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In a previous study by the authors, it was found that benzene-ethanol extraction (BAE) could remove some of the lignin fragments from pretreated bamboo substrate and therefore cause a decrease in the lignin content of the substrate. In the present study, lignin was isolated by dioxane extraction of enzyme hydrolysis of ball-milled substrate (CEL method), and the effects of BAE on lignin molecular weight (MW) were determined. Results indicated that hydrothermal pretreatment could facilitate lignin isolation substantially when CEL method was used. Lignin fragments with low MW were more sensitive to BAE, and therefore, evident increase in both weight average molecular weight (Mw) and number average molecular weight (Mn) was observed; especially, the increase rate of Mn was more than 2.5 times of Mw when degradation prevailed. However, subtle changes were found in Fourier transform infrared spectroscopy and carbon-13 nuclear magnetic resonance spectra between the lignin samples with and without BAE; the solubility of lignin in benzene-ethanol solution was more dependent on the MW level than on the functional groups in lignin structure. [ABSTRACT FROM AUTHOR]