Incomplete enzymatic hydrolysis of lignocellulosic biomass is well known.  Moreover, it has been observed that enzymatic hydrolysis residues are even more difficult to be saccharified, although half of its component is carbohydrate.  High lignin content and/or high crystallinity index might be the most critical factors contributing to the recalcitrant nature of the residues from enzymatic hydrolysis.  However, which factor contributes more to the recalcitrance is unclear and a systematic study for the recalcitrant factors of the enzymatic hydrolysis residues is not straightforward due to the coupling effect of each factor. 
In this research, the factors affecting the recalcitrant nature including lignin content and crystallinity index were studied using green liquor pretreated softwood pulp and Avicel.  Samples were collected at various times during the enzymatic hydrolysis and after washing, they were subjected to a second enzymatic hydrolysis.  
It was found for both Avicel and softwood pulp that the carbohydrate conversion yields increased with enzymatic hydrolysis time.  It was found that cellulose crystallinity index of Avicel changed little during enzymatic hydrolysis and the second enzymatic hydrolysis had almost the same carbohydrate conversion yield regardless of the history of previous enzymatic hydrolysis.  This suggests that the enzymatic hydrolysis of Avicel is a kinetics issue.  However, it was found for softwood pulp that cellulose crystallinity and lignin content increased during enzymatic hydrolysis and the conversion yield of second enzymatic hydrolysis decreased.  These observations indicate that both properties affect the carbohydrate conversion yield of second enzymatic hydrolysis.