ISBN : 978-602-17761-0-0
13
© 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 Forty microliter of the reaction medium was withdrawn at different times in order
to determine the progress of the reaction which was stopped by incubating the withdrawn sample at 90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
