Isolation and Identification of Cellulase-Producing Thermophilic Bacteria from Oil Palm (Elaeis guineensis)

i

ISOLATION AND IDENTIFICATION OF CELLULASE-PRODUCING
THERMOPHILIC BACTERIA FROM OIL PALM (Elaeis guineensis)

RIRIN MASRINA

DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

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ABSTRACT
RIRIN MASRINA. Isolation and Identification of Cellulase-Producing Thermophilic Bacteria
from Oil Palm (Elaeis guineensis). Supervised by ANTONIUS SUWANTO and ESTI
PUSPITASARI.
Cellulase is an enzyme catalyzing hydrolysis of cellulose which usually consists of
endoglucanases (EC.3.2.1.4), exoglucanases (EC.3.2.1.91) and β-glucosidases (EC.3.2.1.21).

Cellulase is a very important enzyme due to its numerous industrial applications. The aim of this
research were to isolate and identify cellulase-producing thermophilic bacteria that can be used to
increase value added in oil palm industries. Samples of soil, empty fruit bunch compost and palm
kernel meal were collected from oil palm plantation for obtained thermophilic bacteria. The
samples were screened of cellulase-producing thermophilic bacteria by using a Congo Red method
were made on carboxymethyl cellulose (CMC) agar plates. The bacterial cultures were incubated
in a shaking incubator (140 rpm) at 50oC for 24 hours. The assay for the enzymatic activity was
based on the release of glucose that was detected using 3,5-dinitrosalicylic acid (DNS). In this
research, 19 isolates of bacteria were isolated. It was found that 11 isolates of bacteria showed
positive results with clear zone around the cultures by using Congo Red method. The result
showed that isolates for CK1, EM4 and CK3 possesed the highest enzyme activity of 13.56, 12.50
and 10.49 U mL-1 for cellulase was detected at pH 7, respectively. The results of strain
identification based on 16S rRNA showed that strain CK1, EM4 and CK3 were identified as
Bacillus subtilis.
Key words: isolation, identification, cellulase, thermophilic bacteria, oil palm

ABSTRAK
RIRIN MASRINA. Isolasi dan Identifikasi Bakteri Termofilik Penghasil Selulase dari Kelapa
Sawit (Elaeis guineensis). Dibimbing oleh ANTONIUS SUWANTO dan ESTI PUSPITASARI.
Selulase merupakan enzim untuk mengkatalisasi hidrolisis selulosa yang pada umumnya

terdiri atas endoglukanase (EC.3.2.1.4), eksoglukanase (EC.3.2.1.91), dan β-glukosidase
(EC.3.2.1.21). Selulase sangat penting untuk diterapkan pada banyak industri. Tujuan dari
penelitian ini adalah untuk mengisolasi dan mengidentifikasi bakteri termofilik penghasil selulase
yang dapat digunakan untuk meningkatkan nilai tambah pada industri kelapa sawit. Sampel tanah,
kompos tandan kosong, dan bungkil inti sawit diperoleh dari perkebunan kelapa sawit untuk
memperoleh bakteri. Sampel ditapis untuk memperoleh isolat selulolitik dengan menggunakan
metode Congo Red pada media padat carboxymethyl cellulose (CMC). Kultur bakteri diinkubasi
pada inkubator bergoyang (140 rpm) pada suhu 50oC selama 24 jam. Assay aktivitas enzim
didasarkan atas pelepasan glukosa yang dideteksi dengan menggunakan 3,5-dinitrosalicylic acid
(DNS). Di dalam penelitian ini, 19 isolat bakteri telah diisolasi. Ditemukan 11 isolat bakteri yang
menunjukkan hasil yang positif dengan adanya zona bening disekitar koloni dengan menggunakan
metode Congo Red. Hasil menunjukkan bahwa isolat CK1, EM4, dan CK3 memiliki aktivitas
enzim tertinggi, yaitu 13.56, 12.50, dan 10.49 U mL-1 untuk selulase yang dideteksi pada pH7
untuk masing-masing isolat. Hasil identifikasi strain berdasarkan 16S rRNA menunjukkan bahwa
strain CK1, EM4, dan CK 3 telah diidentifikasi sebagai Bacillus subtilis.
Kata kunci: isolasi, identifikasi, selulase, bakteri termofilik, kelapa sawit

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ISOLATION AND IDENTIFICATION OF CELLULASE-PRODUCING
THERMOPHILIC BACTERIA FROM OIL PALM (Elaeis guineensis)

RIRIN MASRINA

Minithesis
In partial fulfillment of the requirement for Bachelor Degree of Science in
Department of Biology Faculty of Mathematics and Natural Sciences
Bogor Agricultural University

DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

viii

Title

: Isolation and Identification of Cellulase-Producing Thermophilic Bacteria from Oil
Palm (Elaeis guineensis)

Name

: Ririn Masrina

NIS

: G34070018

Approved by,

Prof. Dr. Ir. Antonius Suwanto, M.Sc.

Esti Puspitasari, M.Si.

Supervisor I

Supervisor II

Endorsed by,
Head of Department of Biology
Bogor Agricultural University

Dr. Ir. Ence Darmo Jaya Supena, M.Si.

Graduation Date:
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ACKNOWLEDGMENTS
All praises and thanks to Allah SWT the Almighty for His bless and Who is entire source
of knowledge that I’m able to finish my minithesis entitled Isolation and Identification of
Cellulase-Producing Thermophilic Bacteria from Oil Palm (Elaeis guineensis).
I would like to express my appreciation to Prof. Dr. Ir. Antonius Suwanto, M.Sc. and Esti
Puspitasari, M.Si. for their advices, knowledges and supports during my research. My sincere
thanks to Dr. Ir. Ence Darmo Jaya Supena, M.Si. as the head of Biology Department, Faculty of

Mathematics and Natural Sciences, Bogor Agricultural University. My gratitude to PT Wilmar
Benih Indonesia that served all my research, also all staff in there, especially for Ludovika Jessica
Virginia, S.Si. and Griselda Herman Natadiputri, S.Si. for helping me completed my research.
Deppest thanks to my parents, my sisters (Teteh Yani ang Umi), and my brother (Alfi)
for all prays, loves, supports and sacrifices. I also take this opportunity to say thanks to all of my
friends specially for LASPATI, TANDA BACA community (Mas Eko, Mas Jay, Agra), IKC,
FORCES (Mba Sari, Tiko, Riska, Ayu, Amin), my housmate Ar-Riyadh (Gita, Mba Ria, Zia, Pito,
Tari, Maya, Lili, Laswi, Vyras, Indi, Putri, Arum, Achi, Nisa, Fira) and Biology 44 IPB for the
encouragement, especially to Rita Handayani, Gita Kusuma Rahayu, Irwanto Adhi Nugroho,
Faizal Kurnia Syavitri and Muhammad Irfan for the supports, cheerfulness and friendships that
they given to me this far.
Life is not measured in the number of breathes we take, but in the moments that take our
breaths away, this minithesis is a small tribute to all of the moments. Hope this minithesis will be
usefull.
Bogor, November 2011
Ririn Masrina

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CURRICULUM VITAE

Author was born in Cirebon, February 9th 1989 as the second child of Salim and Suaebah.
Author was graduated from SMAN 1 Sumber (Cirebon) in 2007 and accepted in
Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural
University (IPB) through Undangan Seleksi Masuk IPB (USMI).
Author did the field study entitled Isolation of Soil Bacteria-Producing Mannanase and
Indol Acetic Acid (IAA) and field work entitled Process of Carp Hatchery in Technical
Implementing Fish Seed Unit Department of Marine and Fishery in Cirebon.
During the college, author assisted the practical class for Biology in 2009 and Phisiology
of Procaryot in 2010. Author was also active in many organization, such as Forum for Scientific
Studies in 2007-2010, IPB Debating Community in 2007-2010, Madani Foundation 2008-now,
Tanda Baca Community as secretary 2008-now, Techno Magazine as Secretary 2009-2010, writer
of book Modal Guyur Air Untung Besar Mengalir with Dr. Elang Ilik Martawijaya and member
of Ikatan Kekeluargaan Cirebon (IKC) in 2007-2011.
Author was also active in many event, such as are Gebyar Inovasi Pemuda Indonesia,
Pesta Sains, IPB goes to School, Kompetisi dan Inovasi Agroteknologi, Seminar Ilmiah Nasional
and many others.
Beside that, author has also taking part on many competitions, recorded her
accomplishment becoming the best student of Department of Biology Bogor Agricultural
University in 2010, 1st winner of essay contest on NDC (National Debating Competition) 2010 in
Bandung, 2nd winner of writing student competition about environmental category of biotis in

2008, finalist of writing students competition about health in Airlangga University in 2009, finalist
of scientific writing competition about biodiversity in Brawijaya University in 2009, finalist of
Intensive-Student Technopreneurship Program (I-STEP) IPB by Government of Indonesia and
Lemelson Foundation, and students creativity program by Directorate of Higher Education
Indonesia in 2008-2011 and others.

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CONTENT

Page

FIGURES..............................................................................................................................................

vii

APENDIXES........................................................................................................................................

viii

INTRODUCTION
Background ....................................................................................................................................
Objectives ......................................................................................................................................

1
1

MATERIALS AND METHODS
Time and Place................................................................................................................................
Materials ........................................................................................................................................
Methods...........................................................................................................................................
Isolation of cellulase-producing thermophilic bacteria..........................................................
Screening of cellulase producers............................................................................................
Cellulase production...............................................................................................................
Preparation of crude enzyme..................................................................................................
Enzyme assays........................................................................................................................
Strain identification................................................................................................................

1
2
2
2
2
2
2
2
2

RESULTS AND DISCUSSIONS
Isolation and selection of cellulase-producing thermophilic bacteria.............................................
Enzyme assay..................................................................................................................................
Identification of cellulase-producing thermophilic bacteria...........................................................

3
4
4

CONCLUSION....................................................................................................................................

5

REFERENCES.....................................................................................................................................

5

APENDIXES........................................................................................................................................

9

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FIGURES
Page
Figure 1 Screening for cellulase-producing thermophilic bacteria by clear zone detection using
Congo Red...........................................................................................................................
Figure 2 Cellulase activity results using DNS method after 24 h incubation time in cellulase
production medium at 50oC on rotary shaker 140 rpm ( : 546 nm)..................................
Figure 3 Agarose gel anlaysis of PCR amplified partial 16S rRNA . Line 1 : 1kb DNA size
marker (Promega). Line 2 1800 bp PCR product with a. CK3, b. EM4 and c. CK1 are
three the best isolate that producing highest cellulase crude enzyme with DNS method...

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3
4

5

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APPENDIXES
Page
Appendix 1 Screening for cellulase-producing bacteria using agar medium containing 0,5%
(w/v) CMC. Samples were incubated at 50oC for 3 days. The plates were stained
with Congo Red and destained with 1M NaCl solution. Clear zone indicated the
hydrolysis of CMC as a result of cellulases production................................................

9

Appendix 2 Glucose standard curve..................................................................................................

9

Appendix 3 Cellulase activity results using DNS method after 24 h incubation time in cellulase
production medium at 50oC on rotary shaker 140 rpm at  546 nm..............................

10

Appendix 4 Result of spectrophotometric measurment of absorbance by DNS method ( : 546
nm).................................................................................................................................

11

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1

INTRODUCTION
Background
The oil palm (Elaeis guineensis) has
become the most important economic
plantation crop in the world. Based on the
data from National Agriculture Department
of Indonesia in 2009, Indonesia palm oil
production reached 18,64 billion tons for oil
palm and 3,47 billion tons for palm kernel
oil with the large of area is 7,51 billion ha
(Deptan 2010). The oil palm consists of
huge amount of cellulosic materials such as
oil palm fronds, trunks and empty fruit
bunches (Deraman 1993).
Cellulose is the primary product of
photosynthesis in plant biomass and the
most abundant renewable bioresource
produced in the biosphere (Jarvis 2003;
Zhang & Lynd 2004). Cellulose is a linear
polymer consisting of D-anhydrogluco
pyranose molecules joined together by β-1,4
glicosidic bond with a degree of
polymerization (Lynd et al. 2005; Zhang et
al. 2006).
Cellulose is commonly degraded by an
enzyme called cellulase (Kotchoni et al.
2006). Cellulases are inducible enzymes
synthesized by microorganisms during their
growth on cellulosic materials (Cai et al.
1999; Lee & Koo 2001). The complete
enzymatic hydrolysis of cellulosic materials
needs at least three different types of
cellulase,
there
are
endoglucanase
(Carboxymethylcellulase or CMCase; EC
3.2.1.4), exoglucanase (EC 3.2.1.91) and βglucosidase (EC 3.2.1.21) (Yi et al. 1999;
Saha 2000; Bhat 2000; Holker et al. 2004).
The endoglucanase randomly hydrolized
the β-1,4 bonds in the cellulose molecule
and the exoglucanase in most cases release a
cellobiose unit showing a recurrent reaction
from chain extremity. Lastly, the cellobiose
is converted to glucose by β-glucosidase
(Beguin & Aubert 1994; Ibrahim & Eldiwany 2007).
With the recent development of
biotechnology, there has been vast interest to
use cellulose digestive microorganisms to
convert cellulosic biomass to glucose that
can be used in different applications such as
production of fuel ethanol, use in waste
treatment (Thambirajah et al. 1995),
brewing industry, for bio-polishing of
fabrics and producing stonewashed look of
denims, animal feeds for improving the
nutritional quality and digestibility (Kasana
et al. 2008), for improving fabric softness,

brightness and anti-deposition (Ibrahim &
El-diwany 2007).
Many mesophilic and thermophilic
bacteria and fungi have been investigated
with respect to the bioconversion of
agricultural and forest biomass into fuels and
valuable chemicals (Tomme et al. 1988;
Meinke et al. 1991; Wang et al. 1993;
Wittmann et al. 1994). The most common
producer is fungi (Lee & Koo 2001; Ariffin
et al. 2006). But, bacteria, which has high
growth rate as compared to fungi has good
potential to be used in cellulase production
(Ariffin et al. 2008).
Bacteria, due to their extremely high
natural diversity have the capability to
produced highly thermostable, alkalistable
enzyme complement and may serve as
highly potent sources of industrially
important enzyme. One of the prerequisite
for the enzymes to be employed for
industrial applications is that they must be
robust enough and highly stable under
hostile conditions of industrial processes like
extremes of temperature and pH. For
instance, for the successfull application of
cellulases in detergent industry, enzymes
must have alkaline pH optima, similarly for
lignocellulose transformation, in pulp and
paper industry or in feed industry, highly
thermostable cellulases with acid or
alkalistability
are
desirable.
Many
resesearchers have documented production
of thermostable and alkalistable cellulases
from different microorganism (Bhat 2000).
There are limited studies on bacteria that
reported as cellulase producers e.g.
Ruminococcus albus (Wood et al. 1982;
Ohara et al. 2000; Schwarz 2001), Bacillus
(Robson & Chambliss 1984), Clostridium
thermocellum (Lamed & Bayer 1988),
Clostridium cellulyticum (Belaich et al.
2002), Thermoactinomycetes sp. (Amritkar
2002), a mutant of Bacillus pumilus
BpCR16 and Bacillus pumilus EB3 (Ariffin
et al. 2006).
Objectives
The objectives of this research were to
isolate and identify cellulase-producing
thermophilic bacteria from oil palm.
.
MATERALS AND METHODS
Time and Place
This research was conducted on
February-September 2011 in Research and
Development (R&D) Laboratory of PT
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Wilmar
Indonesia.

Benih

Indonesia-Cikarang,

Materials
Materials that were used for this research
were soil, empty fruit bunch compost
(EFBC), palm kernel meal (PKM) of oil
palm, carboxymethyl cellulose (CMC),
Congo Red (CR), Gram staining, 3.5-dinitro
salicylic acid (DNS) reagent and other
materials for routine laboratory analysis and
identification.
Methods
Isolation and screening of cellulaseproducing thermophilic bacteria
Bacteria were isolated from soil, EFBC
and PKM samples. The samples were
suspended and serially diluted in
physiological NaCl up to 10-5, 100 μL of
each dilution were spread on 0.5%
carboxymethyl cellulose (CMC) agar
medium (Ruijssenaars & Hartman 2001)
with some modification. CMC agar medium
containing 0.1% (NH4)2SO4, 0.5% KCl,
0.5% yeast extract, 0.5% Tryptone, 0.1%
K2HPO4, 0.05% MgSO4.7H2O, 0.01%
FeSO4.7H2O and 2% agar bacteriological.
The pH was adjusted to 7.0 with 1 M NaOH.
The bacteria were grown on CMC agar
plates were incubated at 50oC for 24 h.
Single colonies on the plates were isolated
and purified by transferring them several
times onto CMC agar plates. The isolated
colonies were further incubated at 50°C for
3 days to allow for the secretion of cellulase.
At the end of the incubation, to visualized
the hydrolysis zone, the agar medium was
flooded with an aqueous solution of Congo
Red (0,1% w/v) for 15 minutes. The Congo
Red solution was then poured off, and the
plates were further treated by flooding with
1 M NaCl for 15 minutes. The formation of
a clear zone of hydrolysis indicated cellulose
degradation by microorganism (Lee 2007;
Baharudin et al. 2010).
Cellulase production
The medium used for production of the
cellulase
contained
the
following
components 0.25% yeast extract, 0.5%
K2HPO4, 0.1% NaCl, 0.02% MgSO4.7H2O,
0.06% (NH4)2.SO4 and 2% CMC was used
as carbon source. The pH was adjusted to
initial pH 7.0 by 1M NaOH. These medium
was inoculated with one single colony from
CMC agar plates into 5 ml CMC broth
production medium and reinoculated with 1
ml bacterial suspensions in 25 ml CMC

broth production medium. Two replicates
were used for each bacteria isolates and the
standard strain Escherichia coli BL 21 for
negative control. The inoculated flasks were
incubated at 50oC on rotary shaker at 140
rpm under aerobic condition as stationary
culture for 24 hours (Kim et al. 2009). After
incubation, all of culture broths were
withdrawn and centrifuged at 4000 rpm for
40 min at 4oC and the supernatant served as
the source of crude enzymes. The crude
enzyme solution
was utilized for
determination
of
enzyme
activities
(Kotchoni et al. 2003; Immanuel et al.
2006). Then, the crude enzyme or free cell
supernatant was obtained and cellulase was
assayed. The supernatans were used for
determination of reducing sugars (Samira et
al. 2011).
Enzyme assays
Cellulase activity was assayed using a
modified method described by Wood and
Bhat (1998) with some modifications. The
cellulase activtity was measured by mixing
0,1 mL of the crude enzyme supernatant
incubated with 0,1 ml of 1% (w/v) CMC in
0,01M sodium phosphate buffer solution pH
7.0 at 50oC for 60 minutes. The reaction was
terminated by adding 1 ml DNS reagent. The
mixture was boiled for 10 minutes and
cooled in ice, then its optical density at 546
nm was determined (Samira et al. 2011).
The cellulase activity was measured by
using a calibrationed curve for glucose. One
unit of cellulase was defined as the amount
of enzyme that released 1 µmol of glucose
per minutes. The resulted reducing sugars
were determined according to DNS method
(Miller 1959).
Strain identification
Bacteria strain molecular identification
used pure culture DNA sequencing method
on 16S rRNA gene (Frothingham et al.
1991). DNA templates were obtained from
pure culture used by steril tooth stick and
inserted into 100 µl ddH2O. 16S rRNA gene
amplified used 63f primer (5’-CAG GCC
TAA CAC ATG CAA GTC-3’) and 1387r
primer (5’-GGG CGG WGT GTA CAA
GGC-3’) (Marchesi et al. 1998). Polymerase
chain reaction (PCR) cycle used C1000TM
Thermal Cycle (Bio Rad) with these
condition: pre-denaturation at 96oC (5),
denaturation at 96oC (30), annealing at
50oC (30), elongation at 72oC (130) and
post-elongation for 7 min. Denaturation,
annealing and elongation passed off for 30
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cycle, while pre-denaturation and postelongation passed off for one cycle. PCR
reaction was done with these composition:
10 µl GoTaq Green Mastermix (Promega,
USA), 7 µl nuclease free water, 1 µl 63f
primer (10 pmol/µl), 1 µl 1387 primer (10
pmol/µl), and 1 µl template. PCR result
purified by EXOSAP-IT® PCR Purification
Kit (USB Corporation, Ohio, USA).
Sequencing cycle used BigDye® XTerminator Cycle Sequencing Kit (Applied
Biosystem, Foster City, California) and
amplified by Bio Rad C1000TM Thermal at
95oC (5), denaturation at 95oC (30),
annealing at 55oC (30), elongation at 60oC
(130) and post-elongation for 5 min.
Denaturation, annealing and elongation
passed off for 25 cycles, while predenaturation and post-elongation passed of
for one cycle. Result from sequencing cycle
purified with Big Dye® X-Terminator
Purification Kit
(Applied
Biosystem,
Foster City, California) and sequenced by
ABI PRISMTM 3130 Genetic analyzer
(Applied
Biosystem,
Foster
City,
California). Then, the 16S rRNA sequences
of the isolates obtained were compared
directly with sequences in the NCBI
(National
Centre for
Biotechnology
Information) database using Basic Local
Alignment
Search
Tool
(BLAST)
(http://ncbi.nlm.gov/BLAST). Then, the best
isolates were stained with Gram staining
procedure.
RESULTS AND DISCUSSION
Isolation and selection of cellulaseproducing thermophilic bacteria
The results indicated after 24 h
incubation, from 19 bacterial isolates grew
on CMC agar plates. There were four
isolates from palm kernel meal (PKM), ten
isolates from empty fruit bunch compost
(EFBC), and five isolates from soil of oil
palm. In this research, screening of bacteria
was conducted by using the CR method as a
preliminary study for identifying cellulase
producers. Eleven isolates exibited clearing
zone around their colonies on CMC agar
plates (Table 1).
Table 1 Eleven isolates showed clear zone
by Congo Red
Source
Isolate Code
Soil
CK5, TH, TC5, TF2
EFBC
CK3, CK4, EM4
PKM
OP, P4, NP, CK1

In this research, clear zone around the
bacteria colonies after staining with CR
indicated the hydrolisis of CMC as a result
of cellulases production (Fig 1). Although
the CR method was sensitive enough for
primary isolation and screening of
cellulolytic bacteria, but the clear zone width
was not implied the amount of cellulase
activity. In 2000, a report showed that
among 77 thermotolerant bacterial isolates
grown on CMC agar, an isolate CMU4.4
exhibited the highest enzyme activity
whereas its clear zone was smaller than
others isolates (Krootdilaganandh 2000).

Bacteria
colony

Clear
zone

Fig 1 Screening for cellulase-producing
thermophilic bacteria by clear zone
detection using Congo Red.
Decolorization or clear zone made by
bacteria showed secretion of cellulase
enzymes by bacteria in order to degrade
cellulose structure of CMC. Some
polysaccaharides interact non-covalently
with dyes. This interaction can be used to
make the polysaccaride visible in plate
medium. The plates are flooded with a dye
solution and incubated to allow the
interaction to take place. After washing off
of unbound dye, stained areas will be visible
that contain the intact polysaccharide,
whereas unstained spots will appear where
the polysaccharide has been degraded
(Ruijssenaars & Hartmans 2001) and this
phenomenon has been reported by Ibrahim
and El-diwany (2007), Samira et al. (2011),
Acharya and Chaudhary (2011), Bakar et al.
(2010), El-Sersy et al. (2010), and
Baharudin et al. (2010). Moreover, it was
reported by Sirisena and Manamendra
(1995) that Geobacillus strain was capable
in hydrolising cellulose. As detected from
CR method, the isolated strain had endo-β1,4-glucanase activity. This was one of the
enzymes required for the conversion of
cellulose to glucose. According to Ariffin et
al. (2008) cellulolytic bacteria, Bacillus
pumilus EB3 was successfully isolated from
EFB. The strain produced clear zone around
the colony after staining with CR on CMC
agar plates.
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Enzyme assay
The results of enzyme assay indicated
eleven isolates as the isolates were able to
decompose CMC detection by CR method
showed enzyme activity. The measurement
of enzyme activity in this research also
support Wood and Bhat (1998) and Miller
(1959). The mentioned researchers, by using
colorimetery method with DNS reagent and
drawing calibration curve by D-glucose,
investigated cellulolytic isolates activities.
The enzymatic hydrolysis produced
sugar with reducing ends that reacted to 3,5dinitrosalicylic acid showing the high
absorbance in 546 nm (Samira et al. 2011).
In order to show cellulase activity, it is
necessary to measure the glucose
concentration which released by enzymatic
hydrolysis. Therefore, calibration glucose
curve was drawn. Then, enzymatic activity
based on U mL-1 stated.
Figure 2 illustrated cellulase activity in
the broth for cellulase production in 100 ml
Erlenmeyer flasks. Cellulase activity for
CK1, EM4 and CK3 was obtained after 24 h
incubation with 13.56, 12.50 and 10.49 U
mL-1, as three the best isolates, respectively.

Fig 2 Cellulase activity results using DNS
method after 24 h incubation time in
cellulase production medium at 50oC
on rotary shaker 140 rpm ( : 546
nm).
These results are in agreement with those
of Narashima et al. (2006) and Niranjane et
al. (2007) who found that carboxymethyl
cellulose was the best carbon source
followed by cellulose for cellulase
production. A higher production of cellulase
when CMC served as substrate may be as a
result of induction of the enzyme since
cellulose is known to be a universal inducer
of cellulase synthesis. Then, This results is
higher than results from Meryandini et al.
(2009). Meryandini et al. (2009) isolated 4
isolates of C4-4, C5-1, C5-3 and C11-1.
The researchers observations showed that
maximum cellulase activity were 3.17, 1.50,

0.17, and 3.33 U mL-1 at 50oC (pH 7.0) after
24h incubation on CMC production medium.
A duplicate of experiment was
performed to verify the optimization result
in order to validate the developed optimized
medium. To be able to control whether CMC
production media works or not, positive
control with commercial cellulase (0,2%
concentration), blank sample and negative
control with E.coli BL 21 known as
cellulase-negative.
The colorimetric assay used for the
determination of cellulase activity was the
dinitrosalicylic acid (DNS) method (Miller
1959). The amount of enzyme production
stage of the organism largely depends upon
the type of microbial strains and their
genetic make up and on cultural and
enviromental conditions employed during
growth of the organism (Bajaj et al. 2009).
Many microorganisms are capable of
degrading and utilizing cellulose and
hemicellulose as carbon and energy sources
(Baharudin et al. 2010).
Cellulases yields appear to depend on a
complex relationship involving a variety of
factors like inoculum size, pH value,
temperature, presence of inducers, medium
additive, aeration, growth time, etc
(Immanuel et al. 2006).
Identification of the cellulase-producing
thermophilic bacteria
To identify the experimental strain
exactly according to 16S rRNA sequence
analysis as well as taxonomical studies,
genomic DNA of the strain was used as
template to amplify partial 16S rRNA using
63f primer and 1387r primer. The result
from gel electrophoresis obtained the
expected length of fragment (~1800 bp) was
observed in 1.5% gel electrophoresis (fig 3).
CK1, CK3 and EM4 were identified
based on 16S rRNA sequence analysis.
Phylogenetic analysis of these strains
showed that strain CK1 had higest homology
(98.5%) with Bacillus subtilis strain DZ029
(access code: DQ408587), CK3 showed
95.9% similarity with Bacillus subtilis strain
C1CC 10088 (access code: AY787000) and
EM4 showed 93.3% similarities with
Bacillus subtilis strain CH19 (access code:
HQ651887). Considering its stability under
high temperature (50oC) as well as neutral
condition (pH 7), the isolated strain maybe
useful
for
the industrial purpose.
Thermophilic bacteria strains, B. subtilis
strain CK1, CK3 and EM4 were successfully
4

5

isolated from palm kernel meal and empty
fruit bunch compost of oil palm and all of
them are Gram positive bacteria.
2
1 2
1
a b

Eleven isolates showed clear zone on CMC
supplemented with Congo Red. Based on
DNS method, three isolates showed the
highest cellulase activity and were identified
as Bacillus subtilis.

c

Fig 3 Agarose gel anlysis of PCR amplified
partial 16S rRNA . Line 1 : 1kb DNA
size marker (Promega, USA). Line 2
1800 bp PCR product with a. CK3, b.
EM4 and c. CK1 are three the best
isolate that producing highest
cellulase crude enzyme with DNS
method.
Since most industrial processes are
carried out at high temperature, there is a
clear need for thermophilic enzymes (Haki
& Rakshit 2003). Application of bacteria in
producing cellulase is not widely used
except for some reports, once of them is
Bacillus sp. (Baird et al. 1990; Immanuel et
al. 2006).
Bacillus subtilis species are commonly
found both in soil and water and they have
great
scientific
and
technological
importance. Bacillus subtilis species have
the ability to use various simple and
complex organic compounds so they are
involved in biodegradation of natural or
man-made chemical compounds. Moreover;
the bacterial genus Bacillus is the most
important producer of extracellular enzymes
like cellulases. Thermophilic bacterial
cellulases have been frequently reported
from Bacillus sp. (Hala & Priset 1994;
Mawazda et al. 2000). Obtained data
confirmed the findings reported by Ray et al.
(2007) who mentioned that pH 7 more
suitable for optimization of cellulase
production by Bacillus subtilis. Furthermore,
the cellulolytic enzyme, endoglucanase
obtained from some baceria including
Bacillus hydrolyzed substrate in the pH
range of 4.0 to 9.0, with maximum activity
transpiring at pH 7 (Immanuel et al. 2006).
CONCLUSION
A total of 19 cellulase producing
thermophilic bacteria was successfully
isolated from soil, palm kernel meal and
empty fruit bunch compost of oil palm.

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APPENDIXES

P4

NP

TC5

TF2

TH
OP

CK1

CK5
CK3
CK4

EM4

Appendix 1 Screening for cellulase-production bacteria using agar medium containing 0,5% (w/v) CMC.
Samples were incubated at 50oC for 3 days. The plates were stained with Congo Red and
destained with 1M NaCl solution. Clear zone indicated the hydrolysis of CMC as a result of
cellulases production

9

10

Appendix 2 Glucose standard curve
Concentration (w/v %)

Absorbance

0,025

0,007

0,050

0,117

0,075

0,302

0,100

0,474

0,125

0,633

0,150

0,771

0,175

0,909

Appendix 3 Cellulase activity results using DNS method after 24 h incubation time in cellulase
production medium at 50oC on rotary shaker 140 rpm at  546 nm

y

x (%)

gr/ml

µgr/ml

µmol/ml

µmol/ml/menit
(U/ml)

0,108

4,3338%

0,0433

43338,17

240,77

4,01

0,007

2,7066%

0,0271

27066,22

150,37

2,51

0,748

14,6448%

0,1464

146447,56

813,60

13,56

EM 4

0,677

13,5009%

0,1350

135008,86

750,05

12,50

CK 3

0,542

11,3259%

0,1133

113259,22

629,22

10,49

TH

0,504

10,7137%

0,1071

107137,10

595,21

9,92

P4

0,500

10,6493%

0,1065

106492,67

591,63

9,86

CK 4

0,489

10,4720%

0,1047

104720,48

581,78

9,70

TC 4

0,445

9,7632%

0,0976

97631,71

542,40

9,04

TC 5

0,418

9,3282%

0,0933

93281,78

518,23

8,64

OP

0,399

9,0221%

0,0902

90220,72

501,23

8,35

NP

0,067

3,6733%

0,0367

36732,72

204,07

3,40

TF 2

0,064

3,6249%

0,0362

36249,40

201,39

3,36

Sample
Positive
Control
Negative
Control
CK 1

10

11

Appendix 4 Result of spectrophotometric measurment of absorbance by DNS method ( : 546 nm)

Sample
Positive
control
Negative
control
CK1

EM4

CK3

TH

P4

CK4

TC4

TC5

OP

Part 1

Part 2

Stdev

Average

Average per
Isolate

0,108

1

2

3

1

2

0,109

0,085

0,104

0,115

0,121

0,01

0,107

0,076

0,097

0,122

0,130

0,121

0,02

0,109

0,021

0,040

0,000

0,000

0,000

0,02

0,012

0,000

0,010

0,000

0,000

0,000

0,00

0,002

0,756

0,772

0,788

0,758

0,771

0,01

0,769

0,759

0,763

0,725

0,765

0,771

0,02

0,757

0,719

0,718

0,728

0,755

0,786

0,03

0,741

0,712

0,729

0,705

0,757

0,727

0,02

0,726

0,719

0,691

0,711

0,684

0,701

0,01

0,701

0,699

0,640

0,639

0,702

0,696

0,03

0,675

0,654

0,658

0,652

0,651

0,637

0,01

0,650

0,714

0,723

0,726

0,627

0,623

0,05

0,683

0,530

0,513

0,530

0,601

0,570

0,04

0,549

0,519

0,620

0,540

0,557

0,570

0,04

0,561

0,570

0,530

0,601

0,493

0,513

0,04

0,541

0,479

0,550

0,479

0,545

0,530

0,04

0,517

0,478

0,486

0,538

0,491

0,535

0,03

0,506

0,445

0,517

0,565

0,453

0,597

0,07

0,515

0,481

0,522

0,477

0,528

0,503

0,02

0,502

0,481

0,529

0,501

0,416

0,533

0,05

0,492

0,415

0,431

0,487

0,522

0,519

0,05

0,475

0,407

0,401

0,452

0,486

0,471

0,04

0,443

0,535

0,605

0,576

0,475

0,491

0,06

0,536

0,550

0,563

0,605

0,518

0,483

0,05

0,544

0,555

0,479

0,480

0,506

0,506

0,03

0,505

0,529

0,495

0,470

0,519

0,466

0,03

0,496

0,515

0,436

0,479

0,466

0,481

0,03

0,475

0,527

0,424

0,451

0,481

0,519

0,04

0,480

0,502

0,453

0,374

0,668

0,336

0,13

0,467

0,482

0,452

0,365

0,496

0,441

0,05

0,447

0,487

0,422

0,365

0,419

0,408

0,04

0,420

0,490

0,387

0,381

0,409

0,571

0,08

0,448

0,385

0,384

0,405

0,466

0,497

0,05

0,427

0,386

0,341

0,342

0,46

0,529

0,08

0,412

0,34

0,379

0,34

0,414

0,496

0,06

0,394

0,372

0,413

0,393

0,516

0,506

0,07

0,440

0,317

0,293

0,313

0,528

0,578

0,14

0,406

0,289

0,307

0,326

0,490

0,564

0,12

0,395

0,007
0,748

0,677

0,542

0,504

0,500

0,489

0,445

0,418

0,399
11

12

NP

TF2

0,224

0,350

0,344

0,578

0,509

0,14

0,401

0,220

0,335

0,337

0,548

0,521

0,14

0,392

0,023

0,087

0,097

0,040

0,03

0,03

0,055

0,033

0,089

0,129

0,04

0,074

0,04

0,073

0,028

0,113

0,123

0,049

0,033

0,05

0,069

0,047

0,113

0,123

0,038

0,040

0,04

0,072

0,074

0,063

0,088

0,043

0,069

0,02

0,067

0,072

0,076

0,064

0,031

0,105

0,03

0,070

0,072

0,075

0,036

0,096

0,039

0,03

0,064

0,051

0,044

0,035

0,105

0,046

0,03

0,056

0,067

0,064

12