AbstractEnzymes still exhibit activities after hydrolysis of biomass according to previous studies. Recycling the enzymes and use them in subsequent hydrolysis cycles can further utilize their remaining activities. Previous studies have mainly discussed enzyme recycling processes up to three cycles, in which the processes did not reach steady state. Steady state investigation is essential for the guidance of the real life process. Four cycles of processing have usually been considered enough to bring the system to steady state in process engineering. In this work, hydrolysate was used as the source of recycled enzymes to fresh substrate for five cycles. Because a large amount of enzymes remained on the pulp, surfactant was introduced to recycle the enzymes that remained with the residue. Recycled hydrolysate from previous enzymatic hydrolysis usually carries a high concentration of sugars, which can inhibit the new round of hydrolysis. To remove sugar from the recycling stream, a wash with fresh buffer was performed. Sugars were removed, while enzymes still remain on the fresh substrates. Six recycling strategies were evaluated for enzyme recycling percentage and enzymatic hydrolysis efficiency with both green-liquor pretreated softwood and hardwood in this investigation. Hydrolysis efficiency increased by about 40% for softwood at 30 mg/g enzyme dosage and about 25% for hardwood at 7.5 mg/g when a washing stage was applied with addition of surfactant.