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• In-situ lignin modification improved enzymatic hydrolysis of acid-pretreated larch. • Lignin modification with 2-naphthol suppressed lignin repolymerization. • Lignin modification with PEGDE lowered the enzyme non-productive binding on lignins. • 2-Naphthol and PEGDE modification synergistically facilitated enzymatic hydrolysis. To overcome the recalcitrance of residual lignins in acid-pretreated larch (AL), a combined acid and alkali pretreatment with in-situ lignin modification was developed in this study. The results showed that introducing in-situ lignin modification with 2-naphthol to acid pretreatment (160 and 180 oC) improved the enzymatic digestibility of AL by 12.7-14.4%, through suppressing lignin repolymerization. The obviously higher improvement (57.8-88.3%) was achieved by applying alkali post-treatment (90 oC) with poly (ethylene glycol) diglycidyl ether (PEGDE) on AL, mainly due to the function of in-situ lignin modification with PEGDE for reducing enzyme non-productive binding on lignins. More importantly, the synergism of 2-naphthol and PEGDE modification facilitated the enzymatic hydrolysis of AL more significantly. Its beneficial mechanism was explored by investigating the effects of in-situ lignin modification on lignin properties, including extraction yields, functional groups, and enzyme affinity of lignins. Results will give insights into establishing an efficient pretreatment of softwood biomass. [ABSTRACT FROM AUTHOR]