J. Gen. Appl. Microbiol.

, 46, 263–267 (2000)

Short Communication

Screening and isolation of a cellulolytic and amylolytic Bacillus

from sago pith waste

Kasing Apun,* Bor Chyan Jong, and Mohd. Azib Salleh

Molecular Biology and Biotechnology Core Group, Faculty of Resource Science and Technology,
University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia

(Received June 26, 2000; Accepted October 13, 2000)

Key Words——amylolytic; Bacillus amyloliquefaciens; cellulolytic; sago pith waste

The final waste product in the extraction of starch degradation of cellulose and starch components
from the sago palm is the starchy fibrous pith residue. (Coughlan, 1985). The breakdown of these compo-
This residue is abundantly and cheaply available espe- nents produce simple sugars that find many uses in
cially in the state of Sarawak, Malaysia. It is usually the feed and fermentation industries.
washed off into drains or nearby streams together with The first important step in this approach is to isolate
wastewater, thus contributing to pollution load, or de- and identify the microorganisms with the necessary
posited in the factory’s compound, which can lead to characteristics as active cellulolytic and amylolytic mi-
serious environmental problems. It has no significant croorganisms. Habitats that contain these substrates
industrial or commercial use, except to a small extent are the best sources in which to find these microorgan-
as animal feed mainly for pigs and poultry (Yeong and isms. This paper described the screening and isolation
Ali, 1982) or as a soil conditioner (Bintoro and Siana- of an indigenous Bacillus that is capable of degrading
par, 1993). Studies have shown that sago pith waste is the substrates. The isolate was identified as Bacillus
composed mainly of starch and fiber, including a fair amyloliquefaciens. To our knowledge, this is the first
amount of minerals. The crude starch and crude fiber report of a B. amyloliquefaciens that can utilize the
contents range from 41.7% to 65.0 and 14.8%, respec- waste of sago palm.
tively (Wina et al., 1986). When the fibrous and starch Three types of sago waste samples were collected
component of the pith waste are considered, it is logi- for this study. First, pith residue, the final waste prod-
cal to find ways to utilize this waste. The cellulose and uct from starch extraction, was obtained from a sago
starch components both have a good potential for bio- processing factory in the district of Pusa, Sarawak.
conversion into value-added products. An attractive Samples were collected around the discharge point at
and efficient means is through a biotechnological ap- a distance of about 3 m from the factory. Second, par-
proach in which microbial strains are employed to de- tially decomposed fibrous waste comprising pith
grade the sago waste. Microorganisms such as fungi residue and bark was collected from around the same
and bacteria are known to play a major role in the factory’s compound. Samples were collected by using
a clean spade and placed in separate sterile plastic
* Address reprint requests to: Dr. Kasing Apun, Molecular Bi- bags. Third, waste effluent samples were collected
ology and Biotechnology Core Group, Faculty of Resource Sci- from a sago processing plant in Mukah, Sarawak. The
ence and Technology, University Malaysia Sarawak, 94300 wastewater containing a large amount of pith residue
Kota Samarahan, Sarawak, Malaysia. and some unseparated starch was collected from sev-
264 APUN, JONG, and SALLEH Vol. 46

eral points along an open drain. The effluent samples termined by using the dinitrosalicylic acid (DNS)
were collected in sterile bottles and kept at 4°C until method (Miller, 1959). Cellulase activity as measured
analyzed. by carboxymethylcellulase activity was determined as
A 10% suspension (w/v) of waste samples was pre- described by Mandels et al. (1974), using CMC as the
pared in sterile distilled water, and 0.1 ml aliquots were substrate, and amylase activity was determined by
plated on nutrient agar (NA) for the isolation of bacte- using starch as substrate. The amount of reducing
ria. Four replicates were prepared for each sample, sugars released from the assay was detected by the
and the plates were incubated at 30!2°C. The bacte- DNS method. To detect the sugar component released
rial colonies recovered were subcultured on NA until into the culture medium from the hydrolysis of the sago
pure cultures were obtained. pith waste, the method of high-performance liquid
The bacterial isolates were initially identified by chromatography (HPLC) was used. The components
means of morphological examination and some bio- were separated by using a Hitachi HPLC system
chemical characterization. The parameters investi- (Model L-6000) equipped with a refractive index (RI)
gated included colonial morphology, Gram reactions, detector (ERC-7515A, ERC Inc., Tokyo, Japan), au-
endospore formation, catalase production, anaerobic tosampler (Model AS-2000), and chromato-integrator
growth, Voges-Proskauer (V-P) reactions, starch hy- (Model D-2500). The unit was fitted with a fermenta-
drolysis, citrate utilization, lecithinase, and growth in tion monitoring column (150 mm"7.8 mm, Bio-Rad,
the presence of 7.5% NaCl and at 50°C. Further iden- U.S.A.) and a guard column (Cation H, Bio-Rad). Before
tification was conducted by using two commercial injection, the eluent was degassed and filtered through
identification systems, the API 50 CHB test kit (Bio- a 0.45 mm filter membrane. Exactly 20 ml of the sample
Merieux, MarcyI’Etoile, France) and the Biolog system were injected into the column with the autosampler.
(Hayward, U.S.A.). All procedures were conducted as The analysis conditions were performed at 60°C and
suggested by the manufacturers. To screen for cellu- at a flow rate of 0.6 ml/min. The mobile phase was
lolytic organisms, the isolates were grown in a minimal 1.0 mM H2SO4, and the refractive index was set at
agar plate consisting of yeast extract (0.2%), KH2PO4 attenuation 3.
(0.1%), MgSO4 (0.5%), and a soluble form of cellulose, A total of 84 bacterial isolates was isolated from the
carboxymethylcellulose (0.5%). Negative control plates pith residue, effluent samples, and partially decom-
inoculated with laboratory E. coli strain (HB101) were posed fibrous waste. Of these isolates, 52 were Gram
included in all tests. The test plates were incubated at positive and 32 Gram negative. Among the Gram-posi-
30!2°C for 2 days. To visualize the hydrolysis zones, tive isolates, 43% were rod shaped and spore formers,
the plates were flooded with an aqueous solution of and the rest were cocci. Screening for the isolates with
Congo-red (1 mg/ml) for 15 min and washed with 1 M cellulolytic and amylolytic activity revealed that the
NaCl (Teather and Wood, 1982). Cellulolytic organ- spore formers were more prolific producers of both en-
isms produced a clear zone around the colonies zymes compared with the cocci-shaped isolates. How-
because of the digestion of carboxymethylcellulose ever, screening for both cellulolytic and amylolytic ac-
(CMC). To screen for amylolytic organisms, the organ- tivities on agar plates revealed only one isolate (UMAS
ism was inoculated on minimal agar plates (as above) 1002) having both activities. It is interesting that this
containing soluble starch (0.5%, w/v) in place of CMC. isolate was also found to be the most active as re-
Degradation of the starch would result in the formation vealed by the size of clearing zones on both types of
of a clear zone around the colonies after flooding the agar plates. The diameters of clear zones on CMC and
plates with Lugol’s iodine (Hyun and Zeikus, 1985). starch plates were 1.7 and 1.45 cm, respectively. On
The diameters of the clear zone produced on CMC NA plates, UMAS 1002 produced creamy colonies with
and starch plates were measured and used as an indi- circular motions. A microscopic examination of the iso-
cation of the cellulolytic and amylolytic activities of the late showed that it is a spore former with an oval-
organisms. Besides the clearing zones on plates, a shaped spore in the center. Further identification using
more quantitative assay method was used to deter- standard biochemical tests showed positive reactions
mine the cellulase and amylase activity of the selected in the catalase test and in the V-P, ONPG, starch hy-
bacterial isolate in liquid medium. The reducing sugar drolysis, gelatin, oxidase, and nitrate tests. From these
content from the hydrolysis of sago pith waste was de- morphological and biochemical reactions, the isolate
2000 A cellulolytic and amylolytic Bacillus from sago pith waste 265

was tentatively identified as B. amyloliquefaciens hydrolyzed the pith substrate into reducing sugars.
(Table 1). Further identification, using the API 50 CHB After 24 h, reducing sugars were detected in the su-
kit, also revealed the isolate to be B. amyloliquefa- pernatant at an estimated amount of 1.03 mg/ml (Table
ciens. Another commercial identification system, the 2). Detection of the components of the reducing sug-
Biolog automated system, which is based on biochem- ars by HPLC revealed the presence of two sugars,
ical characterization for 95 different compounds, also maltose and glucose. The isolate also produced the
confirmed the isolate to be B. amyloliquefaciens. enzymes cellulase and amylase, based on the activi-
When the isolate was grown on sago pith waste, it ties detected in the culture filtrate. The cellulase and
amylase activities measured in the filtrate after 24 h
Table 1. Biochemical and growth characteristics of the bacte- were 0.63 and 0.38 I.U., respectively (Table 2). To de-
rial isolate (UMAS 1002). termine the pattern of production of reducing sugars
and the cellulase and amylase enzymes by the isolate
Characteristics/biochemical tests Observation
on sago pith waste, growth of the isolate in sago pith
Gram reaction Gram positive waste medium was extended up to 72 h. Reducing
Endospore forming/spore location Oval central endospores sugars and enzymatic activity in the crude filtrate were
Motility # assayed at an interval of 6 h for the first 24 h, followed
Catalase # by every 24 h until 72 h of incubation. Table 2 demon-
Starch hydrolysis # strates the level of reducing sugars and the cellulolytic
Lecithinase $ and amylolytic activities over a period of 72 h as mea-
Growth at 50°C #
sured by the ability of the culture filtrate to degrade
Growth in 7% NaCl #
CMC and starch, respectively. The maximum content
Voges-Proskauer (V-P) reactions #
Citrate utilization #
of reducing sugars was 1.03 mg/ml at 24 h, which rep-
NO3 reduction into NO2 # resents the highest level of production. After 24 h, the
reducing sugars declined and the lowest level
#, positive reaction; $, negative reaction. (0.07 mg/ml) was detected at 72 h. The cellulolytic ac-

Table 2. Production of reducing sugars and the cellulolytic and amylolytic activities of
B. amyloliquefaciens (UMAS 1002) in liquid medium.

Reducing sugars Cellulolytic (CMCase) Amylolytic activity

Time (h)
(mg/ml)a activity (I.U./ml)a (I.U./ml)a

0 0.007 0.0 0.0

6 0.06 0.13 0.21
12 0.53 0.52 1.07
18 0.55 0.45 0.85
24 1.03 0.63 0.38
48 0.18 0.27 1.72
72 0.07 0.17 2.30

a
Each value is mean of triplicates.
Overnight culture (1.0% at O.D.600%1.5) was inoculated into 100 ml of sago pith waste medium in a 250 ml flask, which was then
incubated with shaking (200 rpm) for 24 h at 30°C. The sago waste was dried at 65°C and milled into 40-mesh size before being
added into the medium at a concentration of 0.5% w/v. The culture was then centrifuged at 2,500 rpm, and the supernatant was used
as crude enzyme for the enzyme assays and also analyzed for its content of reducing sugars. The reducing sugars content from the
hydrolysis of sago pith waste was determined by using the dinitrosalicyclic acid (DNS) method (Miller, 1959). Cellulase activity was
determined as described by Mandels et al. (1974), using CMC as the substrate, and amylase activity was determined by using
starch as a substrate. The reaction mixture consisting of 2 ml of 1.0% substrate solution (soluble starch or CMC dissolved in 0.1 M
citrate-phosphate buffer, pH 5.8) and 1.0 ml of crude enzyme filtrate was incubated for 1 h at 50°C. The reducing sugar released was
measured by the (DNS) reagent method (Miller, 1959). A standard calibration curve was constructed by using D-glucose. One Inter-
national Unit (I.U.) of each enzyme was defined as the activity that produced 1 µmol of glucose equivalents per minute under the
above assay conditions.
266 APUN, JONG, and SALLEH Vol. 46

tivity continued to increase and reached the maximum zyme can act on the cellulose component while the
(0.63 I.U./ml) at 24 h, although a slight decline amylolytic activity acts on the starch component of the
(0.45 mg/ml) was observed at 18 h. After 24 h, cellu- sago pith waste. In this way the cooperative action of
lolytic activities declined, and at 72 h very little activity both the cellulolytic and amylolytic enzymes will lead to
(0.17 I.U./ml) remained. The pattern of cellulolytic ac- better or even complete hydrolysis of the sago pith
tivities was found to follow the trend of reducing the waste. This kind of synergistic effect has been re-
production of sugar. In contrast, amylolytic activity was ported by Haska and Ohta (1993), who observed that
very low (0.38 I.U./ml) at 24 h, but it sharply increased the hydrolysis of sago starch by raw starch digesting
at 48 h and continued until 72 h (2.30 I.U./ml). On the amylase was greatly improved with the addition of cel-
basis of the highest production of reducing sugars de- lulase. It was reported that cellulase promoted the ac-
tected at 24, it shows that a high amount can be tivity of the amylase leading to increased hydrolysis of
achieved in just 24 h. This suggests that a high pro- the starch granule into the final product, glucose. On
duction of reducing sugars can be achieved within the basis of the results of this work, it is highly proba-
24 h, thus eliminating the need for extended incuba- ble that B. amyloliquefaciens UMAS 1002 could be ef-
tion. fectively used in the bioconversion of sago pith waste.
From this study it is clear that the indigenous isolate Further studies on optimizing the conditions for en-
identified as B. amyloliquefaciens has the ability to hy- zyme production will undoubtedly lead to an improve-
drolyze sago pith waste into reducing sugars. Based ment in the activity of both enzymes. There is also a
on the maximum value of 1.03 mg/ml production of great potential for genetic manipulation of the isolate to
reducing sugars, it is calculated that approximately increase the enzyme activity which can lead to im-
315 mg of reducing sugars can be obtained from every proved hydrolysis of the sago waste substrate.
one gram of sago pith waste used. On this basis, if 1
ton of sago pith waste is used in a batch fermentation Acknowledgments

using the bacterial isolate UMAS 1002, about 0.315 We thank Assoc. Prof. Dr. Son Radu from the Faculty of Food
ton of reducing sugars can be obtained after 24 h. In Science & Biotechnology, University Putra Malaysia (UPM), for
the state of Sarawak, it has been estimated that each assisting us in the identification of bacteria, using the Biolog
sago factory produced approximately 7 tons of sago Identification system. The project was funded by UNIMAS re-
pith waste every day (Bujang et al., 1996). This is a search grant No. 3/94.
huge amount, and if the bacterial isolate UMAS 1002
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