**2.1. Raw material and microorganism**

The steam-exploded pulp obtained from oil palm trunk was prepared by steam explosion treatment. An amount of 150 g of dry oil palm trunk chip sample was placed in 2.5 L batch digester (Nitto Koatsu Company, Japan). Heating was accomplished by direct steam injection into the digester and the temperature of steam at 214°C for 2 min. This condition was previous work by Punsuvon *et al.* [9]. It could briefly explained that oil palm trunk chip was steamed at temperatures varying between 214 and 220ºC for 2 and 5 minutes. The optimization of the pretreatment condition was 214ºC and 2 minutes that gave the highest glucose yield after enzyme hydrolysis. In this studied, the explosive discharge of the digester contents into a collecting tank was actuated by rapidly opening a value. The combined pulp slurry was collected and washed with hot water (80°C) at total volume of 2 L for 30 min. The pulp was filtered and dried at room temperature for using as raw material in alcohol production study.

Saccharomyces cervisiae TIRS 5339 obtained from TISTR, Thailand was used in this study. It was maintained on a medium containing 20.0 g/l glucose, 20.0 g/l peptone and 10.0 g/l yeast extract at 4ºC and subcultured every month at 30ºC. The growth medium of the yeast consisted of 10.0 g/l yeast extract, 6.4 g/l urea, 2.0 g/l KH2PO4, 1.0 g/l MgSO4-7H2O and 2.0 g/l glucose at pH 5.5 [18].

#### **2.2. Alkaline delignification of steam-exploded pulp**

The water-insoluble cellulose pulp obtained from steam explosion was delignified with potassium hydroxide. The reactions were carried out in a beaker under various maintained temperature. Before RSM was applied on alkaline delignification, approximate conditions for glucose content in pulps, namely concentration of pulp, concentration of alkaline solution, reaction time and temperature were determined by varying one factor at time while keeping the other constant. The initial step of the preliminary experiment was to select an appropriate amount of concentration of pulp. Five different concentrations of pulp (3, 6, 9, 12, 15 %w/v) were examined. The other three factors, concentration of alkaline solution, reaction time and temperature, were kept constant at 20%w/w, 60 min and 80ºC, respectively. Based on the glucose content in pulp after delignification, the optimum concentration of pulp was chosen. The second step of the preliminary experiment was to determine the concentration of alkaline solution. The glucose content in pulp was analyzed using the optimum condition of pulp chosen in the previous step. The alkaline solution concentration varied from 2 to 30% w/w while holding reaction time and temperature at 60 min and 80ºC. The third step of the preliminary experiment was to determine the reaction time. Using the concentration of pulp, concentration of alkaline solution, reaction time from the previous steps, alkaline delignifica‐ tion was studied under various reaction times from 15 to 90 min. The final step was to select an appropriate temperature by using the concentration of pulp, concentration of alkaline solution, reaction time from the previous step. The temperature varied from 30 to 100ºC. Based on these results the five level of each process variable were determined for RSM. The inde‐ pendent variables of RSM experiments were shown in Table1. Delignified pulp was recovered by filtration, washed several times with distilled water, died and then analyzed for glucose content. These pulps were ready to be used as the substrate for enzymatic hydrolysis.

**Independent variable Symbol**

were performed to allow the estimation of the pure error.

Temperature, º

delignified pulp

**2.5. Experimental design**

**2.6. Statistical analysis**

Y=bo + ∑ i=1 4 bi xi + ∑ i=1 4 bii xi <sup>2</sup> + ∑ i< *j*=1 3 ∑ 4 bij xij

second-order polynomial equation:

**2.7. Glucose determination**

Where Y is the response (percent glucose, %), xi

the stationary point and changing the other two variables.

Reaction time, h X1 10 30 50 70 90

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

Enzyme loading, Filter Paper Unit (FPU)/g substrate X3 5 30 55 80 105 Concentration of pulp, %w/v X4 1 2 3 4 5

**Table 2.** Independent variables and their levels for central composite design in optimization of enzyme hydrolysis of

A central composite design was employed the response, namely percentage of glucose for alkaline delignification, and percentage of glucose yield for enzyme hydrolysis. The inde‐ pendent variables of alkaline delignification were X1, X2, X3 and X4 representing concentration of pulp, %w/v, and concentration of alkaline solution, % w/w, reaction time, min and temper‐ ature, °C, respectively. The independent variables of enzyme hydrolysis were X1, X2, X3 and X4 representing reaction time, h, temperature, °C, enzyme loading, FPU/g substrate and concentration of pulp, %w/v, respectively. Each variable to be optimized was coded at five levels: - α, -1, 0, +1 and + α. This gives a range of these variables of alkaline delignification (Table 1) and enzyme hydrolysis (Table2). Six replication runs at the centre (0, 0, 0) of the design

The data obtained by carrying out the experiment according to central composite design were analyzed by SPSS package (version 12.0). The response surface was expressed at the following

bo is constant, bi is linear term coefficients, bii is quadratic term coefficients. bij is cross-product term coefficients. SPSS package was used for regression analysis of variance (ANOVA) and response surface methodology was performed using STATISTICA Software. Response surface plots were developed using the fitted second order polynomial equation obtain from regres‐ sion analysis holding one of the independent variables at a constant value corresponding to

The glucose content of hydrolysis liquid was analyzed by High Performance Liquid Chroma‐ tography (HPLC, Shimadzu, Kyoto, Japan) with refractive index detector. An AMINEX HPX-87C carbohydrate analysis (Bio-Rad, Hercules, USA) was used as column. The mobile phase

C X2 28 35 42.5 50 57.5

**Coded variable levels α -1 0 1 α**

http://dx.doi.org/10.5772/54691

163

and xij are uncoded independent variables,

#### **2.3. Enzyme hydrolysis**

Delignified pulps were hydrolyzed by cellulase (Celluclast 1.2L, Novozymes A/S Denmark) in flasks. The hydrolysis was performed in 0.05M sodium citrate buffer (pH 4.8) at 150 rpm of shaking. The dependent variables of experiments were shown in Table 2. All enzymatic hydrolysis liquor was analyzed for glucose content by High Performance Liquid Chromatog‐ raphy (HPLC).

#### **2.4. Inoculums and ethanol fermentation**

S. cerevisaie was initiated in the maintenance medium at 30ºC. The yeast was grown for 48 h at 170 rpm on a rotary shaker at 30° C. A 2.5 % w/v inoculums was used for subsequent subcultures. Ethanol fermentation was evaluated at 30° C in 150–ml Erlenmeyer flasks con‐ taining 100 ml fermentation media. The yeast fermentation medium consisted of the hydrolysis liquor containing 50g/l glucose, 2.0 g/l KHPO4, 1.0 g/l MgSO4.7H2O, 10.0 g/l yeast extracts and 6.4 g/l urea at pH 5.5. The flasks were sealed with a one-hole rubber stopper, which a glass tube was connected to an air lock filled with 40% sulfuric acid solution. Ethanol content from fermentation was analyzed by Gas Chromatography (GC).


**Table 1.** Independent variables and their levels for central composite design in optimization of alkaline delignification of steam-exploded pulp


**Table 2.** Independent variables and their levels for central composite design in optimization of enzyme hydrolysis of delignified pulp

#### **2.5. Experimental design**

temperature, were kept constant at 20%w/w, 60 min and 80ºC, respectively. Based on the glucose content in pulp after delignification, the optimum concentration of pulp was chosen. The second step of the preliminary experiment was to determine the concentration of alkaline solution. The glucose content in pulp was analyzed using the optimum condition of pulp chosen in the previous step. The alkaline solution concentration varied from 2 to 30% w/w while holding reaction time and temperature at 60 min and 80ºC. The third step of the preliminary experiment was to determine the reaction time. Using the concentration of pulp, concentration of alkaline solution, reaction time from the previous steps, alkaline delignifica‐ tion was studied under various reaction times from 15 to 90 min. The final step was to select an appropriate temperature by using the concentration of pulp, concentration of alkaline solution, reaction time from the previous step. The temperature varied from 30 to 100ºC. Based on these results the five level of each process variable were determined for RSM. The inde‐ pendent variables of RSM experiments were shown in Table1. Delignified pulp was recovered by filtration, washed several times with distilled water, died and then analyzed for glucose

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

content. These pulps were ready to be used as the substrate for enzymatic hydrolysis.

Delignified pulps were hydrolyzed by cellulase (Celluclast 1.2L, Novozymes A/S Denmark) in flasks. The hydrolysis was performed in 0.05M sodium citrate buffer (pH 4.8) at 150 rpm of shaking. The dependent variables of experiments were shown in Table 2. All enzymatic hydrolysis liquor was analyzed for glucose content by High Performance Liquid Chromatog‐

S. cerevisaie was initiated in the maintenance medium at 30ºC. The yeast was grown for 48 h

taining 100 ml fermentation media. The yeast fermentation medium consisted of the hydrolysis liquor containing 50g/l glucose, 2.0 g/l KHPO4, 1.0 g/l MgSO4.7H2O, 10.0 g/l yeast extracts and 6.4 g/l urea at pH 5.5. The flasks were sealed with a one-hole rubber stopper, which a glass tube was connected to an air lock filled with 40% sulfuric acid solution. Ethanol content from

Concentration of pulp, %w/v X1 3 6 8 12 15 Concentration of alkaline solution, %w/w X2 2 8 14 20 26 Reaction time, min X3 15 30 45 60 75 Temperature, ºC X4 35 50 65 80 95

**Table 1.** Independent variables and their levels for central composite design in optimization of alkaline delignification

C. A 2.5 % w/v inoculums was used for subsequent

C in 150–ml Erlenmeyer flasks con‐

**Coded variable levels α -1 0 1 α**

**2.3. Enzyme hydrolysis**

**2.4. Inoculums and ethanol fermentation**

subcultures. Ethanol fermentation was evaluated at 30°

fermentation was analyzed by Gas Chromatography (GC).

**Independent variable Symbol**

at 170 rpm on a rotary shaker at 30°

raphy (HPLC).

of steam-exploded pulp

A central composite design was employed the response, namely percentage of glucose for alkaline delignification, and percentage of glucose yield for enzyme hydrolysis. The inde‐ pendent variables of alkaline delignification were X1, X2, X3 and X4 representing concentration of pulp, %w/v, and concentration of alkaline solution, % w/w, reaction time, min and temper‐ ature, °C, respectively. The independent variables of enzyme hydrolysis were X1, X2, X3 and X4 representing reaction time, h, temperature, °C, enzyme loading, FPU/g substrate and concentration of pulp, %w/v, respectively. Each variable to be optimized was coded at five levels: - α, -1, 0, +1 and + α. This gives a range of these variables of alkaline delignification (Table 1) and enzyme hydrolysis (Table2). Six replication runs at the centre (0, 0, 0) of the design were performed to allow the estimation of the pure error.

#### **2.6. Statistical analysis**

The data obtained by carrying out the experiment according to central composite design were analyzed by SPSS package (version 12.0). The response surface was expressed at the following second-order polynomial equation:

$$\mathbf{Y} = \mathbf{b}\_{\mathbf{o}} + \sum\_{i=1}^{4} \mathbf{b}\_{i} \mathbf{x}\_{i} + \sum\_{i=1}^{4} \mathbf{b}\_{\mathrm{ii}} \mathbf{x}\_{i}^{2} + \sum\_{i$$

Where Y is the response (percent glucose, %), xi and xij are uncoded independent variables, bo is constant, bi is linear term coefficients, bii is quadratic term coefficients. bij is cross-product term coefficients. SPSS package was used for regression analysis of variance (ANOVA) and response surface methodology was performed using STATISTICA Software. Response surface plots were developed using the fitted second order polynomial equation obtain from regres‐ sion analysis holding one of the independent variables at a constant value corresponding to the stationary point and changing the other two variables.

#### **2.7. Glucose determination**

The glucose content of hydrolysis liquid was analyzed by High Performance Liquid Chroma‐ tography (HPLC, Shimadzu, Kyoto, Japan) with refractive index detector. An AMINEX HPX-87C carbohydrate analysis (Bio-Rad, Hercules, USA) was used as column. The mobile phase was deionied water with flow rate 0.6 ml/min. The injection volume was 20 µl and the column temperature was maintained at 80° C. The glucose content of the solid residue was determined 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 autoclaved at 121° C for 1 h. This hydrolysis liquid was then analyzed for glucose content as described above. All analytical determinations were performed in duplication.

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

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

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

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

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

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‐

. The R2 for the two obtained equations

http://dx.doi.org/10.5772/54691

165

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

evaluated based on the correlation coefficient R2

good fit of the models.

the range of studied variables.

production.
