## 3 Results and Discussion
### Evaluation of Drying Effect Using Water Retention Value and Simons' Staining Method  
The water retention value (WRV) has been used to characterize the drying effect on enzymatic hydrolysis induced by fiber hornification because it can easily measure the pore volume of pulp in a wet state by measuring the amount of water in cell wall [22]. It was previously found that the WRV of the OD pulp decreased significantly due to collapse and shrinkage of pores and the decrease in the WRV correlated well with enzymatic digestibility [22, 32]. In this study, the OD pulp with lower enzymatic digestibility, as shown above, revealed a significantly lower WRV, which decreased by 36.4% and 33.3% based on the ND and FD pulps, respectively (Table 3). The decrease in WRV related to the lower enzymatic digestibility was also caused by less enzyme accessibility to cellulose, induced by the decrease in pore volume [22]. However, as enzymatic hydrolysis progresses, new pores are formed through cellulose conversion to sugars and the pore formation increases the pore volume in cellulose. Therefore, the WRV of the OD pulp hydrolyzed for 24 h increased drastically from 105.7 to 203.0%. As described, the kinetics of enzymatic hydrolysis showed that the bigger drying effect at initial stages, which is due to less pore volume caused by the collapse of pores and pores formed by cellulose conversion during hydrolysis, can supply enzymes with space to access cellulose. Therefore, the difference in enzymatic digestibility between the ND and OD pulps will decrease. The Simons' staining method can be used to evaluate the enzyme accessible surface area on cellulose using adsorption and desorption of dyes [26, 30]. The pores are segregated depending on the diameter of two different dyes, which have a different affinity for hydroxyl groups in cellulose, and thereby, enzyme accessible surface area of cellulose can be measured in a wet state. The large orange-colored dye molecules can only penetrate larger pores and adsorb in preference to the small blue-colored dye molecules as a result of their greater affinity for hydroxyl groups in cellulose [29]. The large orange-colored dye molecule is mainly composed of two fractions that have hydrodynamic diameters of 5-7 and 12-36 nm [29]. The 5-7 nm diameter is very similar to that of the catalytic core of _Trichoderma reseei_ endoglucanase [21]. Therefore, enzyme accessible surface area can be evaluated by the amount of adsorbed orange dye [26], even though the adsorption behavior onto the cellulose surface may be different to enzymes, and also the drying effect on enzymatic hydrolysis has been assessed effectively by the Simons' staining [21]. As result of the Simons' staining, we found that the amount of orange dye adsorbed in the ND and OD pulps were 154.4 and 75.9 mg per g of cellulose, respectively, and the drying process decreased more than half of the accessible surface area in larger pores by pore collapse (Table 4). However, the amount of blue dye, which adsorbed on the surface area of smaller pores, increased from 36.7 to 62.1 mg per g of cellulose. The increase in the adsorption of blue dye was likely caused by the change of larger pores to smaller pores due to the collapse of larger pores, and thus, the surface area of smaller pore increased [21]. The increase in enzyme accessible surface area enhanced enzymatic digestibility through better enzyme adsorption, and it was more remarkable at the initial stage. The total amount of adsorbed dye decreased from 191.0 to 138.0 by drying, which suggested that the formation of the smaller pores and disappearance of larger pores by pore collapse in cellulose decreased the total surface area. It is well known that most pretreatment processes open up the cell wall structure by mechanical shearing force and/or chemical removal of components and this forms new pores in