**3. Results and discussion**

#### **3.1. Chemical components of oil palm trunk and steam exploded oil palm trunk pulp**

The Figure 2 (a) showed oil palm trunk chip before steam explosion pretreatment and Figure 2 (b) showed steam exploded oil palm trunk pulp obtained after steam explosion.

**Figure 2.** Oil palm trunk chip (a) and steam exploed oil palm trunk pulp (b)

The chemical components of steam exploded oil palm trunk pulp were 40.54% of cellulose, 9.36% of hemicelluloses, 38.46% of lignin and 8.56% of extractive in ethanol/benzene. This pulp obtained after steam explosion was used as raw material for optimization study in ethanol production.

#### **3.2. Model fitting for optimization of alkaline delignification**

was deionied water with flow rate 0.6 ml/min. The injection volume was 20 µl and the column

based on monomer content that was measured after two steps of acid hydrolysis. The first step hydrolysis was performed with 72% (w/w) H2SO4 at 30ºC for 60 min. In the second step, the reaction mixture was diluted to 4% (w/w) H2SO4 with distilled water and subsequently

The ethanol content in fermented solution was analyzed by gas chromatography (GC). GC analysis was carried out with an Agilent Technologies (Santa clara, CA) 6890 gas chromato‐ graph equipped with a flame ionization detector and a HP-5 (Bonded 5%phenyl, 95% dime‐ thylpolysiloxane) capillary column (30 m x 0.32 mm ID, 0.25 µm film thickness). The temperature program was an initial temperature of 150ºC, increased to 190ºC at 10ºC/min then 15ºC/min to 250ºC, and held for 15 min. The injector and detector temperature were 250 ºC and 300ºC, respectively. Standard and sample were injected by using the split mode ratio of 50:1.

**3.1. Chemical components of oil palm trunk and steam exploded oil palm trunk pulp**

2 (b) showed steam exploded oil palm trunk pulp obtained after steam explosion.

**Figure 2.** Oil palm trunk chip (a) and steam exploed oil palm trunk pulp (b)

The Figure 2 (a) showed oil palm trunk chip before steam explosion pretreatment and Figure

described above. All analytical determinations were performed in duplication.

164 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

C. The glucose content of the solid residue was determined

C for 1 h. This hydrolysis liquid was then analyzed for glucose content as

temperature was maintained at 80°

autoclaved at 121°

**2.8. Ethanol determination**

**3. Results and discussion**

The complete design matrix together with the values of both the experimental and pre‐ dicted responses is given in Table3. Central composite design was used to develop corre‐ lation between the NaOH and KOH delignification variables to the percentages of glucose yield. The percentages of glucose yield were found to range between 41.4-49.8% for KOH delignification and 38.0-49.9% for NaOH delignification. Runs 17-23 at the cen‐ ter point were used to determine the experiment error. For both reponses of NaOH de‐ lignification and KOH delignification, the quadratic model was selected, as suggested by used software. The final empirical models in terms of coded factors are given by equa‐ tion (1) and Equation (2) in Table 4. Where X1, X2, X2 and X4 were the coded values of test variables that represented pulp concentration, concentration of NaOH or concentra‐ tion of KOH, reaction time and temperature, respectively. The variables X1X2, X1X3, X1X4, X2X3, X2X4 and X3X4 represented the interaction effects of pulp concentration and concen‐ tration of NaOH or concentration of KOH, pulp concentration and reaction time, pulp concentration and temperature, respectively. The quality of the model developed was evaluated based on the correlation coefficient R2 . The R2 for the two obtained equations were found to be 0.875 and 0.890 in NaOH and KOH delignification, respectively. This indicates that 87.5% and 89.0% of the total variation in both delignifications were attrib‐ uted to experimental variables studied. The R2 of 0.875 and 0.890 were considered as the good fit of the models.

The adequacy of the two models was further justified through analysis of variance (ANOVA). The ANOVA for the quadratic models for the two reponses is listed in Table 5. The Fisher's test (F-test) carried out on experimental data make it possible to estimate the statistical significance of the proposed model. The F-test value of the models being 16.69 and 10.88, respectively for glucose in pulp obtained after NaOH and KOH lignifications, with a low probability value (p<0.01), we can conclude that they were statistically significant at 99.9% confidence level. It should be noted that p-value indicates the statistical significance of each parameter. It is based on hypothesis that a parameter is not significant, thus the more effect is significant. From Table 5, it was showed that the two models (both p-value<0.01) were adequate to predict the glucose in pulp obtained after NaOH and KOH lignifications within the range of studied variables.

Response surface contour plots of the RSM as a function of two factors at the time are helpful in understanding both the main and the interaction effects of these factors. The effects of concentration of pulp and concentration of NaOH on the percentage of glucose are shown in Figure 3 (a). Figure 3 showed that the concentration of pulp and NaOH could increased percent glucose in pulp after NaOH delignification. The concentration of pulp higher than 13.50% (w/w) had no significant effect on the amount of percent glu‐ cose in pulp. Response surface plot indicated the optimized condition at 12.50% w/v of pulp concentration and 21.50% (w/v) NaOH that gave 48.10% of percent glucose re‐ mained in pulp after NaOH delignification. Figure 3 (b) showed that increasing tempera‐ ture and concentration of pulp could increased percent glucose in pulp after NaOH delignification. The temperature higher than 80ºC had no significant effect on the percent glucose in pulp after NaOH delignification. Response surface plot indicated the opti‐ mized condition at 12.5% w/w of pulp concentration and 80ºC that gave 47.95% of per‐ cent glucose remained in pulp after NaOH delignification. Figure 3 (c) showed that increasing time and concentration of pulp could increased percent glucose in pulp after NaOH delignification. The time longer than 67 min had no significant effect on the per‐ cent glucose in pulp after delignification. Response surface plot indicated the optimized condition at 12.5% w/v of pulp concentration and 65 min that gave 50.23% of percent glucose in pulp after NaOH delignification.

**Run X1 X2 X3 X4**

delignification

**Glucose (%), Delignification Experimental Predicted KOH NaOH KOH NaOH**

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167

 1 1 -1 -1 48.3 45.2 48.7 45.1 1 1 -1 1 46.4 49.9 46.6 49.2 1 1 1 -1 42.0 43.6 42.3 43.4 1 1 1 1 49.2 49.0 49.6 49.8 1 -1 -1 -1 48.1 42.3 48.6 42.3 1 -1 -1 1 43.2 40.0 43.2 40.2 1 -1 1 -1 47.9 47.4 47.6 47.4 1 -1 1 1 49.3 49.5 49.3 49.5 -1 1 -1 -1 44.2 38.4 44.7 38.2 -1 1 -1 1 46.4 30.8 46.5 30.6 -1 1 1 -1 39.8 40.8 39.9 40.6 -1 1 1 1 47.2 42.1 47.6 42.7 -1 -1 -1 -1 44.1 48.1 44.6 48.5 -1 -1 -1 1 41.4 49.3 41.5 49.3 -1 -1 1 -1 46.3 38.0 46.7 38.1 -1 -1 1 1 49.0 48.2 49.0 48.7 0 0 0 0 48.3 43.3 48.2 43.6 0 0 0 0 47.4 44.9 47.3 44.3 0 0 0 0 48.6 44.0 48.0 44.5 0 0 0 0 46.4 44.9 46.9 44.6 0 0 0 0 46.3 44.3 46.0 44.3 0 0 0 0 48.5 43.8 48.2 43.2 0 0 0 0 48.1 43.2 48.3 43.6 α 0 0 0 49.5 39.8 49.6 39.7 -α 0 0 0 44.3 47.2 44.3 47.9 0 α 0 0 48.4 48.9 48.2 48.4 0 -α 0 0 45.7 43.9 45.0 43.7 0 0 α 0 49.8 48.6 49.5 48.8 0 0 -α 0 47.3 42.7 47.4 42.7 0 0 0 α 49.4 49.0 49.7 49.9 0 0 0 -α 43.7 44.7 43.3 44.9

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ...

**Table 3.** Design and response of the central composite design for glucose (%) obtained from NaOH and KOH

Response surface contour plots of the RSM on the effects of concentration of pulp and concentration of KOH on the percentage of glucose are shown in Figure 4 (a).Figure 4 (a) a showed that increasing concentration of pulp and KOH concentration could in‐ creased percent glucose in pulp. The pulp concentration higher than 12.5% (w/v) had no significant effect on percent glucose in pulp. Likewise, the KOH concentration higher than 23.4% (w/w) had no significant effect on percent glucose in pulp after KOH deligni‐ fication. Response surface plot indicated the optimized condition at 10% (w/v) of pulp concentration and 23.5% (w/w) of KOH concentration that gave 49.04% of percent glu‐ cose in pulp after KOH delignification.

Figure 4 (b) showed that increasing concentration of pulp and temperature could in‐ creased percent glucose in pulp. The temperature higher than 80°C had no significant ef‐ fect on percent glucose in pulp. Likewise, the concentration of pulp higher than 12% (w/v) had no significant effect on percent glucose in pulp after KOH delignification. Re‐ sponse surface plot indicated the optimized condition at 78°C of glucose in pulp after KOH delignification.

Figure 4 (c) showed that increasing time and concentration of pulp could increased percent glucose in pulp. The time longer than 60 min had no significant effect on percent glucose in pulp. Likewise, the concentration of pulp more than 12% (w/v) had no significant effect on percent glucose in pulp after KOH delignification. Response surface plot indicated the optimized condition at 12% (w/v) of pulp concentration and 70 min of reaction time that gave 48.35% of percent glucose in pulp after KOH delignification.

The summary result from combination of each response surface plot showed that the op‐ timum condition for NaOH delignification was obtained from 11% (w/v) pulp concentra‐ tion, 21% (w/w) NaOH concentration, 65 min reaction time and 78ºC temperature with the maximum glucose at 47.50% remaining in the pulp. The optimum condition for KOH delignification was 12% (w/v) pulp concentration, 23% (w/w) KOH concentration, 65 min of reaction time and 80ºC of temperature with the maximum glucose at 49.50% remain‐ ing in the pulp.

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ... http://dx.doi.org/10.5772/54691 167

cose in pulp. Response surface plot indicated the optimized condition at 12.50% w/v of pulp concentration and 21.50% (w/v) NaOH that gave 48.10% of percent glucose re‐ mained in pulp after NaOH delignification. Figure 3 (b) showed that increasing tempera‐ ture and concentration of pulp could increased percent glucose in pulp after NaOH delignification. The temperature higher than 80ºC had no significant effect on the percent glucose in pulp after NaOH delignification. Response surface plot indicated the opti‐ mized condition at 12.5% w/w of pulp concentration and 80ºC that gave 47.95% of per‐ cent glucose remained in pulp after NaOH delignification. Figure 3 (c) showed that increasing time and concentration of pulp could increased percent glucose in pulp after NaOH delignification. The time longer than 67 min had no significant effect on the per‐ cent glucose in pulp after delignification. Response surface plot indicated the optimized condition at 12.5% w/v of pulp concentration and 65 min that gave 50.23% of percent

166 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Response surface contour plots of the RSM on the effects of concentration of pulp and concentration of KOH on the percentage of glucose are shown in Figure 4 (a).Figure 4 (a) a showed that increasing concentration of pulp and KOH concentration could in‐ creased percent glucose in pulp. The pulp concentration higher than 12.5% (w/v) had no significant effect on percent glucose in pulp. Likewise, the KOH concentration higher than 23.4% (w/w) had no significant effect on percent glucose in pulp after KOH deligni‐ fication. Response surface plot indicated the optimized condition at 10% (w/v) of pulp concentration and 23.5% (w/w) of KOH concentration that gave 49.04% of percent glu‐

Figure 4 (b) showed that increasing concentration of pulp and temperature could in‐ creased percent glucose in pulp. The temperature higher than 80°C had no significant ef‐ fect on percent glucose in pulp. Likewise, the concentration of pulp higher than 12% (w/v) had no significant effect on percent glucose in pulp after KOH delignification. Re‐ sponse surface plot indicated the optimized condition at 78°C of glucose in pulp after

Figure 4 (c) showed that increasing time and concentration of pulp could increased percent glucose in pulp. The time longer than 60 min had no significant effect on percent glucose in pulp. Likewise, the concentration of pulp more than 12% (w/v) had no significant effect on percent glucose in pulp after KOH delignification. Response surface plot indicated the optimized condition at 12% (w/v) of pulp concentration and 70 min of reaction time that gave

The summary result from combination of each response surface plot showed that the op‐ timum condition for NaOH delignification was obtained from 11% (w/v) pulp concentra‐ tion, 21% (w/w) NaOH concentration, 65 min reaction time and 78ºC temperature with the maximum glucose at 47.50% remaining in the pulp. The optimum condition for KOH delignification was 12% (w/v) pulp concentration, 23% (w/w) KOH concentration, 65 min of reaction time and 80ºC of temperature with the maximum glucose at 49.50% remain‐

glucose in pulp after NaOH delignification.

cose in pulp after KOH delignification.

48.35% of percent glucose in pulp after KOH delignification.

KOH delignification.

ing in the pulp.


**Table 3.** Design and response of the central composite design for glucose (%) obtained from NaOH and KOH delignification


**Table 4.** The linear regression of dependent for alkaline delignification of glucose


(a) (b)

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ...

(c)

**Figure 3.** Response surface plots of glucose as a function of concentration of pulp and concentration of NaOH (a),

concentration of pulp and temperature (b), time and concentration of pulp (c), other fixed variables

)

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169

**Table 5.** Analysis of variance (ANOVA) for the fit of experimental data to response surface models

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ... http://dx.doi.org/10.5772/54691 169

**Dependent variable Predictive R2**

168 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

**Table 4.** The linear regression of dependent for alkaline delignification of glucose

**Source Degree of**

**freedom**

Total 30 10733.87

Total 30 8768.35

**Table 5.** Analysis of variance (ANOVA) for the fit of experimental data to response surface models

R2 0.875

R2 0.890

37.112 - 0.166 X1 - 0.959X2 + 0.511X3 + 0.104X4 - 0.074X12 - 0.005X22 + 2.706X32 + 001X42 + 0.133X1X2 - 0.011X1X3 + 0.001X1X4 - 0.005X2X 3 - 0.005X2X4 - 0.006X3X4

60.266 + 0.574X1 – 1.398X2 -0.320X3 + 0.091X4 – 0.030X12 - 0.001X22 + 0.006X32 – 002X42+ 0.133X1X2 – 0.008X1X3 + 0.006X1X4 - 0.004X2X 3 + 0.015X2X4 + 0.013X3X4

> **Sum of square**

Lack of fit 10 530.43 53.04 1.60 -

Pure error 6 198.21 33.04 - -

Lack of fit 10 523.45 52.34 1.01 -

Pure error 6 309.61 51.60 - -

NaOH Model 14 10005.23 714.65 15.65 0.0078

delignification Residual 16 728.64 45.54 - -

KOH Model 14 7935.29 566.81 10.88 0.0071

delignification Residual 16 833.06 52.07 - -

**Mean square** 0.875

0.890

**F-value P-value**

Glucose (%) NaOH (Eqs.1)

Glucose (%) KOH (Eqs.2)

**Figure 3.** Response surface plots of glucose as a function of concentration of pulp and concentration of NaOH (a), concentration of pulp and temperature (b), time and concentration of pulp (c), other fixed variables

loading, temperature and pulp concentration and enzyme loading and pulp concentration, respectively. The quality of the model developed was evaluated based on the correlation

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ...

hydrolysis of pulp obtained after NaOH and KOH delignification. This indicated that 86.70% and 93.50% of total variation in both enzyme hydrolysis were attributed to the experimental

The adequacy of the two models was further justified through analysis of variance as the same as delignification reaction in previous studied. The statistical result showed that the two models had p-values less than 0.01 that indicated these two models were adequate to predict the percentage of glucose yield in enzyme hydrolysis within the range of the studied variables.

In addition, response surface contour plots of the RSM on the effects of reaction time and concentration of pulp on the percentage of glucose in enzyme hydrolysis are shown in Fig‐ ure 5 (a). Figure 5 (a) showed that increasing time and pulp concentration could increased percent glucose yield in hydrolyzed solution. The pulp concentration higher than 4.5% (w/v) had no significant effect on percent glucose yield in hydrolyzed in solution. Response sur‐ face plot indicated the optimized condition at 3% (w/v) of pulp concentration and 65 h of reaction time that gave 81.01% of percent glucose yield in hydrolyzed solution obtained af‐ ter NaOH delignification. Figure 5 (b) showed that increasing time and temperature could increased percent glucose yield in hydrolyzed solution. The temperature higher than 50ºC had no significant effect on percent glucose yield in hydrolyzed solution. Response surface plot indicated the optimized condition at 44ºC of temperature and 65 h of reaction time that gave 81.33% of percent glucose yield hydrolyzed solution obtained after NaOH delignifica‐ tion. Figure 5 (c) showed that increasing time and enzyme loading could increased percent glucose yield in hydrolyzed solution. The enzyme loading higher than 65 (FPU/g substrate) had no significant effect on percent glucose yield in hydrolyzed solution. Response surface plot indicated the optimized condition at 54 (FPU/g substrate) of enzyme loading and 50 h of reaction time that gave 91.36% of percent glucose yield in hydrolyzed solution obtained after NaOH delignification. Figure 6 (a) showed that increasing time and pulp concentration could increased percent glucose yield in hydrolyzed solution. The reaction time longer than 75 h had no significant effect on percent glucose yield in hydrolyzed solution. Response sur‐ face plot indicated the optimized condition at 3% (w/v) of pulp concentration and 65 h of reaction time that gave 92.08% of percent glucose yield in hydrolyzed solution obtained af‐ ter KOH delignification. Figure 6 (b) showed that increasing time and temperature could in‐ creased percent glucose yield in hydrolyzed solution. The temperature higher than 50ºC had no significant effect on percent glucose in hydrolyzed in solution. Response surface plot in‐ dicated the optimized condition at 50ºC of temperature and 65 h of reaction time that gave 88.91% (w/v) of percent glucose yield in hydrolyzed solution obtained after KOH delignifi‐ cation. Figure 6 (c) showed that increasing time and enzyme loading could increased per‐ cent glucose yield in hydrolyzed solution. The enzyme loading higher than 85 (FPU/g substrate) had no significant effect on percent glucose yield in hydrolyzed solution. Re‐ sponse surface plot indicated the optimized condition at 54.5 (FPU/g substrate) of enzyme loading and 66 h of reaction time that gave 88.44% of percent glucose yield in hydrolyzed in

for the two obtained equations were found to be 0.867 and 0.935 in enzyme

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171

of 0.867 and 0.935 were considered as the good fit of the models.

coefficient R2

. The R2

solution obtained after KOH delignification.

variables studied. The R2

**Figure 4.** Response surface plots of glucose as a function of concentration of pulp and concentration of KOH (a), con‐ centration of pulp and temperature (b), time and concentration of pulp (c), other fixed variables

#### **3.3. Model fitting for optimization of enzyme hydrolysis**

The complete design matrix together with values of both experimental and predicted respons‐ es is given in Table 6. Central composite design was used to develop correclation between enzyme hydrolysis of both pulps obtained after NaOH or KOH delignification to percentage of glucose yield were found to range between 6.30-80.10% for pulp obtained after KOH delignification and 10.30-89.80% for pulp obtained after NaOH delignification. Runs 17-23 at the center point were used to determine the experiment error. For both responses of enzyme hydrolysis, the quadratic model was selected, as suggested by used software. The final empirical models in terms of coded factors are given by Equation (3) and Equation (4) in Table 7. Where X1, X2, X2 and X4 were the coded values of test variables reaction time, temperature, enzyme loading and pulp concentration, respectively. The variables X1X2, X1X3, X1X4, X2X3, X2X4 and X3X4 represented the interaction effects of reaction time and temperature, reaction time and enzyme loading, reaction time and pulp concentration, temperature and enzyme loading, temperature and pulp concentration and enzyme loading and pulp concentration, respectively. The quality of the model developed was evaluated based on the correlation coefficient R2 . The R2 for the two obtained equations were found to be 0.867 and 0.935 in enzyme hydrolysis of pulp obtained after NaOH and KOH delignification. This indicated that 86.70% and 93.50% of total variation in both enzyme hydrolysis were attributed to the experimental variables studied. The R2 of 0.867 and 0.935 were considered as the good fit of the models.

The adequacy of the two models was further justified through analysis of variance as the same as delignification reaction in previous studied. The statistical result showed that the two models had p-values less than 0.01 that indicated these two models were adequate to predict the percentage of glucose yield in enzyme hydrolysis within the range of the studied variables.

(a) (b)

(c)

**Figure 4.** Response surface plots of glucose as a function of concentration of pulp and concentration of KOH (a), con‐

The complete design matrix together with values of both experimental and predicted respons‐ es is given in Table 6. Central composite design was used to develop correclation between enzyme hydrolysis of both pulps obtained after NaOH or KOH delignification to percentage of glucose yield were found to range between 6.30-80.10% for pulp obtained after KOH delignification and 10.30-89.80% for pulp obtained after NaOH delignification. Runs 17-23 at the center point were used to determine the experiment error. For both responses of enzyme hydrolysis, the quadratic model was selected, as suggested by used software. The final empirical models in terms of coded factors are given by Equation (3) and Equation (4) in Table 7. Where X1, X2, X2 and X4 were the coded values of test variables reaction time, temperature, enzyme loading and pulp concentration, respectively. The variables X1X2, X1X3, X1X4, X2X3, X2X4 and X3X4 represented the interaction effects of reaction time and temperature, reaction time and enzyme loading, reaction time and pulp concentration, temperature and enzyme

centration of pulp and temperature (b), time and concentration of pulp (c), other fixed variables

170 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

**3.3. Model fitting for optimization of enzyme hydrolysis**

)

In addition, response surface contour plots of the RSM on the effects of reaction time and concentration of pulp on the percentage of glucose in enzyme hydrolysis are shown in Fig‐ ure 5 (a). Figure 5 (a) showed that increasing time and pulp concentration could increased percent glucose yield in hydrolyzed solution. The pulp concentration higher than 4.5% (w/v) had no significant effect on percent glucose yield in hydrolyzed in solution. Response sur‐ face plot indicated the optimized condition at 3% (w/v) of pulp concentration and 65 h of reaction time that gave 81.01% of percent glucose yield in hydrolyzed solution obtained af‐ ter NaOH delignification. Figure 5 (b) showed that increasing time and temperature could increased percent glucose yield in hydrolyzed solution. The temperature higher than 50ºC had no significant effect on percent glucose yield in hydrolyzed solution. Response surface plot indicated the optimized condition at 44ºC of temperature and 65 h of reaction time that gave 81.33% of percent glucose yield hydrolyzed solution obtained after NaOH delignifica‐ tion. Figure 5 (c) showed that increasing time and enzyme loading could increased percent glucose yield in hydrolyzed solution. The enzyme loading higher than 65 (FPU/g substrate) had no significant effect on percent glucose yield in hydrolyzed solution. Response surface plot indicated the optimized condition at 54 (FPU/g substrate) of enzyme loading and 50 h of reaction time that gave 91.36% of percent glucose yield in hydrolyzed solution obtained after NaOH delignification. Figure 6 (a) showed that increasing time and pulp concentration could increased percent glucose yield in hydrolyzed solution. The reaction time longer than 75 h had no significant effect on percent glucose yield in hydrolyzed solution. Response sur‐ face plot indicated the optimized condition at 3% (w/v) of pulp concentration and 65 h of reaction time that gave 92.08% of percent glucose yield in hydrolyzed solution obtained af‐ ter KOH delignification. Figure 6 (b) showed that increasing time and temperature could in‐ creased percent glucose yield in hydrolyzed solution. The temperature higher than 50ºC had no significant effect on percent glucose in hydrolyzed in solution. Response surface plot in‐ dicated the optimized condition at 50ºC of temperature and 65 h of reaction time that gave 88.91% (w/v) of percent glucose yield in hydrolyzed solution obtained after KOH delignifi‐ cation. Figure 6 (c) showed that increasing time and enzyme loading could increased per‐ cent glucose yield in hydrolyzed solution. The enzyme loading higher than 85 (FPU/g substrate) had no significant effect on percent glucose yield in hydrolyzed solution. Re‐ sponse surface plot indicated the optimized condition at 54.5 (FPU/g substrate) of enzyme loading and 66 h of reaction time that gave 88.44% of percent glucose yield in hydrolyzed in solution obtained after KOH delignification.

**Dependent variable Predictive R2**

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ...

(a) (b)

(c)

**Figure 5.** Response surface plots of glucose yield as a function concentration of pulp and time (a), temperature and

time (b), enzymatic loading and time (c), other fixed variables

)

**Table 7.** The linear regression of dependent for enzyme hydrolysis of glucose yield


> -51.956 + 2.654X1 + 20.332X2 + 1.410X3 - 6.477X4 - 0.021X12 - 0.224X22 - 0.012X32 - 1.835X42 - 0.004X1X2 + 0.001X1X3 + 0.003X1X4 + 0.002X2X 3 + 0.225X2X4 + 0.003X3X4

0.867

173

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0.935

Glucose yield (%) NaOH (Eqs.3)

Glucose yield (%) KOH (Eqs.4)


**Table 6.** Design and response of the central composite design for glucose (%) obtained from enzymatic hydrolysis

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ... http://dx.doi.org/10.5772/54691 173


**Run X1 X2 X3 X4**

**Glucose (%), Enzyme hydrolysis Experimental Predicted KOH NaOH KOH NaOH**

 1 1 -1 -1 80.1 86.2 79.8 86.0 1 1 -1 1 69.5 75.4 69.0 75.3 1 1 1 -1 74.2 80.0 74.5 80.0 1 1 1 1 54.4 63.9 55.4 64.2 1 -1 -1 -1 37.6 46.7 37.4 46.2 1 -1 -1 1 16.8 23.0 16.1 23.2 1 -1 1 -1 30.3 35.4 30.9 35.1 1 -1 1 1 14.5 20.6 14.3 20.0 -1 1 -1 -1 74.4 80.3 74.4 80.8 -1 1 -1 1 63.8 71.6 63.3 70.2 -1 1 1 -1 70.1 76.4 70.4 76.3 -1 1 1 1 53.8 58.6 53.6 58.2 -1 -1 -1 -1 34.9 41.4 34.7 41.3 -1 -1 -1 1 12.2 18.8 12.5 18.8 -1 -1 1 -1 20.1 27.3 20.1 27.3 -1 -1 1 1 4.4 10.5 4.5 10.9 0 0 0 0 74.2 89.8 74.2 89.2 0 0 0 0 73.3 79.3 73.3 79.2 0 0 0 0 74.8 80.7 74.8 80.7 0 0 0 0 75.2 80.4 75.2 80.1 0 0 0 0 75.3 81.3 75.3 81.6 0 0 0 0 75.9 81.6 75.4 81.9 0 0 0 0 75.3 81.4 75.2 81.8 α 0 0 0 77.0 83.7 77.3 83.4 -α 0 0 0 6.3 10.3 6.3 10.0 0 α 0 0 11.7 15.4 11.7 15.4 0 -α 0 0 45.1 50.5 45.1 50.5 0 0 α 0 73.0 77.6 73.0 77.6 0 0 -α 0 15.2 21.3 15.3 21.3 0 0 0 α 74.4 80.2 74.8 81.4 0 0 0 -α 62.2 79.8 62.4 79.7

172 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

**Table 6.** Design and response of the central composite design for glucose (%) obtained from enzymatic hydrolysis

**Figure 5.** Response surface plots of glucose yield as a function concentration of pulp and time (a), temperature and time (b), enzymatic loading and time (c), other fixed variables

**3.4. Ethanol fermentation**

**Time (h)**

*3.4.1. Production of ethanol from pure glucose*

production was shown in Table 7.

Ethanol yield= ethanol from experiment

*nondelignified pulp*

shown in Table 9.

**Residual glucose (g/l)**

S. Cerevisiae TISTR 5339 was grown in YMB containing 50 g/l glucose, 20 g/l peptone and 10 g/l yeast extract. The experiment was performed at room temperature. The result of ethanol

Optimization of Delignification and Enzyme Hydrolysis of Steam Exploded Oil Palm Trunk for Ethanol Production ...

**Ethanol (g/l)**

**Ethanol yield (%)**

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175

**Consumed glucose (g/g)**

**Table 8.** Production of ethanol from 50 g/l of pure glucose by S. Cerevisiae TISTR 5339

Theoretical ethanol ×100

 50.00 0.00 0.00 0.00 40.52 9.48 4.62 18.12 33.21 16.79 8.05 31.57 25.16 24.84 12.15 47.65 17.38 32.65 16.08 63.06 10.30 39.70 19.46 76.31 3.21 46.79 21.92 85.96 0.00 50.00 21.81 85.53 0.00 50.00 21.75 85.29 0.00 50.00 21.04 82.51

Table 8 showed the production of ethanol from 50 g/l of pure glucose by S. Cerevisiae TISTR 5339. The result showed that within 36 h of fermentation, the highest ethanol concentration was obtained at 21.92 g/l. The ethanol yield from calculation was 85.96%. This result indicated that S. Cerevisiae TISTR 5339 has 85.96% in capability to change pure glucose to ethanol.

The hydrolyzed solutions from three samples of pulp were concentrated to 50 g/l of glucose concentration. S. Cerevisiae TISTR 5339 was applied in the same amount as production of ethanol from pure glucose but pure glucose was replaced with the three hydrolyzed solutions. The fermentation solutions were analyzed by GC for ethanol concentration. The results were

*3.4.2. Production of ethanol from hydrolyzed solution of NaOH and KOH delignified pulp and*

**Figure 6.** Response surface plots of glucose yield as a function concentration of pulp and time (a), temperature and time (b), enzymatic loading and time (c), other fixed variables

The summary result from combination of each response surface plots showed that the optimum condition of enzyme hydrolysis for NaOH and KOH delignification were 54 FPU/g substrate, 65 FPU/g substrate enzyme concentation, 50 h, 60 h reaction time, 50ºC reaction temperature and 2.5% pulp concentration, respectively. The maximum glucose contents were 47.50% and 48.00% from NaOH and KOH delignification, respectively. The maximum glucose yield obtained were 85%, 81% and 75% from NaOH and KOH delignification and without delignification respectively. Both optimization conditions in enzyme hydrolysis of delignifi‐ cation pulp by NaOH and KOH used to hydrolyze undelignification pulp, the result showed that percent glucose yield was lower than those from delignified pulp about 8-10%.
