1.
The TSH06 system performs better than other biological agents? (Authors mention that batch results are similar to those obtained by clostridium species: ~12 g/L).
TSH06 possesses the capability to synthesize butanol under non-strict anaerobic conditions. Therefore, all the fermentation processes were carried out without nitrogen gas flushing and oxygen removal. Its butanol production is comparable to that of traditional strict anaerobic Clostridium species (~12 g/L).
Reviewer 2 Report
The authors fermented cassava flour with a mixture from Clostridium acetobutylicum and Bacillus cereus, called TSH06, under micro-aerobic conditions to acetone, butanol and ethanol (ABE). A two stage continuous fermentation process with gas stripping of the products ABE by utilization of CO2 and H2 produced by the bacteria was developed to obtain at the same time high productivity and high ABE concentrations. During the development process, the influence of immobilisation of bacteria on cassava peel residues of different sizes was evaluated.
When reading the article three key points arise, which the authors do not cover sufficiently:
a) Butanol is the product of interest. However, the bacteria are also producing acetone and ethanol. What application, do the authors have in mind for these chemicals? This is highly important, if butanol should be produced as bulk product alternative to gasoline, the market will be flooded with acetone and ethanol as well, which will decrease their market prize drastically. The authors should elaborate on this in the introduction.
b) The authors describe the amount of ABE they can gain by gas stripping. Unfortunately, it is not clear to the reader, if the gas stripping process expel ABE from the fermentation broth as such or if a fractionation into the single products like butanol is already possible by the gas stripping process. If not an energy intensive fractionation process has to be included in a future industrial application, which should be mentioned by the authors to tell the whole story. Please modify if necessary e.g. p. 2 paragraph 2 near at the end.
c) The authors write on page 1 that cassava does not compete with food crops for land. This can be true for China. However, other parts of the world like Africa or South America are highly depending on cassava as food source. The authors should handle the food vs. fuel debate in the introduction with a differentiated more global view.
In general, I recommend to extend the % data throughout the whole text in order to know e.g. (w/w) or (w/v).
In fig. 5, 10 and 11 dealing with the fermentations the error bars are missing. Hence, the authors are asked to explain the statistical validity of their experiments/results.
The next comments occur in the order of the text:
P. 3: Why is there no pH control installed/necessary during fermentation process? A benefit of fermentation units is normally the high degree of control of fermentation parameters to sustain optimal conditions for the microorganisms.
P. 5: In order to understand chapter “Bacterial cultivation” better, I recommend to explain “Analytical Methods” before the cultivation chapter.
In the chapter “Analytical Methods” are mentioned the samples to be analysed by HPLC. What is the sample size taken from fermentation/gas stripping process, which has to be diluted for HPLC? How much rcf is 12,000 rpm? Was the centrifugation step at room temperature?
P. 7: Figure 5 has included a lot of information and the butanol productivity and yield are hardly distinguishable. Hence, I suggest to use two graphs to transport the information.
P. 8: The “18” at the end of the first paragraph should be in brackets.
End of second paragraph “0.26h-1” has to be in superscript.
“Additive amount” is a misleading expression. I think you mean to have e.g. 5% (w/x?) of cassava peel residue in the fermentation broth. However, it can be interpreted as 5% (w/x?) of cassava peel residue was added on top to the fermentation broth, which would increase the actual fermentation volume. Please explain what you really want to express. Please alter p. 9 (text and fig. 7) accordingly.
The authors should explain why they started the experiments on influence of residue size by applying 5% (w/x?) of cassava peel residue. What was the base for the decision?
P. 9: As the authors start by interpretation of fig. 6 with the beneficial experiments, I suggest the explanation of the results with 10 mesh is unnecessary and can be eliminated.
The control of fig. 7 have to show the same results as the “free cell” experiment of fig. 6. Hence, it would be nice not to copy and paste the sentence on these results from fig. 6 into the beginning of the paragraph below fig. 7 instead to be more creative and connect both results, as they must be the same.
P. 10: Please add to the legend of fig. 8 and 9 what the pictures a)-c) or a)-b), respectively show. You should understand a figure legend without looking at the corresponding text.
Do you have any clue, if the immobilization process favours one of the bacteria of your mixture TSH06?
P. 13: What are the butanol and ABE concentration, respectively, after gas stripping in the one stage process? Comparing this with the two stage process could show the potential of the later.
Fig. 11: The legend should state the word “Comparison”. At the moment the legend is incomplete.
The x-axis on the right side state the ABE and the butanol concentration. Please correct this.
When have the authors started in the one stage process with the gas stripping? This should be indicated in the figure.
a) and b) are overcrowded making it nearly impossible to see the important differences in the graphs. Distribution of the information into more graphs would improve the readability and allow the reader to understand the difference more easily.
P. 14: At least the conclusions should elaborate on the two “side” products acetone and ethanol as well. What is the concentration? How does these values compare to other fermentative routes to obtain both?
In summary, I would recommend this article for publication after the authors have dealt with the points raised above and have included a more critical view on the feedstock and the impact of large scale ABE processes in their introduction section.
Beside one or two expressions, which need some clarification, because they can be interpreted in different ways, I have no complaints concerning the English language.
Author Response
Comments and Suggestions for Authors
The authors fermented cassava flour with a mixture from Clostridium acetobutylicum and Bacillus cereus, called TSH06, under micro-aerobic conditions to acetone, butanol and ethanol (ABE). A two stage continuous fermentation process with gas stripping of the products ABE by utilization of CO2 and H2 produced by the bacteria was developed to obtain at the same time high productivity and high ABE concentrations. During the development process, the influence of immobilisation of bacteria on cassava peel residues of different sizes was evaluated.
When reading the article three key points arise, which the authors do not cover sufficiently:
a) Butanol is the product of interest. However, the bacteria are also producing acetone and ethanol. What application, do the authors have in mind for these chemicals? This is highly important, if butanol should be produced as bulk product alternative to gasoline, the market will be flooded with acetone and ethanol as well, which will decrease their market prize drastically. The authors should elaborate on this in the introduction.
Thank you for your suggestion! The application value of ethanol and acetone has been supplemented in the introduction. It is in the second paragraph of the introduction. “ABE fermentation can simultaneously produce butanol, acetone, and ethanol, with a ratio of 6:3:1 for butanol, acetone, and ethanol, respectively. Butanol, acetone, and ethanol are all important bulk chemicals and can be used as gasoline additives.”
b) The authors describe the amount of ABE they can gain by gas stripping. Unfortunately, it is not clear to the reader, if the gas stripping process expel ABE from the fermentation broth as such or if a fractionation into the single products like butanol is already possible by the gas stripping process. If not an energy intensive fractionation process has to be included in a future industrial application, which should be mentioned by the authors to tell the whole story. Please modify if necessary e.g. p. 2 paragraph 2 near at the end.
Thank you for your suggestion! I didn't explain it clearly. A detailed description of gas stripping has been added in paragraph 4 of introduction. “The method of gas stripping is to introduce gas (usually nitrogen or carbon dioxide) into the fermentation broth, this gas is used to produce bubbles in the fermentation broth, which in turn capture the ABE. The captured ABE is subsequently collected in a condenser. In general, the concentration of butanol in the condensed recovery liquid can reach 70 g/L or higher, and the total concentration of ABE can reach 100 g/L or higher.”
c) The authors write on page 1 that cassava does not compete with food crops for land. This can be true for China. However, other parts of the world like Africa or South America are highly depending on cassava as food source. The authors should handle the food vs. fuel debate in the introduction with a differentiated more global view.
The description of cassava has been revised. It is in the second paragraph of the introduction. “Cassava can grow in poor soil and does not compete for land with other starch crops such as corn and wheat.”
In general, I recommend to extend the % data throughout the whole text in order to know e.g. (w/w) or (w/v).
The % data has been extended.
In fig. 5, 10 and 11 dealing with the fermentations the error bars are missing. Hence, the authors are asked to explain the statistical validity of their experiments/results.
Thank you for your suggestion! Continuous fermentation is a process of long-term constant cultivation. The stability of the process indicates the reliability of the data. Some literature related to continuous fermentation explains this viewpoint. Some of the literature is listed below.
[1] Chang Z, Cai D, Wang Y, et al. Effective multiple stages continuous acetone–butanol–ethanol fermentation by immobilized bioreactors: Making full use of fresh corn stalk[J]. Bioresource Technology, 2016, 205:82-89.
[2] Rasmus, Lund, Andersen, et al. Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses, Waste Biomasses, Molasses and Syrup Using the Anaerobic, Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411. Plos One, 2015.
[3] Dolejš I, Krasnan V, Stloukal R, et al. Butanol production by immobilised Clostridium acetobutylicum in repeated batch, fed-batch, and continuous modes of fermentation. Bioresource Technology, 2014.169:723-730.
The next comments occur in the order of the text:
P3: Why is there no pH control installed/necessary during fermentation process? A benefit of fermentation units is normally the high degree of control of fermentation parameters to sustain optimal conditions for the microorganisms.
Thank you for your suggestion! I didn't explain it clearly. The initial pH of the fermentation medium in this study is 5.5-5.8, which is suitable for bacterial growth. The process of generating ABE is divided into the acidogenic phase and the solventogenic phase.The acidogenic phase is coupled with the logarithmic growth phase of the cell. As acetic acid and butyric acid gradually accumulate, the pH of the fermentation broth gradually decreases. When the pH drops to a certain value (approximately 4.5), bacterial metabolism shifts to the solventogenic phase. After that, the pH no longer decreases. Therefore, in general, there is no pH control or agitation during the process of fermentation. Some relevant literature is listed below.
[1] Liu W, Zha W, Yin H, et al. Integration of rare earth element stimulation, activated carbon adsorption and cell immobilization in ABE fermentation for promoting biobutanol production. Chemical Engineering and Processing - Process Intensification, 2023,184:109306.
[2] Li HG, Wei Luo W, Wang Q, et al. Direct Fermentation of Gelatinized Cassava Starch to Acetone, Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma. Applied Biochemistry and Biotechnology, 2014,172:3330-3341.
[3] Durre P. Biobutanol: An attractive biofuel. Biotechnology Journal, 2007, 2: 1525-1534.
P5: In order to understand chapter “Bacterial cultivation” better, I recommend to explain “Analytical Methods” before the cultivation chapter.
The order of " Bacterial cultivation" and " Analytical Methods " has been adjusted.
In the chapter “Analytical Methods” are mentioned the samples to be analysed by HPLC. What is the sample size taken from fermentation/gas stripping process, which has to be diluted for HPLC? How much rcf is 12,000 rpm? Was the centrifugation step at room temperature?
Thank you for your suggestion. I didn't explain it clearly. Description of sample testing has been supplemented.12000 rpm is 13680 rcf (×g). “The centrifugation step was at room temperature.” “During the fermentation processes, samples were taken at regular intervals, with each sample having a volume of 1 mL.”
P7: Figure 5 has included a lot of information and the butanol productivity and yield are hardly distinguishable. Hence, I suggest to use two graphs to transport the information.
Thank you for your suggestion! Figure 5 has been modified.
P8: The “18” at the end of the first paragraph should be in brackets.
It was my mistake. The brackets have been supplemented.
End of second paragraph “0.26h-1” has to be in superscript.
It was my mistake. It has been correct.
“Additive amount” is a misleading expression. I think you mean to have e.g. 5% (w/x?) of cassava peel residue in the fermentation broth. However, it can be interpreted as 5% (w/x?) of cassava peel residue was added on top to the fermentation broth, which would increase the actual fermentation volume. Please explain what you really want to express. Please alter p. 9 (text and fig. 7) accordingly.
Thank you for your suggestion. I didn't explain it clearly. The % data has been extended. It is w/v.
The authors should explain why they started the experiments on influence of residue size by applying 5% (w/x?) of cassava peel residue. What was the base for the decision?
Some literature has given me inspiration. Some of the literature is listed below.
[1] Vichuviwat R, Boonsombuti A, Luengnaruemitchai A, et al. Enhanced butanol production by immobilized Clostridium beijerinckii TISTR 1461 using zeolite 13X as a carrier. Bioresource Technology, 2014, 172:76-82.
[2] Chang Z, Cai D, Wang Y, et al. Effective multiple stages continuous acetone–butanol–ethanol fermentation by immobilized bioreactors: Making full use of fresh corn stalk[J]. Bioresource Technology, 2016, 205:82-89.
P9: As the authors start by interpretation of fig. 6 with the beneficial experiments, I suggest the explanation of the results with 10 mesh is unnecessary and can be eliminated.
Thank you for your suggestion! The explanation of the results with 10 mesh has been eliminated.
The control of fig. 7 have to show the same results as the “free cell” experiment of fig. 6. Hence, it would be nice not to copy and paste the sentence on these results from fig. 6 into the beginning of the paragraph below fig. 7 instead to be more creative and connect both results, as they must be the same.
Thank you for your suggestion! There is no need to write the same result twice. And what we are concerned about is the promoting role in an immobilization system. So, the sentence has been deleted.
P10: Please add to the legend of fig. 8 and 9 what the pictures a)-c) or a)-b), respectively show. You should understand a figure legend without looking at the corresponding text.
The legend of fig. 8 and 9 has been modified.
Do you have any clue, if the immobilization process favours one of the bacteria of your mixture TSH06?
The microbial composition of TSH06 and the changes in microbial abundance during the fermentation process were analyzed in previous studies, along with potential interactions among different species. TSH06 was composed of Bacillus cereus and Clostridium acetobutylicum, with the latter being capable of synthesizing butanol while the former cannot. During the initial stage of fermentation, Bacillus cereus briefly grew, which led to a rapid decrease in dissolved oxygen in the fermentation broth. Once the dissolved oxygen dropped below 5%, the biomass of Clostridium acetobutylicum gradually increased, becoming the dominant strain. After 12 hours of fermentation, over 99% of the bacteria in the fermentation broth was Clostridium acetobutylicum. In this study, the immobilization process promoted butanol production, indicating that it primarily enhanced the growth and fermentation of Clostridium acetobutylicum. Relevant literature is listed below.
[1] Wu PF, Wang GY, Wang GH, Borresen BT, Liu HJ and Zhang, JA. Butanol production under microaerobic condi-tions with a symbiotic system of Clostridium acetobutylicum, and Bacillus cereus. Microb Cell Fact, 2016, 15(1):82-89.
[2] Wang GY, Wu PF, Liu Y, Mi S, Mai S, Gu CK, Wang GH, Liu HJ, Zhang JA, Børresen B T, Mellemsæther E, Kotlar H K. Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH0. Appl Microbiol Bi-otechnol, 2015, 99(20):8803-8813.
P13: What are the butanol and ABE concentration, respectively, after gas stripping in the one stage process? Comparing this with the two stage process could show the potential of the later.
Thank you for your suggestion! Related descriptions have been added, it is in page 12. “In the single-stage continuous fermentation coupled with online gas stripping, after gas stripping, the concentration of butanol and ABE in the fermentation broth ranged from 6.3-3.1 g/L and 9.2-5.1 g/L, respectively. In this process, the average productivity of butanol was 0.66 g/(L·h), which was more than three times higher than that of batch fermentation. However, the main issue with this system is that it cannot operate steady for a long period of time. the immobilization system was disrupted due to the gases pumped into the fermentation broth. This hindered proper cell adsorption onto the carrier and impeded normal fermentation. Within 200 hours, a noticeable de-cline in the fermentation performance of the cells was observed, indicating that the immobilization system was not compatible with the gas stripping process in the same fermenter. Therefore, it is necessary to further improve this system.”
Fig. 11: The legend should state the word “Comparison”. At the moment the legend is incomplete.
Thank you for your suggestion. I didn't describe it clearly. The legend has been modified. “Figure 11. Two-stage continuous fermentation coupled online gas stripping. (a) Solvent con-centration and sugar concentration in fermenter 1 of the two-stage system (b) Solvent concentration and sugar concentration in fermenter 2 of the two-stage system.”
The x-axis on the right side state the ABE and the butanol concentration. Please correct this.
It has been corrected.
When have the authors started in the one stage process with the gas stripping? This should be indicated in the figure.
It has been marked in figure 10.
a) and b) are overcrowded making it nearly impossible to see the important differences in the graphs. Distribution of the information into more graphs would improve the readability and allow the reader to understand the difference more easily.
Thank you for your suggestion! Figure 11 shows two-stage continuous fermentation coupled online gas stripping. Figure 11- (a) shows solvent con-centration and sugar concentration in fermenter 1 of the two-stage system. Figure 11- (b) shows solvent concentration and sugar concentration in fermenter 2 of the two-stage system. The caption and font size of Figure 11 have been adjusted to make it clearer and easier to understand.
P14: At least the conclusions should elaborate on the two “side” products acetone and ethanol as well. What is the concentration? How does these values compare to other fermentative routes to obtain both?
Thank you for your suggestion! The concentration of total solvents in the condensate collection solution has been supplemented in the “conclusion”.
In the fermentation process of ABE, butanol is considered as the main product, while ethanol and acetone are by-products. The focus is mainly on the output and productivity of butanol, and some studies aim to reduce the production of ethanol and acetone through genetic engineering or metabolic regulation. Relevant literature is listed below.
[1] Zheng J, Tashiro Y, Wand GH, et al. Recent advances to improve fermentative butanol production: Genetic engineering and fermentation technology. J Biosci Bioeng, 2023, 119:1-9.
[2] Hussain A, Liao H, Ahmad K, et al. Bacterial metabolic engineering for the production of second-generation (2 G) bioethanol and biobutanol; a review. J Apppl Microbiol, 2022, 134:1–11.
[3] Moon H G, Jang Y S , Cho C ,et al. One hundred years of clostridial butanol fermentation. Fems Microbiology Letters, 2016, 363(3):1-15.
[4] Re A, Mazzoli R. Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing. Microb Biotechnol, 2022, 16(2): 238-261.
This study primarily focuses on promoting the production of butanol and improving productivity. Therefore, there is no detailed description of the production of ethanol and acetone.
Reviewer 3 Report
Comments to the manuscript fermentation-2640593: Continuous fermentation coupled with on-line gas stripping for effective biobutanol production
The present manuscript deals with fermentation of butanol by a new inocculum. Due to increasing butanol formation by a factor of 6, this work is of high interest in application. The content fits to the journal. Language is fine. Additional comments to improve the quality of the manuscript are:
Pre-comment: As no line numbers exist, description of the aspects in the manuscript is more challenging than having these numbers. But I tried to describe the relevant section.
· Abstract: It would be a good idea to normalize transformation kinetics that way that biomass concentrations are taken into account, i.e. with a unit of g/(g DM * h).
· Abstract: The type of stripping gas should be mentioned in the abstract.
· Introduction: It should be mentioned that n-butanol is also used a disinfectant underlining the aspect of zytotoxicity mentioned before.
· Introduction: The eco-friendly character of n-butanol should be clearly pointed out as it is of high relevance according to discussions of sustainability, restrictions in transport, change in vehicle engine systems… Suggestion to change second sentence of introduction as followed: At the same time, butanol, which can be produced by biotechnological processes out of bioproducts, can be used as an eco-friendly and carbon-neutral fuel instead of gasoline or as a fuel additive, and also its disposal is possible through cost-effective biological and non-biological processes (https://doi.org/10.1016/j.csite.2022.102612; https://doi.org/10.1016/j.ibiod.2016.10.012; https://doi.org/10.1016/j.jece.2017.10.015).
· Introduction 4th paragraph: Listing the technologies of n-butanol separation: What do you mean with vacuum? A vacuum extraction, vacuum destilation….?
· Materials and Methods – Bacterial Cultivation: Please add the info whether static cultivation and subsequent fermentation were done using an inert gas phase for oxygen exclusion or not.
· Same paragraph: As n-butanol has a very low Henry coefficient it tends to stay in the liquid phase or many times higher gas volume flows need to be used for desorption. As mentioned by the authors the self produced digestion gas (H2, CO2) was used. In this context there are a couple of comments, which should be taken into account:
o Which gas volume was generated by volume of cassava fermentated.
o How was the composition of the fermentation gas – is there no methane in the waste gas.
o Is there an applicability of this technology maybe for industrial H2 formation?
· Header cell immobilization doesn´t fit well as ‘only’ addition of cassava particles of defined size is described.
· Chapter analytical methods: The type of HPLC detector for analyzing organic acids should be presented.
· Table 1: The last entry (Total solvent) is also a productivity and should be renamed.
· Page 9 last three lines: Please give further information about estimation of vitality by cell shape for this inoculum. Does Clostridia change length of rod shape depending on nutrient supply?
· Figure 9: What is the difference between Figure a) and b)?
· Second last paragraph on page 10 – last sentence: Listing pros of immobilization an additional very important factor should be added. According to immobilization correlation between growth rate and dilution rate is split, i.e. even poorly biodegradable compounds can be transformed without wash out effects (for reference as example https://doi.org/10.1016/j.clet.2021.100266).
· Table 2: The yields of H2 and CO2 should be also listed.
· According to the factor of 6 higher yield in butanol formation fermentation should be discussed under economical aspects, i.e. comparing formation costs of butanol using present technologies and using TSH06 based systems.
Author Response
Comments and Suggestions for Authors
Comments to the manuscript fermentation-2640593: Continuous fermentation coupled with on-line gas stripping for effective biobutanol production
The present manuscript deals with fermentation of butanol by a new inocculum. Due to increasing butanol formation by a factor of 6, this work is of high interest in application. The content fits to the journal. Language is fine. Additional comments to improve the quality of the manuscript are:
Pre-comment: As no line numbers exist, description of the aspects in the manuscript is more challenging than having these numbers. But I tried to describe the relevant section.
- Abstract: It would be a good idea to normalize transformation kinetics that way that biomass concentrations are taken into account, i.e. with a unit of g/(g DM * h).
Thank you for your suggestion! I agree with you. However, in an immobilized system, the biomass immobilized on the carrier cannot be measured. The biomass in the fermentation broth is not equal to the total biomass. The fermentation process can only be analyzed by product generation and substrate consumption.
- Abstract: The type of stripping gas should be mentioned in the abstract.
Thank you for your suggestion! “Gas stripping” has been added in “Abstract”.
- Introduction: It should be mentioned that n-butanol is also used a disinfectant underlining the aspect of zytotoxicity mentioned before.
It has been added in the third paragraph of “Introduction”. “(In the field of medicine, n-butanol can be used as a topical disinfectant and dissolving agent)
- Introduction: The eco-friendly character of n-butanol should be clearly pointed out as it is of high relevance according to discussions of sustainability, restrictions in transport, change in vehicle engine systems… Suggestion to change second sentence of introduction as followed: At the same time, butanol, which can be produced by biotechnological processes out of bioproducts, can be used as an eco-friendly and carbon-neutral fuel instead of gasoline or as a fuel additive, and also its disposal is possible through cost-effective biological and non-biological processes. (https://doi.org/10.1016/j.csite.2022.102612; https://doi.org/10.1016/j.ibiod.2016.10.012; https://doi.org/10.1016/j.jece.2017.10.015).
Thank you very much for your suggestion! The sentence has been modified. And the literature has been added.
- Introduction 4thparagraph: Listing the technologies of n-butanol separation: What do you mean with vacuum? A vacuum extraction, vacuum destilation….?
Vacuum means Vacuum fermentation. The sentence has been modified. Vacuum fermentations have a reduced pressure in the fementer, causing the ABE to “boil off” at fermentation temperature. Relevant literature is listed below.
[1] Outram V, Lalander CA; Lee JGM, Davies ET, Harvey AP. Applied in Situ Product Recovery in ABE Fermentation. Biotechnol Progr, 2017,33(3):563-579.
Considering that this study focuses on gas stripping, there is no detailed description of vacuum fermentation.
- Materials and Methods – Bacterial Cultivation: Please add the info whether static cultivation and subsequent fermentation were done using an inert gas phase for oxygen exclusion or not.
The description has been supplemented. “All the fermentation processes were carried out without nitrogen gas flushing and oxygen removal.”
- Same paragraph: As n-butanol has a very low Henry coefficient it tends to stay in the liquid phase or many times higher gas volume flows need to be used for desorption. As mentioned by the authors the self produced digestion gas (H2, CO2) was used. In this context there are a couple of comments, which should be taken into account:
- Which gas volume was generated by volume of cassava fermentated.
I didn't describe it clearly. The description has been supplemented. “The gas produced by TSH06 was circulated through the bioreactor and the condenser system using a peristaltic pump. Butanol, ethanol, and acetone were condensed and collected in the condensing recovery bottle. Subsequently, the gas was reintroduced into the fermentation broth.” All the gas volume was generated by fermentation.
- How was the composition of the fermentation gas – is there no methane in the waste gas.
There is no methane in the waste gas.
C.acetobutylicum will ferment carbohydrates in the form of starch or soluble sugars to produce a mixture of acetone, butanol and ethanol, in an approximate ratio of 3:6:1, and copious quantities of carbon dioxide (60%) and hydrogen (40%). It does so in two phases: the initial acid production phase in which the organism produces ethanoic and butyric acids along with a mixture of CO2 and H2, followed by a solvent production phase in which sugars continue to be metabolised but acids are reassimilated and reduced to produce acetone and butanol.
Relevant literature is listed below.
www.misac.org.uk/article-downloads/13.Adams-ABE-2019.pdf
- Is there an applicability of this technology maybe for industrial H2 formation?
Some studies aim to improve the production of H2 through genetic engineering or metabolic regulation. Relevant literature is listed below.
[1] Woon JM, Khoo KS, Al-Zahrani AA, et al. Epitomizing biohydrogen production from microbes: Critical challenges vs opportunities. Environ Res, 2023, 227:115780.
[2] Wu H, Wang C, Chen P, et al. Effects of pH and ferrous iron on the coproduction of butanol and hydrogen by Clostridium beijerinckii IB4. Int J Hydrogen Energ, 2017, 42:6547-6555.
[3] Beschkov V, Parvanova-Mancheva T, Vasileva E, et al. Experimental Study of Bio-Hydrogen Production by Clostridium beijerinckii from Different Substrates. Energies, 2023, 16(6): 2747.
However, this study primarily focuses on promoting the production of butanol and improving productivity. Therefore, the generation of H2 is not described in detail.
- Header cell immobilization doesn´t fit well as ‘only’ addition of cassava particles of defined size is described.
Thank you for your suggestion. The header has been modified. “Preparation of immobilized cell carriers”.
- Chapter analytical methods: The type of HPLC detector for analyzing organic acids should be presented.
A refractive index detector was used. It has been added in “Analytical Methods”.
- Table 1: The last entry (Total solvent) is also a productivity and should be renamed.
Thank you for your suggestion. It has been modified.
- Page 9 last three lines: Please give further information about estimation of vitality by cell shape for this inoculum. Does Clostridia change length of rod shape depending on nutrient supply?
Thank you for your suggestion. The sentence has been modified. “The bacteria primarily existed in the form of rod-shaped cells, and butanol was being synthesized rapidly at this point, indicating the high vitality of the cells.”
Figure 9: What is the difference between Figure a) and b)?
The legend of Fig.9 has been modified. “Figure 9. Microscopic morphology of immobilized cells (a: Immobilized cells at 24h of fermentation; b: Immobilized cells at 48h of fermentation).”
- Second last paragraph on page 10 – last sentence: Listing pros of immobilization an additional very important factor should be added. According to immobilization correlation between growth rate and dilution rate is split, i.e. even poorly biodegradable compounds can be transformed without wash out effects (for reference as example https://doi.org/10.1016/j.clet.2021.100266).
Thank you for your suggestion. The sentence has been added.
- Table 2: The yields of H2 and CO2 should be also listed.
We believe that in the process of continuous fermentation coupled with gas stripping, promoting the growth of bacteria will also enhance the production of H2 and CO2. However, this study primarily focuses on promoting the production of butanol and improving productivity. Therefore, the generation of H2 and CO2 is not described in detail.
- According to the factor of 6 higher yield in butanol formation fermentation should be discussed under economical aspects, i.e. comparing formation costs of butanol using present technologies and using TSH06 based systems.
Thank you for your suggestion. A discussion about economics has been added.
From the perspective of product generation, by utilizing continuous fermentation coupled with online gas stripping, the productivity is nearly six times that of the traditional method. This means that the initial construction scale of the fermentation plant can be reduced to one-sixth while maintaining the same production capacity.From the perspective of product recovery, it required 36 MJ/kg-butanol for conventional distillation from a solution containing ∼10g/L (w/v) butanol, which was equal to the energy content of butanol itself [1]. For gas stripping, the energy consumption was estimated to be 31 MJ/kg-butanol by Oudshoorn’s study [2] while 29.63 MJ/kg-butanol by Rochón’s study [3]. This energy consumption is estimated based on a butanol concentration of 4-5 g/L in the fermentation broth, which was similar to the concentration of butanol in this study. Although the energy consumption of gas stripping is lower compared to traditional methods (reduced by about 15%), further improvements are still needed to make the process more cost-effective.
[1] Xue C, Du GQ, Sun JX, Chen LJ, Gao SS, Yu ML, Yang ST, Bai FW. Characterization of gas stripping and its integration with acetone–butanol–ethanol fermentation for high-efficient butanol production and recovery. Biochem Eng J 83(15):55-61.
[2] Oudshoorn A, Wielen LAM, Straathof AJ. Assessment of Options for Selective 1-Butanol Recovery from Aqueous Solution. Ind Eng Chem Res, 2009, 48: 7325–7336.
[3] Rochón E, Cortizoa G, Cabot MI Cubero MTG, Coca M, Ferrari MD, Lareo C. Bioprocess intensification for isopropanol, butanol and ethanol (IBE) production by fermentation from sugarcane and sweet sorghum juices through a gas stripping-pervaporation recovery process. Fuel, 2020, 281: 118593.
