Lychee-Derived, Thermotolerant Yeasts for Second-Generation Bioethanol Production

Round 1

Reviewer 1 Report

The research objective of the investigation is quite interesting. The manuscript in general is well written. However, there are some problems that need to be resolved before the paper can be accepted for publication. Please consider my comments below.

1. Some improvements of the English are suggested. For example, in the first paragraph, a) a comma should be added after energy sources (line 30); b) "are required" should be deleted (line 31); c) "Nowadays" (line 32) may be changed to another word, e.g. recently, since nowadays was used just two sentences prior; and d) "fossil-driven energies" (line 36) can be changed to "fossil-derived liquid fuels". These are just some examples. The authors can find opportunities for improvements in other places in the manuscript.

2. The last paragraph of the Introduction is not really needed since the statements made in that paragraph are already mentioned in the Abstract. Besides, this last paragraph may be more suitably placed in the Results and Discussion section.

3. In the Introduction, the authors should add some discussion on the recently developed commercial yeast strains that can use both glucose and xylose quite efficiently to produce ethanol. These yeast strains actually have been used for quite a while for commercial ethanol production.

4. In section 2.1, a) please give the particle size of the ground and sieved SCB that was used in the experiments; b) please indicate if the ratio of biomass:sulfuric acid of 1:10 was on dry basis - please note that air drying did not remove all the water contained in the biomass; c) the authors stated that xylose was added to the hydrolysate to achieve a concentration of 5 g/L whereas the xylose concentration in the hydrolysate was already 17.5 g/L- please clarify.

5. In section 2.2, a) the authors stated that the lychees were harvested and "aseptically" moved to the lab - please explain "aseptically"; were they sterilized before they were moved?; b) line 103, please give the size of the cut lychees, also, the quantity of broth used per g of material; c) was the YM broth sterilized before use?; d) line 107, how much YM broth was used to resuspend the pellet?; e) line 114, were the isolated strains stored in glycerol and how much?

6. In section 2.3, a) how were the flasks sealed to allow the CO2 produced to escape without loss of ethanol, which had high volatility?; b) please describe the sampling method.

7. Section 2.5.1, line 149, please clarify the basis of ethanol % (v/v, w/v, etc).

8. Section 2.5.2, last sentence, "The tests were carried out at least in duplicate" - did the authors mean some were performed in duplicate and others in higher replicate? Consistency is needed in analysis.

9. Section 2.6.2, line 182, experiments should be changed to experimental; b) Table 3 in the supplementary materials, please clarify the meaning of the substrate concentration - was it g/L total sugars or g hydrolysate/L solution?; c) line 192, what is the difference between "significant" and "very significant" statistically?

10. Section 3.1, line 215, 4% ethanol v/v, w/v, etc?, also, what was the purpose of this screening? Was it to check for ethanol tolerance? If that was the case, why chose 4%?

11. Section 3.2, Table 1, did the authors measure residual glucose and calculate the ethanol yields (g ethanol produced/g glucose consumed)? Yield is a very important factor in ethanol fermentation, especially in 2G ethanol processes.

12. The authors wanted to find a good candidate for ethanol production from biomass hydrolysate but in the first screening they used glucose as the sole carbon source (Table 1, section 3.2). Based on these results, H1 was selected. I think H12 could also be a potential good candidate. It did not produce as much ethanol from glucose as H1 but it was possible that it could be a more efficient xylose-fermenting organism. The results in Table 2 indicated that H12 also fermented xylose similarly to H1. The authors need a more convincing argument on selecting H1 over H12 for ethanol production using a biomass hydrolysate, which contained much more xylose than glucose.

13. Section 3.4, line 354 and those after, I think "positive" should be changed to "significant" and "negative" to "insignificant".

14. Table 3, please give the unit of substrate concentration e.g., g/L total sugars, g SBH per L total volume, etc.

15. Section 3.5, line 424, please clarify substrate concentration, 40 g/L of total sugars or what?

16. In the optimization study and the model validation study, the authors should have determined the initial and final concentrations of the individual sugars (G, X, A) and calculate the ethanol yields since, as I stated previously, this parameter is very important in biomass ethanol process design.

Author Response

Dear Editor and reviewers,

Thank you for giving us the opportunity to submit a revised version of our manuscript titled “Lychee-derived, thermotolerant yeasts for second-generation bioethanol”.

We thank the reviewers for helpful suggestions regarding the layout of the article and how to better highlight the key points and issues, raised by the manuscript.

This has allowed us to greatly improve the overall structure of the manuscript, which has been revised in accordance with the suggestions and instructions provided by the reviewers and the editor.

Corrections, explanatory points and issues that were raised during the review are shown in red text.

Regards,

Kim Cuc Thi Nguyen

Corresponding author

Author Response File: Author Response.pdf

Reviewer 2 Report

I recommend for figure 2 line 263 to continue with graphic description for each parameter (ethanol; acetic acid; furfural) considered to analyze yeast growth under different stresses.. Figure 2 is not so accurate in description regarding yeast growth under different stresses. Durham test is well described. Pareto chart present clearly the effects of different factors over the fermentation process.

Response surface method is well modelate and present simultaneous effects of (NH4)2SO4, substrate concentration, and initial pH on ethanol production. 

Overall I recommend the publication of the manuscript with my recommendation regarding figure 2. 

Author Response

Dear Editor and reviewers,

Thank you for giving us the opportunity to submit a revised version of our manuscript titled “Lychee-derived, thermotolerant yeasts for second-generation bioethanol”.

We thank the reviewers for helpful suggestions regarding the layout of the article and how to better highlight the key points and issues, raised by the manuscript.

This has allowed us to greatly improve the overall structure of the manuscript, which has been revised in accordance with the suggestions and instruction provided by the reviewers and the editor.

Corrections, explanatory points and issues that were raised during review are shown in red text.

Regards,

Kim Cuc Thi Nguyen

Corresponding author

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Regarding the industrial xylose-fermenting yeast strains, the authors may look at this: https://www.lbds.com/en/united-states/fuel-ethanol-products/cellulosic-yeast/. Some (or possibly all) of these strains have been used commercially, for example, in the D3Max process for ethanol production from corn fiber.