Time course of an enzymatic hydrolysis
The iKnowzyme acid 2XL cellulase used in this study had cellulase activity of 16,000 U ml-1 and xylanase activity of 570 U ml-1 with a protein content of 278.5 mg ml-1. Time course of sugar production from the hydrolysis of APEFB by the iKnowzyme acid 2XL cellulase is shown in Fig. 1. The results show that glucose and xylose concentrations were slightly increased when increasing the hydrolysis time. The maximum glucose (24.65 g/l) and xylose (1.77 g/l) were obtained at 120 h.
Statistical modeling for an enzymatic hydrolysis
The APEFB (5 g) was hydrolyzed by adding to 45 ml 50 mM acetate buffer pH 5.0 with Tween 80 0.3 g/l and potassium metabisulfite 0.02 g/l. The enzyme dose and liquid to solid ratio were parameters used in this experiment. The conditions for hydrolysis were done at 150 rpm at 50°C for 120 h. The results found that the maximum glucose (65.61 g/l) and xylose (2.22 g/l) concentrations were observed in the run number 7 and 3, respectively (Table 2). The quadratic models in terms of coded variables were shown in equation (2) and (3), where Y1 represented glucose concentration and Y2 represented xylose concentration as a function of the enzyme dose (X1) and liquid to solid ratio (X2).
Y1 = 19.85 + 2.90X1 - 2.09X2 - 0.06X1X2 - 0.02X12 + 0.04X22 (2)
Y2 = 2.77 - 0.04X1 - 0.06X2 + 1.05E-003X1X2 + 6.52E-004X12 - 1.89E-004X22 (3)
The response function and experimental data of the regression analysis were performed and the second order model for all responses was evaluated by ANOVA (Table 3 and 4). The greater the F-value indicates that the factors explain adequately the variation in the data about its mean, and the estimated factor effects are real. The results showed that the value of the adjusted R2 determination for glucose and xylose productions was also very high (99.30% and 96.63%, respectively) which indicated a high accuracy of the model .
Estimations of glucose and xylose concentrations over independent variables, enzyme dose (X1) and the liquid to solid ratio (X2) in terms of response surfaces are shown in Fig. 2. The maximum concentrations of glucose (65.61 g/l) and xylose (2.13 g/l) were observed by using the enzyme dose and liquid to solid ratio of 40 U/g APEFB and 10 ml of liquid per g of APEFB (100 g/l), respectively for 120 h. Shamsudin et al.  report that glucose and xylose concentration of 8.75 and 3.75 g/l were obtained after hydrolysis the steam pretreated EFB (50 g/l) with Celuclast 1.5L (25 U/g EFB) at 50°C for 24 h. In another study, glucose concentration (17.5 g/l) was obtained after hydrolysis the water pretreated EFB (25 g/l) with 70 U/g EFB at 50°C for 48 h .
To confirm the results, the hydrolysis of APEFB by the enzyme was carried out in triplicate under the most and least optimized conditions. The results are shown in Table 5. The highest glucose and xylose concentrations obtained were 65.71 g/l (0.66 g/g APEFB) and 2.14 g/l (0.02 g/g APEFB), respectively when hydrolysis using the iKnowzyme acid 2XL cellulase 40 U/g APEFB and the liquid to solid ratio of 10 ml/g APEFB (100 g/l APEFB) with shaking at 150 rpm and 50°C for 120 h. It was found that the sugars concentrations obtained from enzymatic hydrolysis of APEFB of the experimental tests were similar to the sugar concentrations in the prediction models.
Effect of type of acids
The effect of various acids on the hydrolysis of APEFB to produce glucose and xylose was done by using 0.5% (w/v) of acid solutions at 120°C. The results are shown in Fig 3. Glucose, xylose, furfural and HMF concentrations were increased when increasing the time for hydrolysis. The highest glucose concentration (4.93 g/l) and xylose concentration (15.12 g/l) were obtained when hydrochloric acid was used for hydrolysis. The highest furfural concentration was obtained after hydrolysis the APEFB with nitric acid (1.65 g/l), while the highest HMF was obtained with sulfuric acid (0.41 g/l). The results show that the hydrolysis of APEFB with acid gave xylose concentration higher than glucose. Chong et al.  found that the highest yield of glucose (2.75 g/l) and xylose (24.14 g/l) was obtained after hydrolysis the EFB with 6% sulfuric acid at 120°C for 15 min. Rahman et al.  report that the maximum xylose, glucose and furfural concentrations of 29.4, 2.34 and 0.8 g/l were observed after hydrolysis the EFB by using 6% sulfuric acid at 120°C for 15 min. The ultrasonic pretreated EFB hydrolysis using 2% sulfuric acid at 100°C for 45 min provied glucose and xylose of 2.0 and 23.2 g/l, respectively .
Statistical modeling for acid hydrolysis
Since hydrolysis of APEFB by HCl provided highest sugar productions and less furfural and HMF, it was futher used to optimize the sugar productions. The experimental ranges and levels of the independent process variables including the HCl concentration and temperature for hydrolysis of APEFB are shown in Table 5. The design of the acid hydrolysis experiments including dependent variables, Y3 (glucose), Y4 (xylose), Y5 (furfural) and Y6 (HMF) are also given in Table 6. The release of glucose and xylose in the hydrolysate after acid hydrolysis was dependent on experimental operating conditions. The maximum concentrations of glucose and xylose obtained were 10.75 and 15.86 g/l, respectively when the HCl concentration, temperature and reaction time were 6% (w/v) at 110°C and 90 min, respectively. The lowest furfural and HMF concentrations (1.41 and 0.51 g/l) occurred when the reaction was carried out with 6% HCl at 96°C for 90 min.
The quadratic models in terms of coded variables are shown in equation (4) – (7), as a function of reaction acid (HCl) concentration (X3) and temperature (X4).
Y3 = - 223.31 + 17.11X3 + 3.24 X4 - 0.09 X3X4 - 0.57X32 - 0.01X42 (4)
Y4 = - 300.42 + 16.72X3 + 4.70X4 - 0.11X3X4 - 0.45X32 - 0.02X42 (5)
Y5 = 10.87 - 3.33X3 - 0.047X4 + 0.02X3X4 + 0.15X32 - 3.08E-005X42 (6)
Y6 = -15.13 + 0.74X3 + 0.24X4 - 8.99E-04X3X4 - 0.005X32 - 1.03E-003X42 (7)
To fit the response function and experimental data, regression analysis was performed and the second order model for all response was evaluated by ANOVA. The resules are presented in Table 7–10. In this experiment, the value of R2 of glucose, xylose, furfural, and HMF production from APEFB were 93.97, 95.52, 96.85, and 94.16%, respectively. These values indicate a high degree of correlation between the experimental and the predicted values. The results show that the values of the adjusted R2 determination were also very high (87.94%, 91.03%, 93.71% and 88.32% for glucose, xylose, furfural and HMF productions, respectively) which indicate a high accuracy of the model .
Estimations of sugar concentration (glucose and xylose) and by-products (furfural and HMF) from the hydrolysis of APEFB over independent variables (acid concentration (X3) and temperature (X4)) in term of response surfaces are shown in Fig. 4 and 5. The maximum concentration of glucose (10.56 g/l) was obtained by conducting hydrolysis experiment with 5.85% HCl at 114°C for 90 min while the maximum concentrations of xylose (15.86 g/l) was obtained by hydrolysis with 4% HCl at 120°C for 90 min. On the other hand, maximum concentration of furfural (3.68 g/l) and HMF (0.69 g/l) was obtained by hydrolysis with 7.32% HCl at 120°C for 90 min but the minimum concentration of furfural (1.92 g/l) and HMF (0.38 g/l) was obtained by hydrolysis with 4% HCl and at 100°C for 90 min. Thamsee et al.  reported that the maximum glucose and xylose concentrations of 1.80 and 33.16 g/l was obtained when hydrolysis the EFB with 4.0% sulfuric acid at 119°C for 60 min. While the maximum glucose and xylose concentrations (1.2 and 11.3 g/l, respectively) were obtained by using a combined of 0.5% sulfuric acid and 0.2% phosphoric acid at 160°C for 10 min .
Based on the models, the numerical optimization was carried out with considering each value of response in four conditions. The results are shown in Table 11. The best results of glucose and xylose concentrations obtained by the experiment were 10.70 g/l (0.11 g/g APEFB) and 15.30 g/l (0.15 g/g APEFB), respectively after hydrolysis of the APEFB with 5.80% HCl at 114°C for 90 min. The highest concentrations of furfural (3.81 g/l) and HMF (0.74 g/l) were obtained after hydrolysis with 7.32% HCl at 120°C. Most diluted acid and high temperature was found to generate by-products such as furfural and HMF . Furfural and HMF presented in the hydrolysate higher than 0.2 g/l were potential inhibitors to microbial metabolism. They inhibited the fermentation process by causing cell morphological change or ultimate death of the microorganism . To keep the concentration of furfural and HMF in the hydrolysate at a low level it is necessary to run the hydrolysis reaction at less severe conditions.