Download isolation of cellulase producing thermophilic bacterial from hot spring

International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009
Volume- 4, Issue-3, Jul.-2016
ISOLATION OF CELLULASE PRODUCING THERMOPHILIC
BACTERIAL FROM HOT SPRING
1
BALAKRISHNAN KUNASUNDARI, 2YI PENG TEOH, 3IBRAHIM ROSHITA
1,2
Faculty of Engineering Technology, Universiti Malaysia Perlis (UniMAP), P.O Box 77, D/A Pejabat Pos Besar Kangar,
Perlis, 01000, Malaysia..
E-mail: [email protected], [email protected], [email protected]
Abstract— This study focuses on the isolation of thermophiliccellulase producing bacteria from hot spring, HutanLipur
Kuala Woh, Tapah, Perak, Malaysia. The cellulose degrading bacteria were isolated based on their halo zone formation on
carboxymethylcelluloseagar medium stained with congo red indicator.Total 4 isolates were obtained from the primary
screening technique. Only one isolate showed the highest celullase activity with the maximum carboxymethycellulose
hydrolysis capacities in the range of 1.5 to 1.8 cm. From the 16S rDNA analysis, the isolate was identified as
MeiothermusSilvanus strain UniMAP06.
Keywords— Cellulase, Cellulose, Isolation, Thermophile, Meiothermus.
of
anaerobic
cellulolytic
bacteria
are
Bacteroidescellulosolvensand
Clostridium
thermocellum (Sun and Cheng, 2002; Sukumaran et
al, 2005; Kuhad et al., 2010; Saranraj et al., 2012).
Most emphasis has been placed on the use of fungi
because of their capability to produce copious
amounts of cellulolytic enzymes and often less
complex than bacterial cellulase allowing for easy
extraction and purification. It can therefore be more
readily cloned and produced via recombination in a
rapidly growing bacterial host. However, the isolation
and characterization of novel cellulase from bacteria
are now becoming widely exploited. There are
several reasons for these shifts (Miranda et al., 2009).
The higher growth rate of bacteria together with
increased function and synergy of multi enzyme
complexes of bacterial cellulases. Besides, abilities of
bacteria to inhabit various environmental conditions
resulting in production of cellulolytic enzymes that
able to tolerate harsh conditions with improved
bioconversion rate. Researchers are now focusing on
the efficient utilization of these enzymes for
production of various value-added products.
The present study aimed to isolate and screen for
potential thermophilic cellulolytic bacteria for hot
spring.
I. INTRODUCTION
Malaysia is a country located at Southeast Asia,
which has tropical climate and is blessed with
agriculture resources such as oil palm and rubber.
Over the past four decades, agriculture industry has
significantly contributed to the economy (Goh et al,
2010).Lignocellulosic
waste
materials
from
agricultural and forest residues represent the largest
renewable reservoir in Malaysia with potential
generation of more than 104.55 million tonne of
biomass waste annually (Shahabuddin et al, 2012). In
accordance to the concept of waste-to-wealth which
has been widely popular, lignocellulosic waste
materials are potentially viable feedstock for
conversion into valuable chemical compounds which
can lead to the sustainability and advancement of
energy and chemicals industries (Goh et al, 2010).
Lignocellulosic biomass generally consists of
cellulose which is an ample natural biopolymer on
earth and the most dominating agricultural waste. It is
a branched glucose polymer made of hundreds to
thousands of β (1→4) linked D-glucose units
(Sharadha
et
al.,
2013).Lignocellulose
is,
unfortunately, a heterogeneous and recalcitrant
material
which
is
highly
resistant
to
depolymerization. In order to exploit lignocellulosic
materials for production of renewable products, it
must first be broken down into constituent
compounds, such as sugars, that can be more easily
converted using chemical and biological processes
(Kumar et al, 2008; Wackett, 2008; Matt et al, 2012).
A variety of microorganisms take part in cellulose
hydrolysis with an aid of a multienzyme system.
Among the best-characterized cellulase from fungi
systems are as follows: Trichodermakoningii,
Trichodermareesei, Fusariumsolani, Aspergillusniger
and Penicillumfuniculosum. Some of the aerobic
cellulolytic bacteria which are having bestcharacterized cellulase systems are Cellulomonasspp.,
Cellvibrio spp. and Thermomonospora spp. Examples
II. DETAILS EXPERIMENTAL
2.1.Enrichment, Isolation and Screening
Cellulases Producing Thermophilic Bacteria
of
The water samples were collected aerobically in
sterile Schott bottle (500 mL) from the hot spring,
HutanLipur Kuala Woh, Tapah, Perak, Malaysia and
stored at -20ºC until used.The collected water
samples were serially diluted in 10ml of sterile
distilled water. 100 µL of each dilution and water
samples (without dilution) were spread on mineral
agar media with the following composition [KH2PO4,
1.36 g/L; (NH4)2SO4, 1.0 g/L; MgSO4.7H2O, 0.2 g/L;
Isolation of Cellulase Producing Thermophilic Bacterial From Hot Spring
155
International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009
FeSO4, 10.0 mg/L; NaCl, 2.0 g/L; yeast extract, 1.0
g/L supplemented with 0.3% carboxymethylcellulose
(CMC)] (Patel et al., 2006) and then incubated at
55ºC for 72 h.After growth, colonies were analyzed
for morphological differences and repeated streaking
was done at 55ºC for 72 h on respective agar plates
until pure colonieswas obtained. The plateswere
flooded with 1% Congo red and 1 M NaCl in order to
observe for cellulolytic activity of isolated strain.
Theformation of a clear zone of hydrolysis
indicatedthe cellulose degradation. The ratio of the
clearzone diameter to colony diameter was
measuredin order to select for the highest cellulase
producer (Ariffin et al., 2006). The largest ratio was
assumed to contain the highest activity.
Volume- 4, Issue-3, Jul.-2016
identification as it showed highest cellulolytic activity
compared to all other isolates.
Analysis of 16S rDNAgene sequence
The genomic DNA of strain 191 was extracted
following the manufacturer’s instruction of Wizard
Genomic DNA Purification Kit (Promega, Madison,
WI, USA) and used as the template for PCR
amplification of 16S rDNA. A partial 16S rDNA
region was amplified using the two primers F27 (50AGAGTTTGATCCTGGCTCAG-30) and 1492R
(50-GGTTACCTTGTTACGACTT-30). The PCR
amplification was performed for 30 cycles using T100 Thermal Cycler (Bio-Rad Laboratories Inc.,
USA). Each cycle consisted of denaturation at 94° C
for 1 min, annealing at 64°C for 30 s and extension at
72°C for 1 min. The amplified DNA fragments were
sequenced at First Base Sdn. Bhd. (Malaysia) and
analyzed by a similarity search in the GenBank
database using the BLAST.
3.2 Genotypic Identification of the Isolate
Analysis of 16S rDNA gene sequence revealed that
this isolate showed 99% identity to those of several
Meiothermussilvanus strains. This isolate was
designated as UniMAP06. The species of the genus
Meiothermus comprises strictly aerobic, nonhalophilic and moderately thermophilic heterotrophs
with
growth
temperature
ranges
between
approximately 35 and 68°C. Up to date, there are five
species of bacteria belonging to the genus
Meiothermushave been described which are
Meiothermusruber,
Meiothermuschliarophilus,
Meiothermussilvanus, Meiothermuscerbereus, and
MeiothermustaiwanensisThese bacteria are generally
red-pigmented, except for the yellow-pigmented
strains of M. timidus and M. chliarophilus (Luciana et
al., 2010; Mori et al., 2012). Detailed microscopic
observations revealed that the strain UniMAP06
formed Gram negative with rod-shaped cells of
variable length. Besides, when the strain was grown
on broth media, large clump of cells formed which
indicate
the
features
of
the
genus
Meiothermus(Sikorski et al., 2010). This strain
showed highest cell density at 55°C in broth media
even though growth was observed from the range of
40 to 58°C. So far to our knowledge, there is no
information available on cellulase production from
M. silvanus.
III. RESULTS AND DISCUSSION
3.1 Screening for cellulolytic activity
The present study focused on the isolation of
thermophilic cellulolytic bacteria considering the fact
that they are ideal biocatalyst for modern
biotechnology applications in various industries due
to thermostability and better yields under extreme
operational conditions (Acharya and Chaudhary,
2012).
Fourisolates which were morphologically distinct
were obtained and maintained as pure culture in CMC
agar plates. One of the isolates were determined to be
red-pigmented as shown in Figure 1(A). The other
three isolates appeared as opaque and pale yellow
colonies and revealed to be morphologically different
under microscopic observation. The clearing zone
size and colony diameter of these isolates were
measured when incubated aerobically at 55°C. The
results showed that the maximum clearing zone of 2.1
cm [Figure 1(B)] was detected with the redpigmented isolate. Other isolates produced clearing
zone of 0.5 to 1.1 cm demonstrating all the isolates
exhibited cellulolytic activity. However, only the redpigmented isolate was selected for further genotypic
CONCLUSIONS
A new source of thermophilic cellulolytic bacterium
has been identified from hot spring which is
Isolation of Cellulase Producing Thermophilic Bacterial From Hot Spring
156
International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009
designated as M. silvanus strain UniMAP06.
However, further research on the optimization of the
cellulase activity is required to prove the efficiency of
this bacterium in the bioconversion of lignocellulosic
wastes.
[6]
ACKNOWLEDGMENTS
[7]
This study was supported by Fundamental Research
Grant Scheme (FRGS), Grant No: 900300530[FRGS/1/2015/SG05/UNIMAP/02/4] entitled:
Identification of New Cellulolytic Bacterial Strains
from Tropical Mangrove Soil.
[8]
[9]
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