ISBN : 978-602-17761-0-0
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© 2013 Published by Center for Pulp and Paper through REPTech2012
study is to optimize glucose production to produce high value product with high concentration, such as
succinic acid.
2. Materials and Methods 2.1 Raw Material
Oil Palm empty fruit bunch OPEFB iber was collected from local palm oil mill United Oil Palm
Industries Sdn Bhd, Malaysia, sun dried and ground to a particle size 1 mm. The homogenized OPEFB
biomass was then oven dried at 105
o
C for overnight and was analysed following standard method for
determination of its main composition [10].
2.2 Chemicals and Enzymes
Two types of standard cellulose from Sigma- Aldrich were selected for this study. The irst is iber
cellulose and the second microcrystalline cellulose. Solvent and other chemicals were obtained from
R M Chemicals. Celluclast 1.5 L Cellulase and Novozyme 188 Cellobiase were obtained from
Novozymes Malaysia Sdn Bhd.
2.3 Autohydrolysis Pretreatment Deligniication
Autohydrolysis pretreatment was for deligniication process for OPEFB,that is meant to break down the
lignin linkage in the surface of OPEFB. 30 grams of OPEFB were loaded into 4 L stainless steel
Electronic Pressure cooker 98 Kpa, 120
o
C and were supplemented with appropriate amount of deionized
water. The auto hydrolysis was carried out at 120
o
C for 1-2 hours.
2.4 Organosolv Treatment
10 grams of auto hydrolysed OPEFB was milled and mixed with 80 aqueous ethanol EtOHH
2
0: 82 vv and 0.2 ww sulphuric acid as catalyst. The
mixture was heated at 120
o
C for 1 hour and iltered and washed with methanol [11]. This was followed by
treatment with Hydrogen Peroxide H
2
O
2
2 for 4 hours at 50
o
C to obtain cellulose.
2.5 Enzymatic Hydrolysis
Enzymatic hydrolysis experiments were conducted in 100 ml shaking lask at 40
o
C and 145 rpm inside incubator shaker. The cellulase-catalysed hydrolysis
of the different cellulose substrate untreated or pretreated was carried out in a stirred lask. In a typical
hydrolysis reaction, 500 mg of cellulose was added to 9 mL acetate buffer 50 mm, pH 4 and incubated
for 2 hours 40
o
C; 145 rpm. After this preincubation step, hydrolysis was initiated by addition 0.1 ml of
10 mgmL cellulase. Forty microliter of the reaction medium was withdrawn at different times in order
to determine the progress of the reaction which was stopped by incubating the withdrawn sample at 90
o
C for 20 min to deactivate the enzyme [12]. Then, the
sample was diluted in ultra-pure water and iltered 0.2 µm prior to analysis by high performance liquid
chromatography HPLC. The same procedure was followed for the cellulose
extracted from OPEFB. Reaction was carried out at 40
o
C, for 48-94 hours. Different amount of cellulose 300mg-500mg and the total of enzyme 0.3-0.7 mL
were used in this reaction.
2.6 Box-Behnken Design
Box-Behnken design was used in this study to help in planning experiment. According to
Box Behnken design principle, duration of reaction time, amount of samples and total enzymes, which
were identiied to have strong effects on the response in preliminary one-factor-at-a-time experiments, were
taken as the variable tested in a 15-run experiment to determine their optimum levels [13]. As shown in
Table 1, the three factors chosen for this study were designated as X
1
, X
2
, X
3
and prescribed into three levels, coded +1, 0, -1 for high, intermediate and low
value, respectively.
2.6 Analysis Method
Infrared spectra of raw and treated OPEFB ibers were recorded using Perkin Elmer, Fourier Transform
Infrared FTIR model GX. The powdered samples for each type were mixed with KBr and compressed
Table 1- Levels and Code of Variables Chosen for Box-Behnken Design Variables
Symbol Coded Levels
Coded -1
+1 Hydrolysis Reaction time h
X
1
48 76
94 Total of enzymes mL
X
2
0.1 0.2
0.3 Amount of samples g
X
3
0.3 0.5
0.7
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© 2013 Published by Center for Pulp and Paper through REPTech2012
into pellet. Samples were scanned from 400 to 4000 cm
-1
. Thermo gravimetric analysis TGA was carried out using a Mettler Toledo model TGASDTA 851
e
. Samples of approximately 6 mg were placed in alumina
pans and heated from 30 to 800
o
C at 10
o
Cmin
-1
, under dynamic low of nitrogen 50 mLmin
-1
. Morphology of the samples was examined by using Zeiss, Field
Emission Scanning Electron Microscope FESEM. Model Supra 46VP. SEM images were recorded using
an accelerating voltage at 3-5 kV.
Glucose concentration was determined by using High Performance Liquid Chromatography HPLC
and Glucose meter. The system used for HPLC was water HPLC system and evaporative light scattering
detectors ELSD used as detectors. Glucose was determined using a Zorbax NH
2
column and acetonitrile: water 80:20 as mobile phase at a low rate
of 1mlmin. The retention time of glucose is 13.32 min rsd ±0.56. Quantiication was based on calibration
curves established using standard glucose purchased from Sigma-Aldrich.
3. Result and Discussion 3.1 Characterization of Oil Palm Fruit Bunch
