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Research Article

Received: 27 January 2011 Revised: 21 March 2011 Accepted: 2 May 2011 Published online in Wiley Online Library: 14 June 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2668

High efciency bioethanol production from OPEFB using pilot pretreatment reactor
Minhee Han,a Yule Kim,a Seung Wook Kimb and Gi-Wook Choia
Abstract
BACKGROUND: Current ethanol production processes using crops such as corn and sugar cane are well established. However, the utilization of cheaper biomasses such as lignocellulose could make bioethanol more competitive with fossil fuels while avoiding the ethical concerns associated with using potential food resources. RESULTS: Oil palm empty fruit bunches (OPEFB), a lignocellulosic biomass, was pretreated using NaOH to produce bioethanol. The pretreatment and enzymatic hydrolysis conditions were evaluated by response surface methodology (RSM). The optimal conditions were found to be 127.64 C, 22.08 min, and 2.89 mol L1 for temperature, reaction time, and NaOH concentration, respectively. Regarding enzymatic digestibility, 50 FPU g1 cellulose of cellulase was selected as the test concentration, resulting in a total glucose conversion rate (TGCR) of 86.37% using the Changhae Ethanol Multi Explosion (CHEMEX) facility. Fermentation of pretreated OPEFB using Saccharomyces cerevisiae resulted in an ethanol concentration of 48.54 g L1 at 20% (w/v) pretreated biomass loading, along with simultaneous saccharication and fermentation (SSF) processes. Overall, 410.48 g of ethanol were produced from 3 kg of raw OPEFB in a single run, using the CHEMEX 50 L reactor. CONCLUSION: The results presented here constitute a signicant contribution to the production of bioethanol from OPEFB. c 2011 Society of Chemical Industry Keywords: oil palm empty fruit bunches (OPEFB); response surface methodology (RSM); Changhae Ethanol Multi Explosion (CHEMEX); enzymatic hydrolysis; fermentation

INTRODUCTION
Malaysias oil palm industry has grown rapidly over the last four decades and has become a very important agriculture-based industry, so that the country has maintained its position as the world leading palm oil producing country. Malaysia is blessed with favorable weather conditions throughout the year and are advantageous for oil palm plantations. Nevertheless, the industry has also generated vast quantities of palm biomass, mainly from milling and crushing of palm kernels. The main by-product and waste are empty fruit bunches (EFB), palm oil mill efuent (POME), palm ber, and palm kernel shells. Oil palm empty fruit bunches (OPEFB) is a lignocellulosic residue that has potential as a cheap, renewable feedstock for large scale production of bioproduct. Each year, more than 15 million tons of OPEFB are generated by the palm oil industry in Malaysia.1 Bioconversion of lignocellulose to bioethanol employs three major steps, which include pretreatment of OPEFB for breakdown of lignin and to open up the crystalline structure of cellulosic materials; cellulosic hydrolysis using a combination of enzymes for fermentable sugar (polyose) production; and bioconversion of the fermentable sugars (polyoses) produced to bioethanol. Cellulose degradation by enzymatic hydrolysis is a reaction carried out by cellulase enzymes, which are very specic for their substrates. Three major groups of cellulases are involved in the cellulose hydrolysis process:2 -1-4-endoglucanase, which attacks the low crystalline region in the cellulose ber, creating free chain ends;

-1-4-exoglucanase or cellobiohydrolase, which further degrades the molecule by removing cellobiose units from the free chain ends; -glucosidase or cellobiase, which hydrolyzes cellobiose to produce glucose. A variety of physical (comminution, hydrothermolysis), chemical (acid, alkali, solvents, ozone), physico-chemical (steam explosion, ammonia ber explosion), and biological pretreatment techniques have been developed to improve the accessibility of enzymes to cellulosic bers.3 Acid pretreatment includes the use of sulfuric, nitric, or hydrochloric acids to remove hemicellulosic components and expose cellulose to enzymatic digestion.4 Agricultural residues such as corncobs and stovers are particularly well suited to dilute acid pretreatment.5 Alkali pretreatment refers to the application of alkaline solutions for the removal of lignin and various uronic acid substitutions present in hemicellulose that lower enzyme accessibility.6 Generally, alkaline pretreatment is more effective for agricultural residues and herbaceous crops than

Correspondence to: Gi-Wook Choi, Changhae Institute of Cassava and Ethanol Research, Changhae Ethanol Co., Ltd., Palbok-Dong 829, Dukjin-Gu, Jeonju 561-203, Korea. E-mail: changrd@chethanol.com

a Changhae Institute of Cassava and Ethanol Research, Changhae Ethanol Co., Ltd, Jeonju, 561-203, Korea

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b Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Korea

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c 2011 Society of Chemical Industry

www.soci.org for wood materials.7 Peroxide pretreatment enhances enzymatic conversion through oxidative delignication and reduction of cellulose crystallinity.8 Increased lignin solubilization and cellulose availability have been observed during the peroxide pretreatment of wheat straw,9 Douglas r,10 and oak.11 Ozonation is another attractive pretreatment method that does not leave strong acidic, basic, or toxic residues in treated materials.12 The effect of ozone pretreatment is essentially limited to lignin degradation. Specically, hemicellulose is attacked while cellulose is barely affected.13 Further, ozonation has been widely used to reduce the lignin content of both agricultural and forestry wastes.12 To fully utilize OPEFB as a feedstock for ethanol production, pretreatment is required to render the cellulose bers more amenable to the action of hydrolytic enzymes. In this study, response surface methodology (RSM) was used to determine the optimal conditions that produce high concentrations of bioethanol. RSM is a statistical technique used to model and optimize multiple variables, and can be used to determine the optimal conditions by combining experimental design with interpolation of rst- or second-order polynomial equations in a sequential testing procedure. This methodology has been successfully applied to optimize the enzymatic hydrolysis of several substrates, including cellulose.14 18 The optimal conditions for pretreatment and enzymatic hydrolysis were investigated using a lab-scale reactor. Based on these results, the selected conditions were applied using a pilot-scale pretreatment reactor (Changhae Ethanol Multi Explosion; CHEMEX 50 L). In addition, the optimal levels of the enzymes and pretreated biomass dosage for enzymatic hydrolysis and fermentation were determined. Ethanol production was then evaluated in accordance with the selected enzymatic hydrolysis time to reduce process time and cost.

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Table 1. Coded and decoded values for each variable of the central composite rotatable design Coded levels of experimental factors 1.68 1 0 1 1.68 X1 : Temperature ( C) 72.33 100.00 140.00 180.00 207.27 X2 : Time (min) 4.77 15.00 30.00 45.00 55.23 X3 : NaOH concentration (mol L1 ) 0.32 1.00 2.00 3.00 3.68

To investigate the effects of pretreatment on enzymatic digestibility, 1 g of dried biomass was hydrolyzed with an excess of cellulase complex and -glucosidase in 0.05 mol L1 sodium citrate buffer (pH 4.8) in a total liquid volume of 100 mL in an Erlenmeyer ask. Hydrolysis was conducted at 50 C and 150 rpm for 72 h. Experimental design Response surface methodology (RSM) is a collection of mathematical and statistical techniques used to model and analyze problems in which the response of interest is inuenced by several variables, and the objective is to optimize this response.19 In this study, many variables could potentially affect the efciency of the pretreatment process. Central composite rotatable design (CCRD) was employed to determine the effects of independent variables on the response and factor interactions using different combinations of variables. Three independent variables including temperature (X1 ), reaction time (X2 ), and NaOH solution concentration (X3 ), were studied at three levels with three repetitions at the central point, and three replicates at the axial and factorial points (Table 1). For each of the ve variables studied, high (coded +1.68) and low (coded 1.68) set points were selected according to the results obtained in the preliminary tests. The results of each CCRD were analyzed using Design Expert software version 7.1.3 from StatEase, Inc., Minneapolis, MN, USA. The quadratic effects of the ve variables were calculated, as well as their possible interactions with the conversion rate of the biomass to glucose. The signicance of these variables was evaluated using variance analysis (ANOVA). Three-dimensional surface plots were drawn to illustrate the effects of the independent variables on the dependent variables, as described by a modied equation tted to the experimental data. The t of the models was evaluated by determining the R-squared coefcient and the adjusted R-squared coefcient. To verify the models, optimum values for the selected variables were obtained by solving the regression equation using Design Expert software version 7.1.3. Pilot-scale pretreatment The 50 L pilot-scale reactor was manufactured by Changhae Ethanol Co., Ltd, Jeonju, Korea, and designated as Changhae Ethanol Multi Explosion (CHEMEX). The CHEMEX was designed for operation at a maximum 30 L volume, 250 C temperature, and 30 kg cm2 of pressure. The reactor heat-up process utilizes indirect heating by oil. A diagram (a) and photograph (b) of the CHEMEX facility are provided in Fig. 1. The method of operation can be described as follows: the oil heater temperature is set at >1.2 times the pretreatment temperature. At this time, the heated oil is

MATERIALS AND METHODS


Raw materials The biomass used in this study was shredded OPEFB, obtained from Global Bio-Diesel Sdn. Bhd. at Jalan kastan Baru in Datu, Sabah, Malaysia. The raw substrate was kept in a cold room at 4 C to avoid contamination. The OPEFB was soaked in detergent overnight to remove any residual oil, and then sun-dried. The OPEFB was chopped and hammer-milled to a particle size of 13 mm, then stored in sealed plastic bags at room temperature until use. Enzymes were provided by Novozymes, Korea. A cellulase complex (NS50013) and -glucosidase (NS50010) were used to investigate enzymatic digestibility. The cellulase complex had an activity of 70 lter paper units (FPU) g1 cellulose. The glucosidase had an activity of 250 cellobiase units (CbU) g1 . All reagents used in this study were of analytical grade. Lab-scale NaOH pretreatment and enzymatic hydrolysis for optimal conditions Different concentrations of NaOH solutions (30 mL volume) were used to pretreat 5 g of ground EFB samples to determine the optimum pretreatment and enzymatic hydrolysis conditions. The treatments were performed at various temperatures and for different lengths of time in an oil bath. The reaction times were estimated after approaching a pre-set temperature. After cooling, the treated biomasses were washed several times with deionized water, and dried at 45 C to x the moisture for enzymatic hydrolysis.

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dosages, a cellulase activity of 570 FPU g1 cellulose and glucosidase (30% weight of cellulase dosage) were added to 1% (w/v) biomass in buffer (pH 4.8) solution in a working volume of 200 mL. After determination of the optimal enzyme dosage to produce ethanol efciently, a concentration of 130% biomass (w/v) was then added to suitable enzymes for enzymatic hydrolysis and fermentation in a working volume of 300 mL. Hydrolysis was conducted under the same conditions. To estimate the ethanol production at different enzymatic hydrolysis times, S. cerevisiae CHY 1011 was inoculated, and solid caps were replaced with silistoppers to exhaust the CO2 released during fermentation. The bottles were then placed back on the shaker/incubator, and the temperature was reset to 32 C. These bottles were periodically sampled for the next 72 h, after which the nal ethanol concentration was estimated. Analytical methods The total solid, acid soluble lignin and acid insoluble lignin contents of OPEFB were determined by the National Renewable Energy Laboratory (NREL) using Standard Biomass Analytical Procedures.20 The carbohydrate content of OPEFB was estimated by measuring the hemicellulose (xylan, galactan, and arabinan) and cellulose (glucan) derived sugars. The composition of the hydrolysate produced by enzymatic hydrolysis was determined by measuring glucose, xylose, and ethanol concentrations using high performance liquid chromatography (HPLC). The HPLC (Waters, USA) system was equipped with a Bio-Rad Aminex HPX-87P column, a guard column, an automated sampler, a gradient pump, and a refractive index detector. The mobile phase was deionized water at a ow rate of 0.6 mL min1 at 85 C. Prior to HPLC injection, all samples (derived from solids and hydrolysate) were neutralized with calcium carbonate, centrifuged at 5000 g for 10 min, and ltered through 0.2 m syringe lters.
Figure 1. Diagram (a) and photograph (b) of the CHEMEX facility: 1 pretreatment reactor; 2 cyclone; 3 oil heater.

RESULTS AND DISCUSSION


circulated with the inlet line of the pretreatment reactor blocked. During the heat-up time, biomass and pretreatment solution are added to the reactor with stirring, and nitrogen gas is introduced into the reactor to prevent boiling. Upon approaching the preset temperature of the oil heater, the valve of the pretreatment reactor jacket is opened; the oil heat up time is a matter of minutes. The reaction times were measured after reaching a pre-set temperature in the pretreatment reactor. The reactor temperature was controlled by adjusting the oil heater temperature and oil inlet valve of the reactor. Based on the RSM results, pretreatment for enzymatic hydrolysis and fermentation experiments was carried out using 3 kg of ground OPEFB biomass and 15 L of NaOH solution in the 50 L pilot scale reactor. After pretreatment, pretreated biomass was collected in cyclones. The cyclones were replaced with fresh tanks to reduce the space and increase recovery efciency. The collected biomass was washed with deionized water, and dewatered using an oil press and dried at 45 C to x the moisture for enzymatic hydrolysis and fermentation experiments. Enzymatic hydrolysis and fermentation for high efciency ethanol production Two enzyme solutions, the cellulase complex and -glucosidase, were used to investigate the effects of enzyme and biomass concentration on ethanol production. To determine enzyme Characteristics of OPEFB The chemical composition of OPEFB varies according to its growth location, season, harvesting method, and analysis procedure.20 The composition of OPEFB used in this study is listed in Table 2. Based on HPLC carbohydrate analysis, the sugar fraction was 54.16%, the lignin fraction was 26.77% and the ash fraction was 3% of the dry biomass. Glucan, which was derived from both the OPEFB ber and plant cell wall, was the major component (32.74%). Xylan, as the major hemicellulose constituent, constituted up to 21.42%. Lignin is a complex biomass-derived chemical compound that protects against enzymatic attack. Arabinan accounted for only a small portion (<1%) of the biomass; galatan and mannan were not detected. Additionally, the other 16.02% may include some organic compounds such as uronic acid and acetyl groups, and other trace components including minerals, waxes, fats, starches, resins, and gums.21 Glucan and xylan can be converted to ethanol using organisms that ferment pentose and hexose. However, digestion of pentose by microorganisms (S. cerevisiae) is difcult, and therefore, cellulose was retained during pretreatment and utilized to ferment hexoses derived from the biomass. Optimization of pretreatment and enzymatic hydrolysis with central composite rotatable design (CCRD) The change in composition, total solid and recovery of glucan efciency after pretreatment during sodium hydroxide pretreatment

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Table 2.

Major components of OPEFB [%] 32.74 21.42 23.55 3.22 3.05 16.02

Component Cellulose Hemicellulose Acid-insoluble lignin Acid-soluble lignin Ash Others

Table 4. Central composite design for the optimization of three variables in determining the total glucose conversion rate (TGCR) after enzymatic hydrolysis Runs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 X1 1.68 1.00 1.00 1.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 1.68 X2 0.00 1.00 1.00 1.00 1.00 1.68 0.00 0.00 0.00 0.00 0.00 1.68 1.00 1.00 1.00 1.00 0.00 X3 0.00 1.00 1.00 1.00 1.00 0.00 1.68 0.00 0.00 0.00 1.68 0.00 1.00 1.00 1.00 1.00 0.00 TGCR [%] 69.49 66.81 81.24 71.94 84.64 79.09 60.43 85.36 84.24 85.23 85.81 85.45 76.73 75.83 75.69 61.95 49.69

of OPEFB are shown in Table 3. As the severity of pretreatment conditions was increased, glucan increased and lignin showed a relative decrease. Although the pretreatment seems effective, glucan ingredient needs an absolute change as the quantity of total solid decreases. The recovery of glucan showed that increased severity of pretreatment conditions was associated with decreased absolute glucan ingredient. However, the rate of enzymatic hydrolysis is also an important factor, because the goal of pretreatment is to facilitate penetration of enzymes for effective enzymatic hydrolysis. In this regard, pretreatment and enzymatic hydrolysis conditions should be considered simultaneously when attempting to optimize ethanol production. Following pretreatment and enzymatic hydrolysis, the total glucose conversion rate (TGCR) was evaluated as a function of temperature, time, and NaOH solution concentration. The TGCR was expressed as the efciency of simultaneous pretreatment and enzymatic hydrolysis, and was determined according to the following equation: TGCR[%] = SP [%] Gg [%] BL [%] Gg [%] (1)

X1 Reaction temperature, X2 Residual time, X3 NaOH solution concentration. Values indicate the mean of triplicate observations.

in which SP is the solid ratio after pretreatment, GE is the glucose concentration after enzymatic hydrolysis, BL is the pretreated

biomass loading at enzymatic hydrolysis, and GR is the raw biomass glucose concentration. The temperature ranged from 100 to 180 C, time ranged from 15 to 40 min, and the pretreatment solution concentration ranged from 1.0 to 3.0 mol L1 in the optimal CCRD test (Table 4). Variance analysis (ANOVA) was performed to evaluate the effects of the variables and their possible interactions. The coefcients of

Table 3.

Composition of NaOH-pretreated OPEFB, recovery of glucan after pretreatment and enzymatic hydrolysis Composition of solid fraction [g per 100 g initial dry biomass] Glucan [%] 48.46 46.31 50.68 47.85 51.74 50.02 43.82 59.12 57.27 61.66 59.90 60.71 56.87 73.29 59.38 78.02 69.89 Xylan [%] 16.89 20.47 15.89 20.54 14.65 15.77 21.91 14.66 14.39 13.92 13.25 13.63 20.05 6.98 19.17 7.17 8.16 Lignin [%] 23.66 20.95 23.15 19.12 22.36 21.51 19.26 15.16 15.91 15.4 15.97 13.91 8.36 6.95 9.25 6.00 9.17 Total solid [g per 100 g initial dry biomass] 66.97 68.97 62.96 65.64 59.22 61.45 70.86 50.74 50.72 50.75 50.04 48.75 45.95 36.34 46.49 27.25 25.09 Recovery of glucan after pretreatment [%] 98.17 96.62 96.51 95.02 92.68 92.98 93.92 90.74 87.85 94.65 90.67 89.52 79.04 80.56 83.50 64.32 53.04 Enzymatic digestibility [%] 70.79 69.15 84.17 75.71 91.32 85.06 64.35 94.08 95.88 90.05 94.64 95.45 97.07 94.13 90.64 96.33 93.69

Pretreatment conditions Temp. [ C] 72.73 100 100 100 100 140 140 140 140 140 140 140 180 180 180 180 207.27 Time [min] 30 15 15 45 45 4.77 30 30 30 30 30 55.23 15 15 45 45 30 NaOH concentration [mol L1 ] 2 1 3 1 3 2 0.32 2 2 2 3.68 2 1 3 1 3 2

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Bioethanol production from palm oil waste using pretreatment reactor the full model were evaluated using regression analysis, and their signicance was tested. Insignicant coefcients were excluded from the model. The variance analysis performed on the reduced models (Table 5) demonstrated that they were statistically valid with P-values <0.0400. NaOH concentration produced the lowest p-values (<0.016) among all factors, which indicates that NaOH concentration was the dominant factor affecting the TGCR. Equation (2) describes the correlation between the signicant variables and glucose release rate for the pretreated biomass in terms of the decoded values. Ya = 84.72 5.89X1 + 1.89X2 + 7.54X3 2.93X1 X2 5.22X1 X3 1.82X2 X3 8.19X 1 2 0.17X 2 2 3.40X 3 2 1.39X1 X2 X3 2.69X 1 2 X2 5.98X 1 2 X3 + 4.08X1 X 2 2 (2)

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Table 5. ANOVA results for the response of the total glucose conversion rate (TGCR) Source Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X12 X22 X32 X1 X2 X3 X 1 2 X2 X 1 2 X3 X1 X 2 2 Residual Lack of t Pure error Total Sum of squares 1764.64 195.96 20.22 322.02 68.71 217.95 26.55 755.33 0.31 130.58 15.42 23.97 118.57 55.24 39.74 38.99 0.76 1804.39 d.f. 13 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 2 16 Mean of square 135.74 195.96 20.22 322.02 68.71 217.95 26.55 755.33 0.31 130.58 15.42 23.97 118.57 55.24 13.25 38.99 0.38 F-value 10.25 14.79 1.53 24.31 5.19 16.45 2.00 57.02 0.02 9.86 1.16 1.81 8.95 4.17 103.25 P-value (Prob > F) 0.0400 0.0310 0.3046 0.0160 0.1072 0.0270 0.2519 0.0048 0.8873 0.0517 0.3597 0.2712 0.0580 0.1338 0.0095

with X1 : temperature, X2 : time, and X3 : NaOH concentration. The relationship between the response and variables was visualized using the response surface or contour plots. The modied equations (Equation (2)) were described using the response surface plots to estimate the TGCR as a function of two factors while maintaining all the other factors at a xed level of zero. The convex response surfaces suggest that there were well-dened optimal variables. Graphic representation of the response surface shown in Fig. 2 helps visualize the effects of various variables. The proportion of the total variation attributed to each t was evaluated using the R-squared value (an R-square value > 0.75 indicates a suitable model).22 The regression equation for the pretreated OPEFB resulted in an R-squared value of 0.9780, which is in good agreement with the adjusted R-squared of 0.8825. These

R2 = 0.9780; adj. R2 = 0.8825; d.f.= degree of freedom.

results ensure that the theoretical values were well adjusted to the experimental data using this model. Therefore, the model was suitable for predicting the TGCR.

Figure 2. Response surface plots show the effects of temperature and time (a); temperature and NaOH concentration (b); time and NaOH concentration (c). The value of the time variable was xed at the optimal point.

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Table 6. Optimal values of the test variables in decoded units, and the predicted maximum of the total glucose conversion rate (TGCR) from dry biomass at a 95% condence interval Variables X1 : Temperature [ C] X2 : Time [min] X3 : NaOH concentration [mol L1 ] TGCR of predicted response with 95% condence interval Value 127.64 22.08 2.89 89.61 (85.1394.09)

(a) 8

Converted glucose conc. [%]

80

0 0 5 10 15 20 25 30 Biomass loading [ % (w/v) ]

Enzymatic digestibility [%]

60

(b) 100

40

80 Enzymatic digestibility [%]

20

60

0 0 20 40 Time [h] 60 80

40

20

Figure 3. Enzymatic digestibility of pretreated OPEFB at 1% (w/v) biomass loading with cellulase complex and -glucosidase enzyme loadings of 570 FPU g1 cellulose and 30% weight of cellulase, respectively. 5; 10; 20; 30; 40; 50; 60; 70 FPU g1 cellulose.

0 0 5 10 15 20 25 30 Biomass loading [ % (w/v) ]

The optimum values of the selected variables were obtained by solving the regression equation, as shown in Table 6. To validate the model, the optimum values for Equation (2) were used in triplicate sets of experiments with the CHEMEX reactor, and the maximum response is presented in Table 7. The average experimental response for OPEFB was 86.37% of the TGCR. This value is in good agreement with the predicted value of 89.61 (85.1394.09) with a 95% condence interval. This behavior shows that the model could be adapted to the experimental results, conrming the validity and adequacy of the models. Enzymatic hydrolysis of pretreated OPEFB Enzymatic hydrolysis experiments were carried out using the pretreated biomass to determine the appropriate enzyme concentration and biomass loading. Figure 3 shows the enzymatic digestibility of pretreated OPEFB at 1% (w/v) biomass loading using cellulase complex and -glucosidase enzyme loadings of 570 FPU g1 cellulose and a 30% weight of cellulase complex, respectively. The conversion rate was enhanced in accordance with an increase in enzyme dosage. However, there was little difference in enzymatic digestibility when the enzyme dosage was >50 FPU g1 cellulose. Based on these results, an enzyme loading of 50 FPU g1 cellulose was chosen to examine the enzymatic digestibility of pretreated OPEFB, owing to the stability and effectiveness of the reaction at this loading.

Figure 4. Converted glucose concentration (a) and enzymatic digestibility (b) of pretreated OPEFB at 130% (w/v) biomass loading with cellulase complex and -glucosidase enzyme loadings of 50 FPU g1 cellulose and 30% weight of cellulase, respectively.

Figure 4(a), (b) shows the converted glucose concentration and enzymatic digestibility of pretreated OPEFB at various biomass loadings (130% (w/v)) with 50 FPU g1 of cellulose enzyme. Biomass loading >25% was impossible in a practical sense because of stirring. The converted glucose concentration showed an upward trend, but enzymatic digestibility decreased with increased biomass loading. There was little difference in enzymatic digestibility (90%) below 10% (w/v) biomass loading, whereas 20% (w/v) biomass loading resulted in 63% enzymatic digestibility. One explanation is that the enzyme activity decreased in accordance with the increasing converted glucose concentration, signifying that enzyme activity was inhibited by glucose production. Ethanol production according to enzymatic hydrolysis time The fermentative potentials of the pretreated materials were evaluated using S. cerevisiae. Pretreatment and enzymatic hydrolysis conditions were applied based on the results of the RSM. To evaluate the ethanol concentration as a function of enzymatic hydrolysis time to reduce operating cost, the 20% (w/v) loading

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Table 7.

Results for CHEMEX reactor under optimal conditions Glucan [%] Xylan [%] 20.26 19.83 20.36 20.15 Lignin [%] 20.15 20.38 19.96 20.16 Total solid [g per 100 g initial dry biomass] 60.29 60.47 59.85 60.20 Recovery of glucan after pretreatment [%] 92.58 93.57 93.68 93.28 Enzymatic digestibility [%] 93.09 92.12 92.58 92.60 TGCR of experimental response [%] 86.18 86.20 86.73 86.37

1 2 3 Average

45.59 45.95 46.47 46.00

50 Ethanol concentration [ g L-1 ]

Table 8. Ethanol fermentation and fermentation ratio as a function of enzymatic hydrolysis time (048 h) Time of enzymatic hydrolysis [h] Max. converted glucose conc. [g L1 of theoretical] Max. ethanol conc. [g L1 ] Ethanol yield [% of theoretical] 0 6 12 100.96 24 48

40

30

48.54 86.62

47.35 84.51

46.02 82.14

45.92 82.03

45.91 81.93

20

10

0 0 20 40 60 80 100 120 Time (enzymatic hydrolysis + fermentation) [ h ]

Figure 5. Ethanol fermentation as a function of enzymatic hydrolysis time. 0; 6; 12; 24; 48 h of enzymatic hydrolysis time.

of pretreated biomass was mixed with the cellulase complex and -glucosidase for 048 h at 50 C. Fermentation was subsequently carried out for 72 h at 32 C with inoculation of 10% S. cerevisiae. Figure 5 shows ethanol production plotted against enzymatic hydrolysis time. In the case where enzymatic hydrolysis and fermentation S. cerevisiae (Table 8) were performed simultaneously, ethanol production rose to 48.54 g L1 (by 2.63 g L1 ) during 48 h enzymatic hydrolysis. Fermentation ratio showed a lowering tendency as enzymatic hydrolysis time was extended. However, this result contradicted results of the ethanol production process using lignocellulosic biomass. This is because

the ethanol production process is generally completed by fermentation of glucose at 32 C, which was produced by enzymatic hydrolysis at 50 C, where the enzyme activity is optimal. Additionally, the shorter enzymatic hydrolysis time used in this study resulted in greater ethanol production. This means that simultaneous saccharication and fermentation (SSF) is an efcient and economical process that can be applied to produce ethanol. In summary, although it is not an optimal saccharication condition, enzymes can be used to produce glucose free from the product inhibition which occurs during the conversion of glucose into ethanol by enzymatic hydrolysis in yeast.

CONCLUSIONS
The overall process of OPEFB pretreatment for production of bioethanol was examined in this study. Pretreatment is essential for the production of ethanol from lignocellulosic biomass,

3 kg of Raw EFB Moisture: 7% Cellulose: 32.74%

Pretreatment process

Loss of cellulose: 6.72%

842.90 g of glucan

SSF process

Glucan to glucose ratio: 1.1 Glucose to ethanol ratio: 0.5111 SSF yield: 86.62%

410.48 g of Ethanol

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Figure 6. Materials balance of raw OPEFB cellulose to ethanol using the SSF process.

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www.soci.org which was achieved through saccharication by breaking the tangled structure of cellulose, hemicellulose, and lignin, allowing the enzymes to permeate the cellulose and hemicellulose. In this study, NaOH solution was used for pretreatment, and the optimal pretreatment and enzymatic hydrolysis conditions were determined through RSM. Based on the RSM result, bioethanol was produced by enzymatic hydrolysis and fermentation at a maximum amount of 48.54 g L1 . Figure 6 shows the material balance of raw OPEFB cellulose to ethanol used during the SSF process. In summary, 410.48 g of ethanol were produced from 3 kg of raw OPEFB during a single run using the CHEMEX 50L reactor. Overall, the ethanol production process from OPEFB using NaOH pretreatment was effective, and may be feasible for the commercial production of bioethanol in the near future.

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7 8 9 10

11 12

ACKNOWLEDGEMENTS
This study was supported nancially by the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea (No. 309016-5).

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