| Summary: | The objective of this study was to investigate structural changes and lignin redistribution in <i>Eucalyptus globulus</i> pre-treated by steam explosion under different degrees of severity (S<sub>0</sub>), in order to evaluate their effect on cellulose accessibility by enzymatic hydrolysis. Approximately 87.7% to 98.5% of original glucans were retained in the pre-treated material. Glucose yields after the enzymatic hydrolysis of pre-treated material improved from 19.4% to 85.1% when S<sub>0</sub> was increased from 8.53 to 10.42. One of the main reasons for the increase in glucose yield was the redistribution of lignin as micro-particles were deposited on the surface and interior of the fibre cell wall. This information was confirmed by laser scanning confocal fluorescence and FT-IR imaging; these microscopic techniques show changes in the physical and chemical characteristics of pre-treated fibres. In addition, the results allowed the construction of an explanatory model for microscale understanding of the enzymatic accessibility mechanism in the pre-treated lignocellulose.
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