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[Display omitted] • Liquid fraction containing glycerol was used for recycled corn stover pretreatment. • Dry cell weight of P. pastoris reached ∼ 40 g/L with glycerol as sole carbon source. • The titer of human lysozyme reached 1.3 g/L by P. pastoris with methanol induction. • Mixing pretreated corn stover with cassava as co-substrate to produce biobutanol. • Final butanol titer of 13.9 g/L was produced from mixed substrates under SSF. Glycerol organosolv pretreatment (GOP) is considered an efficient method to deconstruct lignocellulose for producing fermentable sugars. Herein, the liquid fraction containing glycerol after GOP was utilized for recycled pretreatment of corn stover (CS) for four cycles. Enzymatic yield of glucose after recycled pretreatment was enhanced by 2.4–3.5 folds compared with untreated CS. Meanwhile, residual glycerol was used as carbon source for cultivation of Pichia pastoris to obtain high cell-density, and a final titer of 1.3 g/L human lysozyme was produced by P. pastoris under low temperature methanol induction strategy. Additionally, the pretreated CS was mixed with cassava as fermentable substrates for butanol production by wild-type Clostridium acetobutylicum ATCC 824. Final butanol production of 13.9 g/L was obtained from mixed substrates (25%:75% of CS/cassava) at 10% solids loading by simultaneous saccharification and fermentation. Overall, integration of residual glycerol utilization and butanol production by microbial fermentation provided an efficient strategy for biorefinery. [ABSTRACT FROM AUTHOR]