High-Titer Bioethanol Production from Steam-Exploded Corn Stover Using an Engineering Saccharomyces cerevisiae Strain with High Inhibitor Tolerance

Round 1

Reviewer 1 Report

General

• In the review of the research article High-titer bioethanol production from steam exploded corn stover by an engineering Saccharomyces cerevisiae strain with high inhibitors tolerance, it was generally identified that it is an original and well-structured writing. Review the use of some words in the writing in general, to improve grammar.

Abstract

1. The abstract is concise and has the necessary elements. However, it could be improved by mentioning the strain modification method used.

Introduction

1. The introduction is adequate, describes the problem and justifies the case study. In line 81, proper citation is missing, as in line 85. It would be appropriate to increase the evidence of the use of the proposed metabolic engineering methods, and mainly ALE.

Materials and methods

1. The methods are adequately described. However, there are some details that need to be corrected.

2. Line 131 place space between Fig.S1 and review the other similar ones

3. Line 132, justify the use of a temperature of 30°C, since the optimal values for E. coli and S. Serevisiae are different.

4. There should be a section focused on the statistical analysis of the data obtained. Please consider it.

Results and discussion

1. The results are clear, however, the tables require adjustments so that the information is displayed as little as possible.

2. In Figure 5.B, there is no considerable statistical difference in ethanol production from 36 to 96 hours, however xylose presents a considerable decrease. Please explain these phenomena.

3. Increase discussion in all sections of the results.

4. Statistically support the differences between the tested processes.

Conclusions

• There are no comments.

References

  Adequate.

Improve writing in terms of double spaces between words, lack of spaces after a period, etc.

Author Response

To Reviewer #1

Thanks for your agreement and helpful comment. We made corrections, and we hope they meet with your approval. We revised the paper according to your specific comments. Detailed explanations for the comments are shown below.

1. General

• In the review of the research article High-titer bioethanol production from steam exploded corn stover by an engineering Saccharomyces cerevisiae strain with high inhibitors tolerance, it was generally identified that it is an original and well-structured writing.

Response:

Thank you!

2. Review the use of some words in the writing in general, to improve grammar.

Response:

Thanks for pointing out the problem of words in the writing and grammar, we were seriously corrected errors in the original manuscript by English-speaking native people, details for the changes are shown in highlighting in the current revised manuscript.

3. Abstract

The abstract is concise and has the necessary elements. However, it could be improved by mentioning the strain modification method used.

Response:

Thanks for the suggestion. According to your suggestion, we have revised the abstract and improved by mentioning the strain modification method in highlighting in the current revised manuscript.

4. Introduction

The introduction is adequate, describes the problem and justifies the case study. In line 81, proper citation is missing, as in line 85. It would be appropriate to increase the evidence of the use of the proposed metabolic engineering methods, and mainly ALE.

Response:

Thanks for your suggestion. According to your suggestion, we increased the proper citation in line 81 and line 85 in the current revised manuscript. In addition, we added evidence of the use of the proposed metabolic engineering methods, and mainly ALE in line 66 and line 91. The following references were cited and added in the reference list in the current revised manuscript.

References

[21] B. Li, N. Liu, X. Zhao, Response mechanisms of Saccharomyces cerevisiae to the stress factors present in lignocellulose hydrolysate and strategies for constructing robust strains, Biotechnol Biofuels Bioprod 15(1) (2022) 28. https://doi.org/10.1186/s13068-022-02127-9.

[22] L. Wang, B. Li, R.R. Su, S.P. Wang, Z.Y. Xia, C.Y. Xie, Y.Q. Tang, Screening novel genes by a comprehensive strategy to construct multiple stress-tolerant industrial Saccharomyces cerevisiae with prominent bioethanol production, Biotechnol Biofuels Bioprod 15(1) (2022) 11. https://doi.org/10.1186/s13068-022-02109-x.

[23] Hemansi, Himanshu, A.K. Patel, J.K. Saini, R.R. Singhania, Development of multiple inhibitor tolerant yeast via adaptive laboratory evolution for sustainable bioethanol production, Bioresour Technol 344(Pt B) (2022) 126247. https://doi.org/10.1016/j.biortech.2021.126247.

[24] H. Li, Y. Shen, M. Wu, J. Hou, C. Jiao, Z. Li, X. Liu, X. Bao, Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production, Bioresour Bioprocess 3(1) (2016) 51. https://doi.org/10.1186/s40643-016-0126-4.

[31] C. Borgstrom, L. Wasserstrom, H. Almqvist, K. Broberg, B. Klein, S. Noack, G. Liden, M.F. Gorwa-Grauslund, Identification of modifications procuring growth on xylose in recombinant Saccharomyces cerevisiae strains carrying the Weimberg pathway, Metab Eng 55 (2019) 1-11. https://doi.org/10.1016/j.ymben.2019.05.010.

[32] M.M. Demeke, F. Dumortier, Y. Li, T. Broeckx, M.R. Foulquié-Moreno, J.M. Thevelein, Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production, Biotechnology for Biofuels 6(1) (2013) 120. https://doi.org/10.1186/1754-6834-6-120.

[35] R. Tang, P. Ye, H.S. Alper, Z. Liu, X. Zhao, F. Bai, Identification and characterization of novel xylose isomerases from a Bos taurus fecal metagenome, Applied Microbiology and Biotechnology 103(23-24) (2019) 9465-9477. https://doi.org/10.1007/s00253-019-10161-1.

5. Materials and methods

The methods are adequately described. However, there are some details that need to be corrected.

Response:

Thanks for the suggestion. We conducted a thorough review of methods and corrected some details in highlighting in the current revised manuscript. Specifically, the sentence “All the yeasts were grown at an optimal ethanol fermentation temperature of 30 °C and 200 rpm for 24 h before inoculation.” has been added in section 2.3. The sentence “All fermentations had three parallel experimental groups.” has been added in section 2.5. The sentence “The software IBM SPSS Statistics 22 was used for statistical analysis to test significant differences between each treatment. All data were presented as means ± standard deviation of duplicate determination.” has been added in section 2.7.

6. Line 131 place space between Fig.S1 and review the other similar ones

Response:

Thanks for the suggestion. According to your suggestion, we had placed space between Fig.S1 and review the other similar ones.

7. Line 132, justify the use of a temperature of 30°C, since the optimal values for E. coli and S. Serevisiae are different.

Response:

Thanks for the suggestion. In contrast to the optimal temperature of E. coli at 37°C, the optimal ethanol fermentation temperature of constructed S. cerevisiae is at 30°C, below which the enzymes related to ethanol fermentation are inhibited, while the growth of S. cerevisiae is gradually inhibited when the cultivation temperature is higher than 30 °C. In the revised manuscript, according to your suggestion, to make it easier for readers to understand and avoid confusion, we justified the use of 30 °C for S. cerevisiae t in the corresponding lines.

8. There should be a section focused on the statistical analysis of the data obtained. Please consider it.

Response:

Thanks for the suggestion. According to your suggestion, we had added a section focused on the statistical analysis of the data obtained in the current revised manuscript.

9. Results and discussion

The results are clear, however, the tables require adjustments so that the information is displayed as little as possible.

Response:

Thanks for the suggestion. According to your suggestion, we had adjusted the tables so that the information is displayed as little as possible.

10. In Figure 5.B, there is no considerable statistical difference in ethanol production from 36 to 96 hours, however xylose presents a considerable decrease Please explain these phenomena.

Response:

Thanks for the suggestion. Actually, there were still some statistical difference in ethanol production from 36 to 96 hours. In Figure 5B, YL13-2 depleted glucose and produced 45.06 ± 2.08 g/L ethanol in 36 h. Subsequently, xylose was assimilated and metabolized by S. cerevisiae to produce ethanol, however, after all, it is a heterologous metabolic pathway in S. cerevisiae, which resulted in a much lower yield of bioethanol production using xylose as a substrate than that of ethanol using glucose as a substrate. Nevertheless, the remaining 18.70 ± 0.47 g/L xylose produced up to 6.06 g/L of bioethanol in the following 60 h. To make it easier for readers to understand and avoid confusion, we had added it more detailed description in the corresponding section of the current revised manuscript.

11. Increase discussion in all sections of the results.

Response:

Thanks for the suggestion. According to your suggestion, we had increased discussion in all sections of the results. Specifically, the sentence “As shown in Fig. 2, the genome of the M3013 strain was modified by metabolic engineering such as heterologous expression, knockout, and genes modification by GTR-CRIPSR system.” has been added in section 3.2. The sentence “while 3.53 ± 0.59 g/L residual xylose was detected in the broth of YL13-1 and without xylose consumption was detected in the broth of M3013.” has been added in section 3.3. The sentence “Despite being negatively affected by higher concentrations of inhibitors, YL13-2 de-pleted glucose and produced 45.06 ± 2.08 g/L ethanol in 36 h, and continued to produce up to 6.06 g/L of ethanol from xylose in the following 60 h.” has been added in section 3.4. The sentence “the initial sugars concentration in substrate was far lower than the ideal titer to-wards >5 wt% bioethanol production [59-61]” has been added in section 3.5. The sentence “Inhibitors including 0.212 g of phenolic compounds and 4.945 g of others were also co-generated after saccharification.” has been added in section 3.6.  

12. Statistically support the differences between the tested processes.

Response:

Thanks for the suggestion. According to your suggestion, we had supported the differences between the tested processes statistically.

13. Conclusions

There are no comments.

Response:

Thank you!

14. Comments on the Quality of English Language

Improve writing in terms of double spaces between words, lack of spaces after a period, etc.

Response:

Thanks for your suggestion. we were seriously corrected grammatical errors in the original manuscript and improved writing by English-speaking native people. Details for the changes were shown in highlighting in the current revised manuscript.

Reviewer 2 Report

The topic is original and relevant for the field of second generation biochemicals, with focus on ethanol as it address the main gap in the field – efficiency of the process. The experimental design is very well elaborated, the team made a tremendous work in pretreatment, hydrolysis, fermentation using wildtype S. cerevisiae M3013, metabolic engineered and ALE yeast strain, gathering information from previous works and discussing the results in the context of the state of the art and comparing with other authors. Important findings are presented here by authors regarding fed-batch hydrolysis & batch fermentation and calculations for mass balance of bioethanol production from corn stover in the approached process. This is an important progress to the second generation biochemicals area compared with other published materials.

Some minor reviews in using English language need to be made, please review expressions such as:

Page 3, Row 129: ” …Wildtype S. cerevisiae M3013 were grown…”

Page 4, Row 161: ” …pre-cultured for twice times in YPX medium…”

Page 9, Row 335: ”… It worthy to be noted here that …”

Page 11, Row 376:”… the chassis strain…”

References are appropriate and the conclusions are consistent with the evidence and arguments presented, addressing the main question posed in this work.

Some minor reviews in using English language need to be made, please review expressions such as:

Page 3, Row 129: ” …Wildtype S. cerevisiae M3013 were grown…”

Page 4, Row 161: ” …pre-cultured for twice times in YPX medium…”

Page 9, Row 335: ”… It worthy to be noted here that …”

Page 11, Row 376:”… the chassis strain…”

Author Response

To Reviewer #2

Thank you very much for your agreement and helpful comment, these constructive suggestions have greatly helped my manuscript. We made correction carefully and thoroughly, and we hope the current revised manuscript could meet with your approval. Detailed explanations for the comments are shown below.

1. The topic is original and relevant for the field of second generation biochemicals, with focus on ethanol as it address the main gap in the field – efficiency of the process. The experimental design is very well elaborated, the team made a tremendous work in pretreatment, hydrolysis, fermentation using wildtype S. cerevisiae M3013, metabolic engineered and ALE yeast strain, gathering information from previous works and discussing the results in the context of the state of the art and comparing with other authors. Important findings are presented here by authors regarding fed-batch hydrolysis & batch fermentation and calculations for mass balance of bioethanol production from corn stover in the approached process. This is an important progress to the second generation biochemicals area compared with other published materials.

Response:

Thank you!

2. Some minor reviews in using English language need to be made, please review expressions such as:

Page 3, Row 129: ” …Wildtype S. cerevisiae M3013 were grown…”

Response:

Thanks for your suggestion. We reviewed the manuscript in detail and corrected the grammatical errors carefully and thoroughly. According to your suggestion, we have changed the sentence " Wildtype S. cerevisiae M3013 were grown" to " Wildtype S. cerevisiae M3013 was grown" in the revised manuscript.

3. Page 4, Row 161: ” …pre-cultured for twice times in YPX medium…”

Response:

Thanks for your suggestion. We have changed the sentence " …pre-cultured for twice times in YPX medium…" to " …pre-cultured for twice in YPX medium…" in the revised manuscript.

4. Page 9, Row 335: ”… It worthy to be noted here that …”

Response:

Thanks for your suggestion. We have changed the sentence " … It worthy to be noted here that …" to "… It was worthy to be noted here that …" in the revised manuscript.

5. Page 11, Row 376:”… the chassis strain…”

Response:

Thanks for your suggestion. We have changed the sentence " … the chassis strain…" to "… the parent strain…" in the revised manuscript.

6. References are appropriate and the conclusions are consistent with the evidence and arguments presented, addressing the main question posed in this work.

Response:

Thank you!