Evaluation of the factors affecting avicel reactivity using multi-stage enzymatic hydrolysis

Multi-stage and single-stage enzymatic hydrolysis of cellulose (Avicel PH-101) were conducted to investigate individual factors that affect the rate-reducing kinetics of enzymatic hydrolysis. Understanding factors affecting enzymatic hydrolysis of Avicel will help improve hydrolysis of various biomasses. Product inhibition, enzyme deactivation, and the changes of substrate are potential factors that can affect the hydrolysis efficiency of Avicel. Multi-stage enzymatic hydrolysis resulted in 36.9% and 25.4% higher carbohydrate conversion as compared to a single-stage enzymatic hydrolysis with an enzyme loading of 5 and 20 FPU/g in a 96 h reaction. However, a decline in carbohydrate conversion of 1.6% and 2.6% was observed through each stage with 5 and 20 FPU/g, respectively. This indicated that the substrate became more recalcitrant as hydrolysis progressed. The decreased reactivity was not due to crystallinity because no significant change in crystallinity was detected by X-ray diffraction. Product inhibition was significant at low enzyme loading, while it was marginal at high enzyme loading. Therefore, product inhibition can only partially explain this decreased conversion. Another important factor, enzyme deactivation, contributed to 20.3% and 25.4% decrease in the total carbohydrate conversion of 96 h hydrolysis with 5 and 20 FPU/g, respectively. This work shows that an important reason for the decreased Avicel digestibility is the effect of enzyme blockage, which refers to the enzymes that irreversibly adsorb on accessible sites of substrate. About 45.3% and 63.2% of the total decreased conversion at the end of the 8th stage with 5 and 20 FPU/g, respectively, was due to the presence of irreversibly adsorbed enzymes. This blockage of active sites by enzymes has been speculated by other researchers, but this article shows further evidence of this effect.