Effect of Oil Palm Empty Fruit Bunch Particle Size on Cellulase Production by Botryosphaeria sp. Under Solid State Fermentation

Abstract

Locally isolated Botryosphaeria sp. showed the ablity to produce cellulases (FPase, CMCase and β-glucosidase) from oil palm empty fruit bunch (OPEFB) as substrate. Different particle sizes (0.25-0.3 mm, 0.42-0.6 mm, 0.84-1.0 mm and 5.0-10 mm) of OPEFB were investigated under solid state fermentation on the cellulase production. The highest production of FPase and β-glucosidase were obtained from OPEFB particle size of 0.42 – 0.60 mm with 3.261 ± 0.011 U/g and 0.115 ± 0.008 U/g, respectively. It was found that among the four different OPEFB particle sizes studied, particle size of 0.84 – 1.0 mm gave the highest activity of CMCase (8.134 ± 0.071 U/g). Highest concentration of reducing sugars produced in this experiment was 4.303 ± 0.095 mg/ml.

Full text

Australian Journal of Basic and Applied Sciences, 5(3): 276-280, 2011
ISSN 1991-8178
Corresponding Author: Suraini Abd Aziz, Department of Bioprocess Technology, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
E-mail: suraini@biotech.upm.edu.my
276
Effect of Oil Palm Empty Fruit Bunch Particle Size on Cellulase Production by
Botryosphaeria sp. Under Solid State Fermentation
Ezyana Kamal Bahrin, Piong Yeau Seng and Suraini Abd-Aziz
Bioprocess Technology Department, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Abstract: Locally isolated Botryosphaeria sp. showed the ablity to produce cellulases (FPase, CMCase
and β-glucosidase) from oil palm empty fruit bunch (OPEFB) as substrate. Different particle sizes
(0.25-0.3 mm, 0.42-0.6 mm, 0.84-1.0 mm and 5.0-10 mm) of OPEFB were investigated under solid
state fermentation on the cellulase production. The highest production of FPase and β-glucosidase were
obtained from OPEFB particle size of 0.42 – 0.60 mm with 3.261 ± 0.011 U/g and 0.115 ± 0.008
U/g, respectively. It was found that among the four different OPEFB particle sizes studied, particle
size of 0.84 – 1.0 mm gave the highest activity of CMCase (8.134 ± 0.071 U/g). Highest
concentration of reducing sugars produced in this experiment was 4.303 ± 0.095 mg/ml.
Key words: substrate particle size, cellulase, solid state fermentation, Botryosphaeria sp.
INTRODUCTION
Oil palm mills produce a large amount of biomass waste from its daily operation. Generally, 17.08 million
tones per annum of oil palm empty fruit bunches (OPEFB) have been produced continuously in 2005 (MPOB
2006). Fully utilization of OPEFB can be achieved by generating value added products such as activated
carbon, enzymes, citric acid and others. OPEFB is categorized as lignocellulosic feedstock since it is rich in
cellulose contents .Moreover the usage of OPEFB as substrate in cellulases production can reduce the operation
cost since substrate cost became one of the major operational costs, representing 30-40% of total production
cost (Tanaka et al., 2006; Zhang et al., 2007).
Insolubility of OPEFB is one of the limitations in submerged fermentation. Solid state fermentation (SSF)
is more capable in producing certain enzymes and metabolites that usually produced with low yield in
submerged fermentation. The bioconversion of OPEFB into polyoses by using SSF resembles the natural
condition of growth for the majority of fungi. Bacteria, yeast and fungi are able to grow on solid substrate but
filamentous fungi are the best adapted for SSF (Krishna, 2005). The hypha of the fungi has the power to
penetrate into the solid substrate.
There are several factors involved in the selection of a suitable substrate for SSF such as macromolecular
structure, particle size and shape, porosity and particle consistency (Krishna, 2005; Tao et al., 1997). The
substrate must be in a limited size range for an optimal production of cellulase. This process can be facilitated
by chipping, milling and grinding the biomass into a fine powder to increase the surface area/volume ratio of
the cellulose particle. An optimal sized particle lead to better nutrient absorption, gas exchange and heat
transfer thus high enzyme production.
The species of Botryosphaeria fungus attacks woody host and it is described as an endophyte (Crous,
2006) and pathogen on plants. The ascomycete fungus Botryosphaeria sp. produces a broad range of
lignocellulolytic enzymes such as laccases (Barbosa et al., 1996), pectinases (Da Cunha et al., 2003) , cellulase
and xylanases (Dekker et al., 2001). These enzymes play an important role in the degradation process of
lignocellulosic materials through a synergistic action (Lynd et al., 2002; Zhang and Lynd, 2004).
Less report are available on cellulases production by Botryosphaeria sp. using lignocellulose material.
Dekker et al. (2001) only reported low activity of filter paper cellulase in the media containing veratryl
alcohol. The main objective of this study is to evaluate the potential of locally isolated fungus, Botryosphaeria
sp. to produce cellulases under solid state fermentation by investigating the effect of different substrate particle
size.

Aust. J. Basic & Appl. Sci., 5(3): 276-280, 2011
277
MATERIALS AND METHODS
Microorganism:
Botryosphaeria sp. was obtained from Biomass Technology Centre, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia. The fungus was grown on potato dextrose agar (PDA) at
30
°
C to a 7 days culture for development.
Substrate:
OPEFB fiber was obtained from Seri Ulu Langat Palm Oil Mill in Dengkil, Selangor, Malaysia. The dry-
mixed substrates were subjected to a sieving procedure employing sieves mesh-size of: 18, 20, 30, 40, 50 and
60 (Mc Cabe et al., 2001). These fibers later were classified into four different diameter sizes according to
which of the sieves mesh-size that the fiber retained. The smallest fiber size 0.25-0.30 mm were collected from
fractions between meshes 50 and 60, followed by the fibers size of 0.420-0.60 mm collected from fractions
between meshes 30 and 40, and fibers size of 0.84-1.0 mm which were collected from fractions between
meshes 18 and 20. Another fiber size that will use as substrate in this experiment is unsieved shredded OPEFB
fiber (5-10 mm).
Substrate Pretreatment
Pretreatment of OPEFB fibers were done by soaking in 2.0 % (w/v) NaOH per 5.0 g of OPEFB followed
by autoclaving at 121 °C for 90 minutes. The pretreated OPEFB was filtered, washed with distilled water until
no traces of alkaline were detected and then dried in oven for 24 hours at 60 °C.
Solid State Fermentation of OPEFB:
Three gram of pretreated OPEFB was placed in 250 ml flasks and then was autoclaved at 121 °C for 15
minutes. Mandel medium (Mandels et al., 1974) was added as nutrient to initial moisture content of 70%. The
composition of the Mandel medium is as followed (g/l): 1.4, (NH
4
)2SO
4
; 2.0, KH
2
PO
4
; 0.3, CaCl
2
·2H
2
O; 0.3,
MgSO
4
·7H
2
O; 0.005, FeSO
4
·7H
2
O; 0.0016, MnSO
4
·H
2
O; 0.0014, ZnSO
4
·7H
2
O; 0.002, CoCl
2
·6H
2
O. Each flask
was inoculated with 3 mycelium plugs (0.5 cm diameter each). The flasks were incubated at 30 °C for 10
days. Sampling was done everyday to measure the cellulase activity and the reducing sugar concentration.
Enzyme Extraction:
Thirty ml of 0.2 M acetate buffer at pH 4.8 was added in each flask and shake at 150 rpm, room
temperature for 30 minutes. For sample analysis, the resulting homogenate was centrifuged at 4000 rpm, 10
minutes and 4°C to remove fungi cells. The cell free supernatant was then used for sample analysis.
Analytical Analysis:
The activity of FPase, CMCase and β-glucosidase was assayed according to the method explained by
Wood and Bhat (1988) with some modifications. One unit of FPase or CMCase activity was expressed as 1
µmole of glucose liberated per ml enzyme per minute. While, one unit of β-glucosidase activity was
determined as 1 µmole of p-nitrophenol liberated per ml enzyme per minute. The cellulases were expressed
as U/g of dry OPEFB. Reducing sugars concentration was assayed with DNS method (Miller, 1959).
RESULTS AND DISCUSSION
Bioconversion of lignocellulosic biomass into useful products is a complex process involving synergistic
actions of three enzymes namely endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and β-glucosidase
(EC 3.2.1.21) (Zhang and Lynd 2004). The results illustrated that the locally isolated Botryosphaeria sp. which
is described as an ascomycete fungus produced cellulases (endoglucanase, exoglucanase and β-glucosidase)
from OPEFB.
Cellulase activities were measured quantitatively based on the individual cellulase production
(exoglucanase, endoglucanase and β-glucosidase). FPase activity is one of the assays to determine the amount
of exoglucanase enzyme present in the sample. Among all of the different sizes investigated, particle size of
0.42 - 0.60 mm produced the highest FPase activity of 3.261 ± 0.011 U/g which almost more than 2 fold
higher compared with particle size between 5.0 to 10.0 mm (Fig 1).

Aust. J. Basic & Appl. Sci., 5(3): 276-280, 2011
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Fig. 1: Cellulases activity (Fpase , CMCase G and B-glucosidase activities) on four different particle
sizes of OPEFB
Higher CMCase activity of Botryosphaeria sp. was observed using 0.84 – 1.0 mm OPEFB particle size
as compared to FPase and β-glucosidase which showed maximal activities with substrate particle size of 0.42
– 0.6 mm (Fig 1). Membrillo et al. (2008) showed that Pleurotus ostreatus CP-50 produced maximum FPase
and CMCase on different particle size of sugarcane baggase. FPase activity was formed on 0.92 mm particles
while CMCase activity was on 1.68 mm substrate particle size. Study by Blandino et al. (2002), shown that
two factors affect the rate of Aspergillus awamori growth are particle size and chemical composition of milled
wheat and wheat grains mixture. The growth of the fungus on different particle size had a significant effect
on cellulase production.
The highest β-glucosidase activity was obtained using particle size of 0.42-0.6 mm, 0.115 ± 0.008 0.148
U/g while the lowest enzyme activity production (0.085 ±0.024U/g) was obtained from OPEFB particle size
of 0.25-0.30 mm (Fig 1). Overall, β-glucosidase activity produced during the fermentation was considered low
(< 0.2 U/g). This may due to the low productivity of β-glucosidase enzyme by Botryosphaeria sp. The highest
reducing sugars concentration was 4.303 ± 0.095 mg/ml using 0.42-0.60 mm OPEFB particle size.
Generally low OPEFB particle sizes (400µm) produced high cellulases due to larger specific surface area
in fine particles but low porosity property (Tao, 1997). Due to the inverse correlation of porosity and surface
area factors, most researchers claimed that 400 µm substrate sized particles contribute to the optimum fungal
growth and cellulases production (Tao et al., 1997; Krishna and Chandrasekaran 1996). The low porosity
caused less penetration of fungus hypha into the pores of the substrate and fungal growth only can be observed
on the surface of the substrate. When larger substrate particle size (> 400 µm) was applied in the fermentation,
a network of aerial hypha grows into the inter-particle space low fungal growth on surface of the substrate
particle and decreased the resulted enzymes.
Highest FPase and CMCase activity was obtained on day 3 (Fig 2). FPase activity was increased
exponentially before reach the optimum day and thereafter the enzyme activity decreased. Fifty percent particle
degradation was reported for the period of 12 to 36 hours of fermentation period. In figure 2, the FPase
activity increased significantly on 2nd day until it reach the maximal value on 3rd day for 0.42-0.60 and 0.84-
1.0 mm particle size. After 3rd
day, the FPase production started to decline thereafter followed by a slight
decreasing pattern until the end of the fermentation.
The study by Nandakumar et al. (1993) and Giese et al. (2008) are in agreements with our observation.
Study by Nandakumar et al. (1993) showed that cellulases, xylanases and reducing sugar production by
Aspergillus niger occurred after 36 hours of fermentation and escalated up to 72 hours. Giese et al. (2008)
reported that Botryosphaeria rhodina grown on orange bagasse (peel,seed and pulp) in solid state fermentation
produced high titers of pectinase was on 72 hours while laccase on 96 hours.
Figure 2 shows that β-glucosidase activity increased rapidly within the first 2 days of fermentation, after
which a more or less stationary phase was observed. The relative distribution of β-glucosidase was quite
different with FPase and CMCase activities. β-glucosidase activity always lagged behind FPase and CMCase.
This is an interesting pattern which seems to be due to the increased levels of disaccharides (the hydrolysis

Aust. J. Basic & Appl. Sci., 5(3): 276-280, 2011
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Fig. 2: FPase (Ë), CMCase (#), β-glucosidase (•) activities and reducing sugars (M) production with 0.42-
0.6 mm particle size of OPEFB
products of glucanases) at this stage. The optimum day to produce β-glucosidase was around day 5 to day 7.
This may due to the substrate (cellobiose) for β-glucosidase is only obtained in large quantity after cellulose
being hydrolysed by exoglucanase and endoglucanase where the optimum day for those enzymes are at day
3.
The level of reducing sugars during this solid state fermentation showed a major increase until it reached
the highest peak on day 3 (Fig 2). Fungi cannot directly absorb polysaccharides, so they are induced by low
molecular weight compounds to synthesize and secrete the enzymes to hydrolyse the macromolecules into
smaller metabolizable compounds (Zhang et al., 2004). However, the reducing sugars content decreased
gradually towards the end of the fermentation.
In a nutshell, Botryosphaeria sp. had shown its ability to convert the OPEFB into cellulases and simple
sugars and the optimal particle size to obtain high yield of cellulases is 0.42-0.6 mm.
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