Multi-Response Optimization of Thermochemical Pretreatment of Soybean Hulls for 2G-Bioethanol Production
Round 1
Reviewer 1 Report
The authors describe in the article the conversion of soybean hulls into 2G-Bioethanol. Based on experimental results a model was developed to derive the optimal preconditioning, sugar mobilization and conversion into ethanol.
The most important question arises when looking at table 2. How often were the treatment experiments listed in table 2 conducted to have a statistically sound base for the model. At the moment, it looks like that each treatment experiment was conducted once except for number 7, which could be a thin base to establish on it the mathematical models of the manuscript. The authors are asked to explain this point in more details.
The authors use chloramphenicol in the enzymatic saccharification. As they declare the process in the manuscript feasible for industrial application, they should elaborate on the question, how the antibiotic can be avoided at industrial scale or if 2.5ppm are still necessary at that scale.
Following the line numbering several other points should be raised:
l. 27: Maybe change “industry” into “industrial”
l. 119: The phrase “Five-carbon monomeric sugar” is right but quite unwieldy.
l. 149/Table 1: The concentration of H2SO4 should be expressed as % weight per volume. The used abbreviation is quite uncommon. I suggest changing m/V to w/v and m/m to w/w. Interestingly, this expression is not used throughout the whole article. Please revise the expression where necessary.
l. 154: It is confusing to the reader that some explanations like in formula 1 Cpentoses are written as subscript and others like gSH/L in l. 154 not. Please unify this, whereby I suggest subscript everywhere.
l. 175: Make numbers better readable with a separator for a thousand.
l. 184-85: To understand the amount of YPD media in the fermentation, the authors should extrapolate the volume of the preculture used.
l. 188: Could you give a literature origin of your “standard” technique?
l. 190 ff: Can you elaborate on the salt concentration in the yeast fermentation as the pH was adjusted by NaOH?
l. 203: Is it possible to state the SH distribution including standard deviations?
l. 213/Table 3: Superscript 2 is before the one of 1 when reading from left to right. Can you change the order? Secondly, I wonder whether the common expression “not determined, ND” is better than “not informed”.
l. 215: Please add an apostrophe to “solids”.
l. 221: Please use improper.
l. 230: Please revise the sentence starting in this line.
l. 232: You can add “remaining” after three to make things more clear. Maybe “higher” should be replaced by “highest”.
l. 237: “to a breaking of intermolecular”
l. 243: released
l. 244: of degradation / Maybe replace five-carbon by C5
l. 253: What kind of percentage is used here?
l. 259: Which literature “informed” the reader before? Please use here and otherwise diluted instead of dilute.
l. 261: Maybe better “A level of 67% solubilisation”.
l. 264: Please erase “and it was”.
l. 283/Equation 4: Equation 4 starts with -81.67. How can this be if ß0 is zero (l. 278)? Why have you changed the order of the variables going from equation 3 to 4? In my point of view it is actual, ß0+ß3+ß1+ß33.
l. 296/Figure 2: The authors should explain, how the non-relevance of the reaction time on the reaction’s outcome could be derived from this figure? What reaction time was used during obtaining the shown data?
l. 309/Figure 3: Looking at figure 4, it is quite obvious that the reactions were carried out many times. If this is true the quality of the data has to be proven by the corresponding standard deviation, which is missing in figure 3. This is important as this figure expresses one of the key massages for the model.
l. 320: monitoring and concentrations should be moved behind furfural
l. 322: please use a phrase with implying and add after depending “on”
l. 336: “Third on the podium” is slang. Please replace it by a more scientific language.
l. 344/Figure 4: Please add to the figure’s legend, that the treatment sequence is running from low to high concentrations and not according to the numbering of table 2. Maybe you can state the significance of some results compared to the others by using small letters that the reader can easily distinguish the treatments.
l. 354/Figure 5: Please use in the legends of b, c, e, f only one decimal. Please include in the figure’s legend: “furans’”, during instead of for (l. 356) and “b + e” after 135°C
l. 372: What kind of percentage is used here?
l. 376: Please erase works and change inform to reports
l. 385/Figure 7: Can you add a straight balance line to the data points?
l. 391: Maybe of or during instead of for.
l. 409: Please erase them.
l. 427: Do you mean here value instead of p-valor?
l. 441: In equation 9, please replace “y” by “and”.
l. 462: Maybe you can revise the sentence including an expression like “while a constant… was applied”.
l. 469: Here and thereafter, I suggest not to speak from evolution. Better will be here “change”.
l. 488/Figure 10: What are the error bars? In part a, it would be nice to better distinguish, which line corresponds to biomass and which to ethanol. Additionally, I suggest to use experiment instead of assay (also at other positions in the manuscript).
l. 499/Table 5: Please change Máx. to Max. It would help some readers, if you can write below the table what is meant by Yet/glu and so on till rP.
l. 502: Please replace the by a.
l. 526: Maybe you can rephrase the first sentence of conclusions, to make your intention with this sentence clearer.
As this list includes some spelling and grammar corrections, I encourage the authors to have a thorough look through the manuscript before handing in again.
After addressing my points beside spelling and grammar, I think this manuscript is interesting and should be published.
As the list of points to be considered for correction includes some spelling and grammar issues, I encourage the authors to have a thorough look through the manuscript before handing in again.
Author Response
April 29, 2023
We would like to take the occasion to extend our gratitude to the Reviewers for their accurate and helpful suggestions and considerations. This peer-reviewing process has been enormously favourable to the improvement of the manuscript. A set of revisions and corrections following the reviewers' recommendations were made, as well as, the English redaction were revised.
In this letter, the reviewer’s comments are written in cursive, while the changes and responses are in the normal format.
We look forward to your favourable response.
Yours sincerely,
Raúl N. Comelli
Corresponding Author
Comments and Suggestions for Authors:
The authors describe in the article the conversion of soybean hulls into 2G-Bioethanol. Based on experimental results a model was developed to derive the optimal preconditioning, sugar mobilisation and conversion into ethanol.
The most important question arises when looking at table 2. How often were the treatment experiments listed in table 2 conducted to have a statistically sound base for the model. At the moment, it looks like that each treatment experiment was conducted once except for number 7, which could be a thin base to establish on it the mathematical models of the manuscript. The authors are asked to explain this point in more detail.
The experiment was conducted in three blocks, with each condition being studied in triplicate, and the order of the assays was completely randomized. In total, 48 experimental runs were performed, including 2 central points per block, resulting in 36 degrees of freedom for error estimation. Factor combinations for pretreatment are detailed in Table 2.
A new sentence to clarify the statistical aspects in relation to the experimental design was added in page 3, Section 2.2 ‘Experimental design’: “”. Moreover, the reference 1 -corresponding to subscript 1- of table 2 was reformulate: “The central point of each block was performed by duplicate in each block, so this condition was evaluated in sextuplicate.”
The authors use chloramphenicol in the enzymatic saccharification. As they declare the process in the manuscript feasible for industrial application, they should elaborate on the question, how the antibiotic can be avoided at industrial scale or if 2.5ppm are still necessary at that scale.
In Section 3.7 (Separate Saccharification and Fermentation), we have included a discussion on the industrial-scale use of antibiotics in bioethanol production: “Concerning the utilisation of antibiotics in low concentrations in 1G bioethanol production at industrial scale, this is a cost-effective strategy and frequently used for controlling bacterial contamination, whereas it has vertiginously increased over the past few years. The use of antibiotics in fermentation at industrial scale has been extensively studied as a tool to stop contaminant microorganisms proliferation, as well as its impact on yeast performance and ethanol yields. In the fuel ethanol industry, control of bacterial contamination has been achieved by using antibiotics such as penicillin G, streptomycin, tetracycline, virginiamycin, monensin, or mixtures of these antibiotics. Lactrol® (virginiamycin) is commonly used in a concentration range of 0.25-2.00 ppm at industrial scale. The enzymatic hydrolysis and fermentation processes were carried out using Lactrol® at 2 ppm too, obtaining the same results (data not shown) as those exposed next in this section using chloramphenicol. A simultaneous saccharification and fermentation (SSF) strategy is an interesting option for reducing or entirely avoiding the use of antibiotics. In this scenario, yeast is present in the fermentation medium at the initial stage of sugar liberation, and it modifies the medium properties (e.g. pH), displacing other microorganisms such as lactic acid bacteria”.
Following the line numbering several other points should be raised:
- 27: Maybe change “industry” into “industrial”
“Industry” was changed into “industrial” as was suggested.
- 119: The phrase “Five-carbon monomeric sugar” is right but quite unwieldy.
“Five-carbon monomeric sugar” was replaced by C5 sugars.
- 149/Table 1: The concentration of H2SO4should be expressed as % weight per volume. The used abbreviation is quite uncommon. I suggest changing m/V to w/v and m/m to w/w. Interestingly, this expression is not used throughout the whole article. Please revise the expression where necessary.
The concentration of H2SO4 was expressed using the abbreviation suggested (w/v).
- 154: It is confusing to the reader that some explanations like in formula 1 Cpentosesare written as subscript and others like gSH/L in l. 154 not. Please unify this, whereby I suggest subscript everywhere.
The use of subscript was unified as was suggested.
- 175: Make numbers better readable with a separator for a thousand.
A separator for a thousand was included as was suggested.
- 184-85: To understand the amount of YPD media in the fermentation, the authors should extrapolate the volume of the preculture used.
Because the cells were harvested by centrifugation and washed twice using distilled water before inoculation, traces of YPD were not incorporated on the reactors. To clarify this topic, the following text was added: “The cells had been previously grown at 30 C for 12–18 h and were then harvested by centrifugation for 5 min at 4500 rpm, washed twice using distilled water and finally resuspended in a small volume of water before the reactor' inoculation”
- 188: Could you give a literature origin of your “standard” technique?
A literature for Volatile Suspended Solids (VSS) quantification was included: Eaton, A.D., Clescerl, L.S., Greenberg, A.E., 2005. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, USA.
The standard technique cited was previously used in previous work (Comelli et al. (2016)). The following text was added: To build the calibration curve, the yeasts were grown on YPD medium at 30°C for 12–18 h and were then harvested by centrifugation for 5 minutes at 1200 g, washed five times using distilled water. Several dilutions on distilled water were made by triplicate. An aliquot of each diluted sample was used for measure of turbidity (at 600 nm) using distilled water as blank. Another aliquot of the well-mixed sample (50-mL) was filtered in vacuum through a weighted standard Whatman GFC glass fiber filter (47 mm diameter and 1.2 mm nominal pore size, Biopore, Buenos Aires, Argentina) and the residue retained on the filter was dried to a constant weight at 103–105°C. The increase in weight of the filter represents the total suspended solids (TSS). The next step was the combustion of the filter at 500°C for 15 minutes and the weight lost after combustion represents the weight of Volatile Suspended Solids (VSS) in the sample
- 190: Can you elaborate on the salt concentration in the yeast fermentation as the pH was adjusted by NaOH?
A new paragraph has been added to Section 3.7 (Separate Saccharification and Fermentation) to provide further clarification regarding the experimental methodology and to discuss the effects of salt added during pH adjustment: “As aforementioned, the pH should be adjusted after preconditioning operations to suitable values for both enzymatic hydrolysis and yeast metabolism. To adjust the pH from 0.7-0.8 to 5.5 of 100 ml of SH hydrolysates with a solid loading of 150 gSH/L was necessary to add 2.5 g of NaOH (final concentration of sodium of 14.363 gNa/L). This represents a quantity of 0.167 gNaOH/gSH or 0.096 gNa/gSH. Conventional strains of S. cerevisiae tolerate a sodium chloride concentration of 2 M (45.978 gNa/L). The final concentration of sodium was almost three times smaller than the maximum tolerance concentration values reported in literature. Moreover, the fermentation performance and evolution of the SH hydrolysates and control (synthetic medium) was very similar for the evaluated conditions. Because the control does not contain sodium added to adjust the pH, it is possible to assume that sodium concentration used was not detrimental for yeast growth and fermentation in the reactors containing hydrolyzed and neutralized SH”.
- 203: Is it possible to state the SH distribution including standard deviations?
The standard deviations were included in the text as was suggested.
- 213/Table 3: Superscript 2 is before the one of 1 when reading from left to right. Can you change the order? Secondly, I wonder whether the common expression “not determined, ND” is better than “not informed”.
The order of the subscripts was changed as was suggested. The phrase “Not Determined” was replaced with “Not Informed".
- 215: Please add an apostrophe to “solids”.
The subscript was added as was solicited.
- 221: Please use improper.
The word “improperly” was replaced with “improper”.
- 230: Please revise the sentence starting in this line.
The sentence “Because the carbohydrate recovery could be affected by the initial solid content, the selection of an adequate loading is a key factor prior to beginning the process.” was replaced by “Since the initial solid content can have an impact on carbohydrate recovery, the selection of an appropriate loading is a crucial factor to consider before commencing the process.”
- 232: You can add “remaining” after three to make things more clear. Maybe “higher” should be replaced by “highest”.
Corrected as suggested.
- 237: “to a breaking of intermolecular”
The sentence was corrected as was suggested.
- 243: released
The sentence was corrected as was suggested.
- 244: of degradation / Maybe replace five-carbon by C5
“Five-carbon” was replaced by C5 as was suggested.
- 253: What kind of percentage is used here?
The percentage corresponds to hemicelluloses solubilized defined in equation 1. To clarify, “(%HC)” was added.
- 259: Which literature “informed” the reader before? Please use here and otherwise diluted instead of dilute.
“Dilute” was replaced by ”diluted”. The sentence was reformulated for clarity.
- 261: Maybe better “A level of 67% solubilisation”.
The sentence was corrected as was suggested.
- 264: Please erase “and it was”.
The sentence was erased as was suggested.
- 283/Equation 4: Equation 4 starts with -81.67. How can this be if ß0is zero (l. 278)? Why have you changed the order of the variables going from equation 3 to 4? In my point of view, it is actual, ß0+ß3+ß1+ß33.
Thank you so much for the valuable commentary. The observation it’s correct, β0 has a value of -81.67. The order of the factors was corrected as was suggested, being: β0+ β1+β3+β33.
- 296/Figure 2: The authors should explain, how the non-relevance of the reaction time on the reaction’s outcome could be derived from this figure? What reaction time was used during obtaining the shown data?
Figure 2 shows that no interaction effects between temperature and acid concentration were detected on the range of conditions selected. With the intention to clarify this aspect, the next paragraph was added before Figure 2: “It was particularly curious for the interaction between the temperature and the sulfuric acid concentration (Figure 2). These findings may be mainly due to the range of conditions tested. A wider range of conditions (100 – 170 °C and 1.4 - 3.2 % m/m H2SO4) lead to obtaining response surface models for the release of xylose and arabinose, in which it is observed a significant interaction between temperature and sulfuric acid concentration [11]. In the range of conditions evaluated in this work, the surfaces resemble planes, presenting curvatures in higher values, and being influenced primarily by the temperature.”
In Figure 3 it can be noted that “time reaction” did not have significant effects on %HC. This is explained in the paragraph before Figure 3: “Figure 3 summarises the effects of the factors studied: bars correspond to the average values of %HC reached at the corner points of the factorial design. It can be seen that at low and high temperature values, 80°C and 125°C respectively, the %HC increased significantly as the acid concentration increased and the reaction time was maintained constant. On the other hand, not significant changes have been observed by increasing reaction time and leaving the other variables fixed. This suggests that a significant fraction of the hemicellulose was solubilized relatively quickly and/or during the transient state (heating stage)....”
- 309/Figure 3: Looking at figure 4, it is quite obvious that the reactions were carried out many times. If this is true the quality of the data has to be proven by the corresponding standard deviation, which is missing in figure 3. This is important as this figure expresses one of the key massages for the model.
Standard deviations bars were added to Figure 3.
- 320: monitoring and concentrations should be moved behind furfural
The sentence was changed to improve writing:
“There are two main reasons why monitoring the concentrations of HMF and furfural is relevant...”
- 322: please use a phrase with implying and add after depending “on”
Corrected as suggested.
- 336: “Third on the podium” is slang. Please replace it by a more scientific language.
The sentence was reformulate using a more scientific language:
“An average of 60.7 mgFurfural/L and 38.2 mgHMF/L was produced in treatment 15 (2.50 % m/V H2SO4, 125 °C, 30 min), which ranked third.”
- 344/Figure 4: Please add to the figure’s legend, that the treatment sequence is running from low to high concentrations and not according to the numbering of table 2. Maybe you can state the significance of some results compared to the others by using small letters that the reader can easily distinguish the treatments.
A sentence was added in the legend to indicate that the treatment sequence is running from low to high concentrations and not according to the numbering of table 2.
- 354/Figure 5: Please use in the legends of b, c, e, f only one decimal. Please include in the figure’s legend: “furans’”, during instead of for (l. 356) and “b + e” after 135°C
Corrected as suggested.
- 372: What kind of percentage is used here?
Here we refer to the percentage of celluloses solubilized, as was defined in equation 2. “(%CEL)” was added to clarify.
- 376: Please erase works and change inform to reports
“Works” was replaced by “reports” as was suggested.
- 385/Figure 7: Can you add a straight balance line to the data points?
In fact, the best adjusted line for the data points arises from an exponential model (A detailed discussion of this topic will be addressed in future work). This tendence line was added in graph of Figure 7.
- 391: Maybe of or during instead of for.
Corrected as suggested.
- 409: Please erase them.
Corrected as suggested.
- 427: Do you mean here value instead of p-valor?
“p-valor” was replaced with “p-value”.
- 441: In equation 9, please replace “y” by “and”.
The reemplace was made as suggested.
- 462: Maybe you can revise the sentence including an expression like “while a constant… was applied”.
The sentence was revised and corrected:
- 469: Here and thereafter, I suggest not to speak from evolution. Better will be here “change”.
“Evolution” was replaced by “change” as was suggested.
- 488/Figure 10: What are the error bars? In part a, it would be nice to better distinguish, which line corresponds to biomass and which to ethanol. Additionally, I suggest to use experiment instead of assay (also at other positions in the manuscript).
The error bars represent the standard deviation of the means of three independent experiments.
The figure 10 was modified for a better distinction of dashed line (biomass change), and the new reference was added in legend.
The term “assay” was replaced with “experiment”, as was suggested.
- 499/Table 5: Please change Máx. to Max. It would help some readers, if you can write below the table what is meant by Yet/gluand so on till rP.
Corrected as suggested.
- 502: Please replace the by a.
The change suggested was done.
- 526: Maybe you can rephrase the first sentence of conclusions, to make your intention with this sentence clearer.
The sentence was rephrased with: “This study demonstrates that dilute sulfuric acid, a well-known method, is a suitable option for pretreating a new feedstock to develop robust second-generation bioethanol processes.”
As this list includes some spelling and grammar corrections, I encourage the authors to have a thorough look through the manuscript before handing in again. After addressing my points beside spelling and grammar, I think this manuscript is interesting and should be published.
English spelling and grammar were revised along the manuscript. Several paragraphs or sentences were re-written.
Reviewer 2 Report
In this paper, the existing dilute acid pretreatment technology was optimized on the pretreatment to achieve efficient conversion of carbohydrates. Nonetheless, through the whole pretreatment process, the authors did not mention the whereabouts of lignin, which supposedly should remain in the residue, a waste product with use value. Therefore, it is suggested that the authors add some data on lignin, so as to provide some feasible models for biorefinery. In addition, the conclusion of this article should be revised, and the outlook should also be put forward.
The language should be further checked and improved.
Author Response
April 29, 2023
We would like to take the occasion to extend our gratitude to the Reviewers for their accurate and helpful suggestions and considerations. This peer-reviewing process has been enormously favourable to the improvement of the manuscript. A set of revisions and corrections following the reviewers' recommendations were made, as well as, the English redaction were revised.
In this letter, the reviewer’s comments are written in cursive, while the changes and responses are in the normal format.
We look forward to your favourable response.
Yours sincerely,
Raúl N. Comelli
Corresponding Author
Comments and Suggestions for Authors
In this paper, the existing dilute acid pretreatment technology was optimized on the pretreatment to achieve efficient conversion of carbohydrates. Nonetheless, through the whole pretreatment process, the authors did not mention the whereabouts of lignin, which supposedly should remain in the residue, a waste product with use value. Therefore, it is suggested that the authors add some data on lignin, so as to provide some feasible models for biorefinery. In addition, the conclusion of this article should be revised, and the outlook should also be put forward.
A new paragraph has been added to Section 3.7 (Separate Saccharification and Fermentation) to expose the future perspective derived from this work: “Finally, another compound still remains in fermentation media after ethanol separation: lignin. The study of this aromatic biopolymer was out of the scope of the present work. However, lignin has the potential to produce various compounds of interest such as lignin-based hydrogels, surfactants, three-dimensional printing materials, electrodes and fine chemicals through biorefinery activities and is expected to benefit the future circular economy. This aspect will be addressed in future works”.
Round 2
Reviewer 1 Report
The authors could improve their manuscript significantly. Especially the experiments’ number and conduction are now more clear, which is very important as the model is based on them and their plausibility.
However, I would suggest before publication to change the following points:
a. Please look through the whole text again, to find all abbreviations like m/V and m/m to replace them by w/v and w/w. This is not only necessary in the text, but also in some graphs, e.g. figure 1.
b. Please extend the usage of subscripts for compound names to e.g. furfural or HMF (like in line 391 or 393 of the corrected version).
c. Please change “temperatura” in graph 9b to “temperature”.
Author Response
We extend our gratitude to the Reviewer 1 for their accurate suggestions and considerations. The reviewer’s comments are found in italics, while the responses are in the normal format.
The authors could improve their manuscript significantly. Especially the experiments’ number and conduction are now more clear, which is very important as the model is based on them and their plausibility.
However, I would suggest before publication to change the following points:
- Please look through the whole text again, to find all abbreviations like m/V and m/m to replace them by w/v and w/w. This is not only necessary in the text, but also in some graphs, e.g. figure 1.
All abbreviations (m/V and m/m) were searched and replaced along the manuscript by the correct form. Figures 1, 5a, 5d and, 6 were also corrected.
- Please extend the usage of subscripts for compound names to e.g. furfural or HMF (like in line 391 or 393 of the corrected version).
Subscripts has been added for compound names as suggested by the reviewer
- Please change “temperatura” in graph 9b to “temperature”.
The Figure 9 was updated and corrected
