Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate

Round 1

Reviewer 1 Report

The authors studied the physiological features of an industrial yeast strain in spruce hydrolysate for simultaneous saccharification and fermentation (SSF), they also provide data on measurement of furaldehyde reduction activity, phenolic compounds and transcription analysis. They conclude that the short-term adaptation involves induction of NADPH-dependent adehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. They also found changes in transcription of critical genes and analyzed the key transcription factors. The major problem is that it lacks a control experiment, for example, if the authors want to specify the short-term adaptation, they may compare with the non-adapted culture. However, the authors compared two different time points, which suggest that the results are not due to short-term adaptation, but only due to different growth phase. 

Other comments and suggestions: 

1. Line 72, short-term adaptation…here it is better to define what is a short-term, how many hours or days? 

2. Line 86-87, …showed excellent growth performance in different lignocellulosic hydrolysates…if the aim of adaptation is to achieve better growth, then seems this strain does not need any adaptation. 

3. Line 92-94, the authors detected furaldehyde reduction activities, but did not mention furfural degradation, then they mentioned phenolic compounds, but did not state the related enzyme activity. Relatedly, in Line 56-59, the author did not mention why phenolic toxicity is important for hydrolysate fermentation. The authors used spruce wood chips, the hydrolysis of which may lead to high phenolic compounds, it is better to mention this background. 

4. Line 110, …trace metals and vitamins. Please provide details here the amount and concentration. 

5. Line 117, I am not clear why the authors performed aerobic fed-batch culture, is it for ethanol production? 

6. Table 1, how about phenolic compound concentration in the hydrolysate? 

7. Table 2, for acetate concentration, is it all from fermentation product or some is from the hydrolysate? Line 323-325, it is confusing, there is acetate in the medium, but why increase? 

8. Fig.2, for the Y-axis, it is better to add the name of the metabolites and biomass. 

9. Line 553-557, I suggest to delete this part because there is no data to support this as a conclusion, also delete such comments in the abstract. I would like to argue that even the transcription factors are activated at post-transcription level, it will not affect that they function in the stress response. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The process of efficient hydrolysis and fermentation of lignocellulosic materials is an issue that has been pursued by numerous research teams for many years. 

In the evaluated work, certainly, the wide and interdisciplinary scope of research with the use of very diverse research methods, both typical methods used in technological research and advanced molecular techniques, deserves to be emphasized. 

Author Response

We would like to thank reviewer 2 for his/her positive feedback on the study.

Reviewer 3 Report

In this manuscript, Almeida and co-authors present a comprehensive study on the rapid adaptive response of a Saccharomyces cerevisiae strain grown on spruce lignocellulosic hydrolysate, shown here to significantly improve the strain fermentation performance under harsh conditions. The authors present interesting and sound data obtained by integrating in vitro and in vivo measurements of conversion of furaldehyde and phenolic compounds with transcriptome analysis. The study is very relevant for the biotechnology of ethanol production from lignocellulose biomass. The manuscript is very well written, following a coherent and logical thread. Except for some very minor issues (e. g., WIS abbreviation should be explained in Table 1 caption; S. cerevisiae has to be italicized in Table 3), the manuscript can be considered for publication

Author Response

We would like to thank reviewer 3 for the positive feedback. We have performed the suggested modifications in the revised version:

• WIS abbreviation is explained in Table 1 caption
• cerevisiae was italicized in Table 3

We have also made minor changes throughout the manuscript (visible as track changes), in connection with the comments from reviewer 1.