**5. Conclusions**

This investigation clarifies correlations between operational conditions during hydrothermal pretreatment and critical aspects of biochemical conversion of wheat straw, including hemicellulose removal, susceptibility to enzymatic saccharification, total sugar yields, byproduct formation, and inhibitory effects on enzymatic saccharification and ethanolic fermentation. Pseudolignin formation in autocatalyzed hydrothermal pretreatment at 205 ◦C and in sulfuric-acid-catalyzed hydrothermal pretreatment at lower temperatures was demonstrated. The importance of the removal of hemicelluloses for achieving high enzymatic digestibility of glucan was shown. Hydrothermal pretreatment without using mineral acids and without exceeding 200 ◦C was found to be the best approach to achieve high sugar yields while minimizing byproduct formation.

**Author Contributions:** D.I., conceptualization, methodology, investigation, data curation, writing original draft preparation; S.S., methodology, validation, formal analysis; L.J.J., conceptualization, methodology, supervision, writing—review and editing, project administration, funding acquisition; C.M., conceptualization, methodology, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Swedish Energy Agency (P41285-1 and P47516-1), Bio4Energy (www.bio4energy.se, accessed on 4 March 2021), and the Kempe Foundations.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The support provided by Guochao Wu in the preparation of the fermentability experiment, Junko Takahashi Schmidt in the Py-GC/MS analysis, and the statistical assistance given by Francesca Pilotto are gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest. The funding agencies had no role in the design of the study, in the interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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