ISOLATION OF CELLULOSE DEGRADING BACTERIA FROM THE GUT OF THE TERMITE COPTOTERMES GESTROI

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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 17, No. (4) : 2015 : 931-936
© Global Science Publications
ISSN-0972-3005
*Corresponding author’s email: ndthang@hus.edu.vn
ISOLATION OF CELLULOSE DEGRADING BACTERIA FROM THE
GUT OF THE TERMITE COPTOTERMES GESTROI
NGUYEN THI PHUONG1, NGUYEN THI LE NA1 AND NGUYEN DINH THANG1,*
1Department of Biochemistry and Plant Physiology, Faculty of Biology,
Vietnam National University, Hanoi, Vietnam
(Received 6 July, 2015; accepted 30 August, 2015)
Key words : Acinetobacter sp., Cellulose-degrading bacteria, Coptotermes gestroi, Hydrolysis capacity,
Termite gut symbionts.
Astract–Cellulose holds the reputation of the most abundant renewable biomass on the earth. Ethanol
produced from cellulosic biomass is one of the most promising renewable fuel source replacing fossil fuels
with the advantage of zero net contribution of carbon dioxide to the atmosphere. Hence, the bioconversion
of cellulose into ethanol plays an important role for sustainable development. Termites, especially
Coptotermes gestroi - “Asian subterranean termite”, considered as the most successful wood-degrading
species on the earth, play a significant role in the degradation of cellulosic biomass and in global carbon
cycling. This is result of synergistic activity of enzymes secreted by the termites and/or their gut symbiotic
protists. In this study, we succeeded in isolating a cellulolytic bacteria strain from the gut of the termite
Coptotermes gestroi. The results of morphological and chemical assays for identifying of bacterial isolate were
showed that bacterial isolate was Gram negative bacteria, fermenter of carbohydrates (glucose, maltose and
mannitol) and positive capacity of catalase, but without ability of starch hydrolysis. The average hydrolysis
capacity (HC) value of the bacterial isolate estimated through the screening on Congo red agar media was
4.66. Cellulase enzyme activity of the bacterial isolate on the basis of filter unit was shown as 1.61 FPU/mL.
The phylogenetic analysis revealed that bacterial isolate was Acinetobacter sp. Our results suggested that
enzyme secreted by this bacteria strain could be a potential source of cellulase enzyme for the development
of hydrolysis cellulase for cellulosic biomass and for a further aid of bioconversion of ethanol.
INTRODUCTION
Cellulose is perhaps the most abundant renewable
organic material on the Earth with the annual
biomass production estimated over 1.5 × 10^11 tons,
and is considered as almost inexhaustible source of
raw material for different products. Cellulose is
synthesized in variable living organisms, from wide
distribution in higher plants to less range in algae,
fungi, bacteria, invertebrates, and even several
marine animals like tunicates (Habibi et al., 2010).
Cellulases are a group of enzymes catalyzing the
hydrolysis of cellulose. They are produced by Fungi,
Bacteria, and Protozoa, also by other organisms like
plants and animals (Lee et al., 2001). In nature,
complete cellulose hydrolysis that breakdowns of
cellulose and subsequent biological conversion to
glucose as end product is facilitated by a synergistic
activity of three major types of cellulases:
endoglucanases (EC 3.2.1.4), exoglucanases,
including cellobiohydrolases (CBHs) (EC 3.2.1.91),
and β-glucosidase (BG) (EC 3.2.1.21). All of these
enzymes hydrolyze the β-1,4 linkage in cellulose
chains.
Cellulases play an extremely important role with
abundant industrial applications like textile
industry, in detergents, pulp and paper industry,
enhancing digestibility of animal feeds, and food
industry (Kumar et al., 2008). Termites are small to
medium-sized, dull white to light and dark brown
color, soft-bodied insects, belonging to the insect
order Isoptera (Hatami et al., 2008). In these castes,
worker termites directly converse raw materials,
cellulose mainly, in their guts. Unlike higher
termites, which lack the symbiotic community,
lower termites need gut symbionts for their
cellulosic decomposition. As a result of the gradient
in oxygen source, there is a significant variation in

932 PHUONG ET AL.
microbial community structure between different
parts of the gut. Aerobic, facultative aerobic and
anaerobic microorganisms can colonize in the gut
(Ohkuma, 2003; Eggleton, 2006). Several bacteria
have been isolated from the pure culture from the
termite gut (Wenzel et al., 2002; Tokuda et al., 2005;
Hatami et al., 2008; Lee et al., 2010), in which, some
bacteria were reported as cellulolytic organisms or
at least participating on the cellulose decomposition.
This reveals the potential of investigating cellulose
degrading bacteria from termite guts (Varma et al.,
1994).
Agriculture still occupies a large fraction of the
economic development in Vietnam, with a
significant amount of residues from agriculture,
forestry. “Isolation of cellulose degrading bacteria
from the gut of the termite Coptotermes gestroi” was
conducted with the aid of investigating cellulose
degrading bacteria for a further objective of
bioconversion of cellulosic biomass into bioethanol.
MATERIALS AND METHODS
Sample collection
Coptotermes gestroi termites were obtained directly,
and then transported to laboratory in sterile Petri
dish. Nine healthy living organisms were sterilized
by submersion in 70% ethanol for 3 min and then
rinsed in sterile water twice prior to dissection. The
collected gut transferred to 1.5-mL microcentrifuge
tubes containing 0.5-mL of distilled water. Tubes
were squeezed several times using a plastic pestle. 1
mL of PBS was added. The sample was centrifuged
at 4000 rpm at 4oC for 10 seconds to separate the
microbia from the gut wall. The supernatant was
collected, and it was the microbia in the termite gut
that we need (Hu et al., 2014).
Isolation of cellulose-degrading bacteria
The supernatant obtained was inoculated in a sterile
basalt salt media containing filter paper as sole
source of carbon for the isolation of cellulolytic
bacteria. These cultures were inoculated for 7 days
in a shaker incubator at 30oC at 100 rpm. Bacterial
colonies having the ability of utilizing cellulose as
the sole source of carbon were isolated on the
cellulose agar media and inoculated in 30oC for 2
days (The negative control was the culture without
containing bacterial colonies) (Gupta et al., 2012).
Screening of cellulose-degrading bacteria
To rapidly confirm the cellulolytic capability,
bacterial isolates were streaked on the cellulose
Congo red agar media in 30oC for 42 hours. After 42
hours, estimate the ability of cellulose hydrolysis
through the discoloration of Congo red around the
colony. This task was conducted through the
calculation of the ratio of diameter of clearing zone
and colony. Hydrolysis capacity (HC) = diameter of
clearing zone – diameter of colony (mm) (Gupta et
al., 2012).
Enzyme production
The selected cellulose-degrading isolates were
inoculated in an enzyme production media at 300C
for 5 days at 150 rpm. The culture was collected to
centrifuge at 5000 rpm for 15 min at 40C. The
supernatant was obtained and stored at 40C as crude
cellulase enzyme for further assays (Gupta et al.,
2012).
Identification of microorganisms
The colonies were sampled for Gram-staining assay,
Oxidation-Fermentation (OF) test, Catalase activity
test, Starch hydrolysis test, DNS test for
determination of producing sugars, and Molecular
and phylogenetic analysis (Barrow et al., 1993).
RESULTS AND DISCUSSION
Isolation of cellulose-degrading bacteria
Cellulose degrading bacteria in the gut of termites
were enriched and isolated in liquid basalt salt
medium containing filter paper used as the sole
source of carbohydrates. Cultural medium indicated
the growth of bacteria with the medium turned
cloudy and maceration of filter paper after 7 days at
at 300C. Colonies of cellulose degrading bacteria was
then streaked on CMC agar medium and showed in
Fig. 1A. The growth rate of colonies on CMC agar
medium was quite slow. The reason might be the
lack of essential nutrients for promoting the growth
of bacterial isolate and interpreted through a faster
growth rate after 5 days at 300C of culturing in starch
agar medium (Fig. 1B).
Screening of cellulose-degrading bacteria
There was one positive bacterial isolate on screening
media (cellulose - Congo red agar media; Congo red
was used as rapid and sensitive indicator for the
appearance of extracellular cellulolytic enzymes
secreted by cellulose degrading bacterial strains in
the media, due to Congo red strongly interacts with
polysaccharides containing continuous β-1,4-linked

Isolation of Cellulose Degrading Bacteria from the Gut of the Termite Coptotermes gestroi 933
D-glucopyranosyl units and β-1,3-D-glucans and
some hemicellulosic galactoglucomannans possibly
(Teather et al., 1982) during aerobic incubation after
42 hours. The positive bacterial isolate generated
clear halo zone around microbial colonies (Fig. 1C).
The colonies forming clear halo zones were utilized
for the determination of the cellulose hydrolysis
capacity (HC) from the bacterial isolate. The
maximum clearing halo zone in the plate after 42
hours in 300C was 20 mm and the average value was
17.75 mm (Table 1). The result also showed that the
HC value ranged largely from 3.2 to 7.2 (Table 1).
The average HC value was 4.66 after 42 hours
inoculation (Table 1).
characteristic played an important role in the
identification of bacterial isolate (Barrow et al., 1993).
Bacterial isolate was able to oxidize carbohydrate
Oxidation-Fermentation (OF)
The of test is one of the most significant tests in the
early stages needed to identify bacteria. Most genera
are composed of bacteria that are either oxidizers or
fermenters of carbohydrates. The results of OF test
were showed in the Figure 3A-C. The bacterial
isolate was fermenter of three types of
carbohydrates (glucose, maltose and mannitol),
which was visibly indicated by yellow color in both
open (lane 1) and sealed (lane 2) tubes by melted
soft paraffin. The speed of carbohydrate hydrolysis
was really high that expressed firstly through the
amount of gas produced in tubes (Fig. 3A-B). This
gas was CO2 generated during the anaerobic
fermentation, associated with the final production of
ethanol. The hydrolysis capacity of carbohydrates of
bacterial isolate after 16 hours of inoculation was
then presented in Figure 3C also indicating that
bacterial isolate can attack on glucose, maltose and
mannitol by fermentation at high speeds. After 16
Table 1. Maximum clearing zone and hydrolytic (HC) value of cellulose degrading bacteria on cellulose Cong red agar
media (HC = clear zone diameter – colony diameter).
Colony Clear zone Colony Hydrolytic
diameter (mm) diameter (mm) capacity
1 18 2.5 7.2
2 17 4 4.25
3 16 5 3.2
420 5 4
Average 17.75 4.66
Fig. 1. Bacterial colonies on Petri plates. A- Bacterial
colonies on CMC agar medium after 5 days of
inoculation at 300C; B- Bacterial colonies on starch
agar medium after 5 days of inoculation at 300C;
C- Zone of clearance on cellulose Congo red agar
media for after 42 hours of inoculation.
Microbial taxonomy
Bacterial isolate was gram negative bacteria
Bacterial colonies on CMC agar medium were
smooth, entire, convex, glistening, and white or
lightly yellow color and circular with diameter of
around 0.5 - 1 mm at 300C after 2-3 days of
inoculation (Fig. 2A). Gram staining of bacterial
isolate was then conducted to clarify whether it was
Gram positive or Gram negative. The result showed
that it was Gram negative bacteria (Fig. 2B). This
Fig. 2. Bacterial isolate morphology. A- Bacterial colonies
isolated from the gut of the termite Coptotermes
gestroi; B- Gram staining of cellulolytic bacterial
isolate (red color) and Gram positive - Bacillus
(purple color) used as control of reaction.

934 PHUONG ET AL.
hours of incubation, the hydrolysis of glucose was
nearly 60%. The speed of fermentation of mannitol
was higher than that of glucose up to around 20%.
Among the hydrolysis of three these carbohydrates,
the peak of hydrolysis capacity reached to over 80%
of maltose fermentation. The total hydrolysis of
carbohydrates occurred only after 22 hours of
incubation. The OF test showed that bacterial isolate
isolated from the gut of the termite Coptotermes
gestroi was a strong fermenter of carbohydrates.
Bacterial isolate had catalase activity
Catalase activity assay was used as a significant test
for classification of bacteria. The interaction between
catalase and peroxide (H2O2) generates the presence
of H2O and O2. H2O2 is a potent oxidizing agent that
might be harmful to cell. Hence, any cell that uses O2
or can live in the presence of O2 must have a way to
fight against the effect of H2O2. One effective way is
the secretion of catalase. The result revealed that
bacterial isolate was positive for catalase activity
with the evaporation of gas after 5 min of reaction
with peroxidase (Fig. 3D). The result obtained was
consistent with the fact that cultural condition of
bacterial isolate was also aerobic.
Bacterial isolate was not able to hydrolyze of
starch
Starch hydrolysis was usually used for taxonomy of
bacteria with the determination extracellular starch
hydrolysis, which was strongly dependent on the
production and secretion of some hydrolytic
enzyme like amylase. It was showed that the
bacterial isolate was negative with the hydrolysis of
starch or amylase enzyme secretion (data not
shown).
Molecular and phylogenetic analysis
Isolated bacterial strain in this study, named C133,
was then analysed phylogenetically of 16S rDNA
sequence with the result presented in the
supplemental information. The phylogenetic tree
was designed to show the relationship between
strain C133 and other related taxa through ClustalX
1.83 software. In the comparision with other species
in gene bank NCBI, bacterial strain isolated had the
Fig. 3. The of test of carbohydrates (glucose, maltose and mannitol) in open tubes (lane 1) and closed tubes
(lane 2) after 16 hours (A) and 22 hours (B) of inoculation at 300C; Speed of hydrolysis of carbogydrates
(C) and Catalase activity after 2 days of inoculation (D).

Isolation of Cellulose Degrading Bacteria from the Gut of the Termite Coptotermes gestroi 935
similarity in 16S rDNA with 5 bacterial strains in
genus Acinetobacter:
Acinetobacter baumannii ATCC 19606(T), 99.65%
(1414/1419 bp)
Acinetobacter venetianus RAG-1(T), 97.96% (1390/
1419 bp)
Acinetobacter nosocomialis NIPH 2119(T), 97.82%
(1388/1419 bp)
Acinetobacter junii CIP 64.5(T), 97.82% (1388/1419
bp)
Acinetobacter gerneri DSM 14967(T), 97.46% (1383/
1419 bp)
Cellulase enzyme activity
The standard graph was plotted with glucose
concentration on x-axis and OD value on y-axis (Fig.
4). Standard curve equation: y = 1.827x + 0.012; R2 =
0.993. The cellulase enzyme activity on filter paper
was found to be 1.61 FPU/mL after 3 days of
cultivation in enzyme-producing liquid media at
300C. In the comparision with the commercial
enzyme preparation, Cellulase 2000L, with enzyme
activity of 10 FPU/mL, cellulase produced from
bacteria isolated from the gut of the termite
Coptotermes gestroi, revealed the lower hydrolysis of
filter paper (Kabel et al., 2006). The reason might due
to cellulase obtained from bacterial isolate still not
be optimized the cultural conditions like
temperature, pH, substrates, etc. These factors might
influence strongly to the efficiency of enzyme
production by microbia.
CONCLUSION
This study was conducted to investigate the
cellulose degrading bacteria from the gut of the
termite Coptotermes gestroi. We succeeded in
isolating a cellulolytic bacteria strain from the gut of
the termite Coptotermes gestroi with the average
hydrolysis capacity (HC) value estimated through
the screening on Congo red agar media was 4.66
after 42 hours inoculation. This HC value of
bacterial isolate was quite similar to the result of
CDB1, CDB2, CDB8 and CDB9 cellulolytic bacterial
isolates also isolated from the gut of the termite in
the previous reported (Gupta et al. 2012), which had
the HC values in the range from 4.32 to 5.49 after 6
hours later of inoculation (48 hours). The cellulose
decomposing bacteria isolated from the gut of
termite Coptotermes gestroi showed stronger
cellulolytic capacity than cellulolytic aerobic
bacterial isolates investigated from soil samples
collected from forest and farming soils conducted in
the previous study (Hatami et al., 2008) with the
mean ratio of clear zone diamter to colony were 1.6
and 2.1 for forest and farming soil, respectively.
We also revealed that bacterial isolate was Gram
negative bacteria, fermenter of carbohydrates
(glucose, maltose and mannitol) and positive
capacity of catalase, but without ability of starch
hydrolysis. Results of morphological and chemical
tests of bacterial strain was consistent with several
general characteristics of Acinetobacter genus, which
was known clearly as aerobic, coccoid, Gram
negative, catalase-positive and oxidase-negative.
Among species of the genus Acinetobacter, the strain
C133 showed the closest sequence identity (99.65%)
with Acinetobacter baumannii, but it has never been
reported cellulolytic capacity. It was similar with
four other strains, Acinetobacter venetianus (97.96%),
Acinetobacter nosocomialis (97.82%) and Acinetobacter
gerneri (97.46%). Only Acinetobacter junii (97.82%)
was documented the ability of cellulose hydrolysis.
Fig. 4. Glucose standard graph.
Fig. 5. Phylogenetic tree, showing the relationship
between bacterial strain C133 with other related
taxa.

936 PHUONG ET AL.
On the other hand, all species of Acinetobacter genus
are closed related with similarity values for the type
strains of the species in range of 96.1 and 97.8%
(Rainey et al., 1994). Due to still lack of overall
genome relatedness, chemotaxonomic data, the
bacterial strain C133 is identified as Acinetobacter sp.
Cellulase enzyme activity of bacterial isolate on
the basis of filter unit was shown as 1.61 FPU/mL.
However, cellulolytic enzyme obtained from
bacterial isolate showed higher hydrolytic efficiency
on filter paper than that of the mixed culture of
Trichoderma reesei and Aspergillus phoenicis (1.54 FPU/
mL) investigated by Wen et al. (2005). Also the
enzyme activity was evaluated as higher value in the
comparision with cellulase production by
Penicillium echinulatum (1.5 FPU/mL), reported by
Sehnem et al., (2006). The comparision with cellulose
hydrolysis capacity by digestive enzymes of
Reticulitermes speratus, a native termite from Korea,
in the study conducted by Lee et al. (2010), only 8.8
× 10-2 FPU/mL, and then expressed considerably
higher value of enzymatic hydrolysis of cellulose.
Taken together, our results suggested that
enzyme secreted by bacteria isolated from the gut of
the termite Coptotermes gestroi (C133) was able to
hydrolyse cellulose into sugars therefore it could be
a potential source of cellulase enzyme for the
development of hydrolysis cellulase for cellulosic
biomass, for a further aid of bioconversion of
ethanol.
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