## 2 Materials and Methods
### Analysis of Pulp Properties  
#### 2.5.1 Porosity by Water Retention Value and Simons' Staining Method  
When pulp fibers are dried, internal fiber volume shrinks [11] and the shrinkage prevents the accurate measurement of porosity due to the pore collapse caused by drying. Thus, the measurement needs to be done in a wet state [27] and two independent methods, water retention value (WRV) and Simon's staining method were selected to evaluate the porosity of pulp in wet state.  
The WRV has been used to estimate the fiber saturation point, which correlates to the amount of water in the cell wall pores or the volume of pores, and it has been found that the drying effect induced by fiber hornification is reflected in a reduction in the WRV [22]. The WRV was determined using the  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline  & & \multic  
TAPPI Useful method with a centrifugal force (Eppendorf North America, Hauppauge, NY, USA) of 900 ref (2400 rpm) for 30 min [28]. The centrifuged wet pulp was first weighed and then oven dried at 105 \({}^{\circ}\)C overnight. The WRV was calculated by the percentage of water retained in the dried pulp, i.e.,  
\[\text{WRV (\%)}=[(\text{W}_{\text{wet-W}}\text{$\text{{}_{\text{dried}}}$})/ \text{W}_{\text{dried}}]\text{ * }100 \tag{1}\]  
in which:  
* \(\text{W}_{\text{wet}}\): weight of wet pulp
* \(\text{W}_{\text{dried}}\): weight of dried pulp  
In addition to the WRV, the Simon's staining method was performed to analyze the porosity, including the enzyme accessible surface area of the pulp, depending on the drying method. For the [PERSON] staining method, the 1:1 staining method were used in this study [29]. Blue dye (DB) and orange dye (DO) were dissolved in nanopure water for the preparation of each dye solution and the final concentrations of DB and DO were 1% (_w/v_), respectively. For the orange dye, only higher molecular fractions were used after the fractionation. The dye solution was then prepared with 1 mL of the DB solution, 1 mL of the DO solution, 10 mL of 10% (_w/v_) NaCl and 70 mL of nanopure water. For staining, a 25 mg of sample (dry weight) in the dye solution was incubated in a 75 \({}^{\circ}\)C shaking incubator at 200 rpm for 48 h. The stained samples were then filtered, rinsed with a minimum amount of distilled water and stripped with 25% (_w/v_) aqueous pyridine at 45 \({}^{\circ}\)C for 18 h. The dye stripping solution was then analyzed using a UV-Vis spectrophotometer to determine the concentration of DO and DB (the maximum absorbance of DB and DO was at 624 and 455 nm, respectively). The concentration of DO and DB dyes in the dye stripping solution (C\({}_{\text{O}}\) and C\({}_{\text{B}}\)) was determined using the following two equations, Equations (2) and (3) (using the Lambert-Beer law for a binary mixture), which were solved simultaneously [30].  
\[A_{455 mm}=\varepsilon_{O455}\ LC_{\text{O}}+\varepsilon_{B455}\ LC_{B} \tag{2}\]  
\[A_{624 mm}=\varepsilon_{O624}\ LC_{\text{O}}+\varepsilon_{B624}\ LC_{B} \tag{3}\]  
in which:  
* A: absorbance
* \(\varepsilon\): absorptivity
* \(\varepsilon_{O455}=35.62\), \(\varepsilon_{B455}=2.59\), \(\varepsilon_{O624}=0.19\), \(\varepsilon_{B624}=15.62\) L g-1 cm and L = 1 cm