Subodh K. Upadhyaya et al.
: Isolation and characterization of cellulolytic bacteria from gut of termite
Isolation and Characterization of Cellulolytic Bacteria from
Gut of Termite
Subodh K. Upadhyaya*, Anuroop Manandhar, Hemanta Mainali, Anaya R. Pokhrel, Anurag Rijal, Barun
Pradhan, Bhabuk Koirala
Abstract-- Nepal is rich in biodiversity with flora and fauna
of various species scattered over different geographical regions.
Termites are found abundantly in Nepal in forests as well as in
household furnitures and wood causing heavy loss. Wood is a
rich source of cellulose which is most abundantly found
biomass. Termites harbor a system of symbionts that can
degrade cellulose in wood, which has been partly degraded in
its foregut by its own enzyme.
Several strains were isolated from the gut of termite in three
different media. Each strain showed significant cellulolytic
activity in congo red assay. Several biochemical tests were
carried out to identify the bacteria. Results showed that they
were similar like Citrobacter, Enterobacter and Cellulomonas.4
Index Terms— Cellulolytic activity, cellulose, termites
I. INTRODUCTION
ERMITES play a great role in terrestrial ecosystem by
T recycling lignocellulosic biomass, which refers to a
mixture of cellulose, hemicellulose and lignin. Termites
are one of the most important soil insects that efficiently
decompose lignocelluloses with the aid of their associated
microbial symbionts to simpler form of sugars, which later
can be fermented to ethanol using yeasts. Termites are said
to dissimilate a significant proportion of cellulose (74-99%)
and hemicellulose (65-87%) components of lignocellulose
they ingest (Ohkuma; 2003). Termites play an important
role in the turnover and mineralization of complex
biopolymers, such as wood and other cellulose and
hemicelluloses containing materials (Wenzel et al; 2002).
Lignocellusoe is the most predominant component of the
woody and dead plant materials, as well as it is the most
abundant biomass on earth, especially in terrestrial
ecosystems. In contrast to cellulose, lignin degradation does
not appear to be important in the gut of wood feeding
termites (Ohkuma; 2003).
Termite (order Isoptera) comprises of a complex
assemblage of diverse species, roughly divided into so-
called higher and lower termites. Lower termites harbor a
dense population of prokaryotes and protists (single celled
eukaryotes) in their gut. Higher termites comprise only one
apical family (Termitidae) but more than three quarters of all
termite species. While they also harbor a dense and diverse
array of prokaryotes, higher termites lack protists (Ohkuma;
2003). This association of gut cellulolytic protists with lower
termites is a well-known example of mutual symbiosis. The
protists endocytose wood or cellulose particles to produce
acetate, which is then absorbed by termites as energy and
carbon source (Ohkuma; 2003)
* Corresponding author: subodh@ku.edu.np
Rentech Symposium Compendium, Volume 1, March 2012
A. Cellulose Degrading Systems
Endogenous cellulase of termite origin (endo-β-1,4-
glucanase and β-glucosidase) which are excreted from the
salivary glands or the gut have been identified and
characterized in both higher and lower termites. Molecular
analysis reveals these endogenous enzymes are members of
glycosyl hydrolase family 9 (GHF9). In higher termites the
endogenous cellulolytic activity meets the metabolic
requirement. In lower termites, substantial cellulolytic
activity is found in the hindgut. Thus the ingested cellulose
can be partially degraded by the endoglucanase of termite
origin, then unhydrolyzed cellulose travels to the hind gut,
where it can be endocytosed and is fermented by the
symbiotic protists in lower termites. Termites grind and
crunch the ingested material, which may enhance digestion
by increasing the exposed surface area.
Two main types of cellulases exist: endo- β-1,4-
glucanases (EGs) and cellobiohydrolases (CBHs) (EC
numbers 3.2.1.4 and 3.2.1.91, respectively; see Enzyme
Nomenclature of the NC-IUBMB,. EGs hydrolyse the
interior parts of the cellulose chain, while CBHs attack the
reducing ends of the cellulose polymer. A wide variety of
bacteria and yeasts including cellulolytic and
hemicellulolytic ones have been isolated from the termite
gut (Prillinger et al, 1996; Schater et al, 1996; Wenzel et al,
2002). Furthermore, the termite gut is expected to be the
source of novel microorganisms with wide ranging industrial
applications (Tokuda et al; 2004).
The term ‘cellulase’ traditionally includes two types of
enzymes, exoglucanase (primarily cellobiohydrolase [EC
3.2.1.91] and occasionally cellodextranase [EC 3.2.1.74])
that hydrolyses cellulose from its nonreducing or reducing
ends, and endo-β-1,4-glucanase [EC 3.2.1.4] that hydrolyses
along the glucan chain of cellulose fibres randomly.
Cellooligosaccharides produced by these enzymes are
further hydrolysed to glucose by β-glucosidase (Tokuda et
al; 2005).
Carboxymethylcellulose, which measures endo- β -1,4-
glucanase activity, is one of the most popular artificial
substrates for measuring cellulase activity because of its
high solubility in water. Thus, carboxymethylcellulose has
been preferentially used in most studies of cellulose
digestion in termites and other insects (Tokuda et al; 2005).
B. Microbial ecology of Gut Symbiotic Systems
In the final step of the decomposition involves microbes
that consume electron equivalents, usually a molecular
hydrogen (H2), produced from intermediate steps. Efficient
elimination of the electron equivalents greatly enhances the
decomposition itself. In the gut, microbial fermentation of
termites, CO2- reducing acetogenesis as an “H2 sink”
14
 Subodh K. Upadhyaya et al.: Isolation and characterization of cellulolytic bacteria from gut of termite
reaction is one of the most characteristic and enigmatic
features due to thermodynamic deficiency of acetogenesis,
Methanogens usually dominate in most anoxic habitats.
Although methanogenesis seems to be common in gut of
termites, acetogenesis dominates methanogenesis,
particularly in wood- feeding termites. The acetate produced
by gut microbiota support up to 100% of the respiratory
requirement of termites. Nitrogen economy affects the
efficient decomposition of lignocelluloses indirectly but
significantly. Nitrogen fixation by the gut symbionts is one
of the crucial aspects of termite symbiotic system, since the
diet of termites is usually low in nitrogen sources. The gut
symbionts also play a role in degradation of disposed
nitrogen waste as uric acid produced during termite
metabolism. Fig. 1 explains the possible roles of gut
symbionts of lower wood feeding termites (Ohkuma; 2003).
Fig. 1: Some of the roles of the gut symbionts of termites
(Ohkuma; 2003).
The protozoans or the protists are able to engulf and
metabolize cellulose into acetate, hydrogen and
carbondioxide . Bacteria then utilize the H2 and CO2 to form
additional acetate. There are also methanogens present that
can use the H2 and CO2 to form methane through inter
species hydrogen transfer to remove excess hydrogen, which
can be toxic to the microorganisms at higher concentrations.
The termite gut must remain completely anaerobic to
accommodate anaerobic microbes. Some studies have shown
the presence of strict aerobic bacteria in termite gut. Oxygen
can penetrate into the gut through the body wall.
C. Cellulolytic Bacteria and Biotechnology
In recent years, increasing gas prices and environmental
concerns have become the driving force for developing
alternative energy sources, especially fuel ethanol for
automobiles. Currently, corn is the primary raw material for
ethanol production in the United States. However,
lignocellulosic biomass has the potential to provide a more
economical feedstock as a result of its widespread
availability, sustainable production and low starting value.
Conversion of lignocelluloses to ethanol employs five of
the following steps which includes of
1) Isolation of potent microbe from termite’s gut
(Isolation)
2) Pretreatment to breakdown the lignin and open the
crystalline structure of cellulose
Rentech Symposium Compendium, Volume 1, March 2012
3) Inoculation of the pre- treated cellulolytic biomass by
microbes present in termite’s gut
4) Treatment with the enzymes synthesized by bacteria
in termite’s gut to release simple sugar
(saccharification) and
5) Microbial fermentation of simple sugar to ethanol
using yeast (fermentation).
Existing pretreatment methods have largely been
developed on the basis of physicochemical technologies
such as steam explosion, dilute acid, alkali, and oxidation or
varied combinations (Moiser et al., 2005). However, typical
physical and chemical pretreatments require high-energy
(steam or electricity) as well as corrosion-resistant, high-
pressure reactors, which increase the cost of pretreatment
and need for specialty equipment. Furthermore, chemical
pretreatments can be detrimental to subsequent enzymatic
hydrolysis and microbial fermentation apart from producing
acidic or alkaline waste water which needs pre-disposal
treatment to ensure environmental safety (Keller et al.,
2003). Microbial pretreatment employs microorganisms and
their enzyme systems to breakdown lignin present in
lignocellulosic biomass.
This environment friendly approach has recently
received increased attention and has potential advantages
over the prevailing physicochemical pretreatment
technologies due to reduced energy and material costs,
simplified processes and equipment, and use of biologically
based catalysts.
D. Objectives of the Research
The objectives of our research study are outlined as
follows:
1. Isolation of cellulolytic bacteria from termites found
in the Terai region of Nepal.
2. Screening for cellulase producing bacteria.
3. Study of morphological and biochemical
characteristics of the isolated strains.
4. Study of enzyme activity of the produced cellulase.
II. MATERIALS AND METHODS
A. Sampling Site
The termites were collected from Sarlahi district, Nepal.
B. Geographical Region
The termites used for the extraction of cellulolytic
bacteria were collected from the Balganga Samudayik Ban
located in Sarlahi district, Nepal. It lies in the central
development region. The district covers an area of
1,259 km², the district is situated between latitude 26.45'N
and 84.41'E longitude. Owing to the high biological
diversity in the Terai region and the abundance of termites
in the district, Sarlahi was selected for the collection of
termites' sample.
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 Subodh K. Upadhyaya et al.: Isolation and characterization of cellulolytic bacteria from gut of termite

TABLE I
RESULTS OF GRAM STAINING OF ALL THE STAINS
Name of the Observation Results
Strain
M(c)1 Pink, Rod Gram Negative
M(c)2 Pink, Rod Gram Negative
M(c)3 Pink, Rod Gram Negative
M(c)4 Pink, Rod Gram Negative
M5 Pink, Rod Gram Negative
M6 Pink, Rod Gram Negative
M7 Pink, Rod Gram Negative
M8 Pink, Rod Gram Negative
M9 Pink, Rod Gram Negative
M10 Pink, Cocci Gram Negative
Sarlahi district N(c)1 Pink, Rod Gram Negative
N(c)2 Pink, Rod Gram Negative
N3 Pink, Rod Gram Negative
N4 Pink, Rod Gram Negative
N5 Pink, Rod Gram Negative
Fig. 2: Map of Central Developmental Region of Nepal Showing N6 Pink, Rod Gram Negative
Sarlahi District. N7 Pink, Rod Gram Negative
N8 Pink, Rod and Gram Negative
C. Extraction from termites Cocci
The termites were taken out of their nests and placed in N9 Pink, Cocci Gram Negative
sterilized petriplates. They were then surface sterilized by N10 Pink, Rod Gram Negative
washing with 70% alcohol. Each termite was separated into P11 Pink, Rod Gram Negative
its head and body. After removing the heads with forceps, P2 Pink, Rod Gram Negative
the bodies were crushed with the help of glass rods. The P3 Pink, Rod Gram Negative
paste obtained from the termites' gut was used for isolation P4 Pink, Rod Gram Negative
of the bacteria with the help of inoculating loops. P5 Pink, Rod Gram Negative
P6 Pink, Rod Gram Negative
D. Media used for Culture P7 Pink, Rod Gram Negative
Three different media were used for inoculation of the P8 Pink, Rod Gram Negative
bacteria P(c)9 Pink, Rod Gram Negative

• Nutrient agar B. Results for Congo-Red Assay

• Potato dextrose agar
• M1 media Congo-red assay of all the strain was done to determine
Each media was supplemented with its cellulolytic activity. The strains that digest cellulose as
Carboxymethylcellulose (CMC) as cellulose source. CMC in the media, produces halo-zones. The diameter of
halo-zones were measured which corresponded to the
Nutrient agar was used for the isolation of general cellulose digesting ability of the respective strains. The
bacteria. Potato dextrose agar was used for the isolation of culture was sub-cultured to 5 spots.
fungus. M1 agar was used because it preferably supports the
growth of cellulolytic bacteria. TABLE II
RESULTS OF CONGO-RED ASSAY
E. Test for Cellulolytic Activity
Name of Diameter of the halozone(cm)
Agar plates were prepared with 1% CMC. Strains were
the 1st 2nd 3rd 4th 5th
streaked and petriplates were incubated at 37⁰C for 48 Strain colo colony colony colony colony
hours. Petriplates were flooded with 0.1% Congo red ny
reagent and left for 20 minutes. Then the plates were washed M(c)1 1 0.95 1.15 .95 0.85
with 1M NaCl. Clearance zones called halo zones are seen M(c)2 1.3 1.25 1.6 1.2 1
against the red color of Congo red for the positive test. M(c)3 1.35 1.35 1.15 1.35 1.1
M(c)4 3.5 3.3 3.6 3.8 3.5
III. RESULTS AND DISCUSSION M5 0.8 0.7 0.7 0.6 0.6
M6 3.6 3.5 3.4 3.6 2.9
A. Results for Gram Staining M7 1.15 0.95 0.8 0.65 0.75
M8 1.2 0.95 1.3 1.25 1
Gram staining of all the cultures were done for all the M9 1.65 1.8 4 3.5 3.7
strains to decide whether they were Gram positive or Gram M10 0.2 0.35 0.2 0.35 -
negative. Also, their morphology was determined. N(c)1 2.75 2.75 5.5 5.7 2.7
N(c)2 3.25 2.5 4 2.55 2.5
N3 4 4 3.7 3.9 -

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 Subodh K. Upadhyaya et al.: Isolation and characterization of cellulolytic bacteria from gut of termite

N5 1.65 1.35 1.6 1.25 1.5 general bacteria, whereas Potato dextrose agar was used for
N6 3.25 3.1 3.5 3.3 2.75 the isolation of fungus. We used M1 media since it supports
N7 2.55 2.4 3 2.7 4.8 the growth of cellulolytic bacteria preferably (Wenzel et al;
N8 2.9 3.35 2.75 3.2 2.75 2002).
N9 0.5 0.6 1 0.8 -
N10 3.25 2.7 2.4 2.8 2.25 After isolating various strains by crushing the gut of
P1 1.65 2.1 2.75 3.1 3.75 termites, and streaking them on plates, we focused on
P2 1.15 1.9 2.25 2.9 - screening the enzymatic activity of the strains. The fact that
P3 1.75 1.75 2.05 2.9 3.5
the isolated strains produced cellulase was supported by
P4 1.6 3.05 2.2 4.25 -
P5 2 2.3 2.65 3.25 3.55
congo red staining on the culture plates which showed halo
P6 1 1.55 2.2 2.6 3 zones due to cellulose or carboxymethylcellulose
P7 1.1 1.5 2.1 2.6 3.1 degradation. The diameters of the halozones produced by
P8 1.1 1.75 2 2.57 3.15 different colonies were used as a basis for comparison
P(c)9 1.6 2 2.5 3 3.6 between various isolated strains. Those colonies that
produced largest diameter of halozones were considered to
have the highest cellulolytic activity.
By comparing and analyzing the results of various
biochemical tests, with that of known species, three genera
are suspected to have been isolated which includes
Citrobacter, Enterobacter and Cellulomonas. All strains we
isolated were Gram negative and oxidase positive. However,
Cellulomanas have been documented as Gram variable
(Bergey’s manual of determinative biology, 7th edition) and
Citrobacter and Enterobacter are documented as oxidase
positive (Wenzel et al; 2002).
The main objective of the research was to screen the
enzymatic activity of the bacterial enzyme followed by
molecular characterization of the isolated strains. The exact
Fig. 3: Clearance zones (halo zones) are seen against the red color identification would require higher molecular methods
of Congo red for the positive test. which involves characterization using 16s rRNA techniques.
But, based upon the biochemical tests we used for the
Termites thrive in a great abundance in the terrestrial purpose of characterization, we could only suspect the above
ecosystem by recycling cellulose. Cellulose is also referred mentioned three genera to have been isolated.
to as the most abundant biomass on earth. Termites are
social insects and live in large colonies with organized caste
IV. CONCLUSION
systems. Worker termites—the most common caste
encountered—are likely to be visible during a home Termites may produce up to two liters of hydrogen from
inspection. Soldier termites, although far less numerous, are digesting a single sheet of paper, making them one of the
also likely to be found. planet's most efficient bioreactors.
Termites harbor a mechanism which enables them to Termites achieve this high degree of efficiency by
degrade cellulose. This mechanism is facilitated by the exploiting the metabolic capabilities of about 200 different
presence of numerous cellulolytic bacteria found in their gut. species of microbes that inhabit their hindguts. The complex
These bacteria are known to have symbiotic relationship lignocellulose polymers within wood are broken down into
with the termites. Part of the cellulose ingested by the simple sugars by fermenting bacteria in the termite's gut,
termites gets hydrolyzed by cellulase of termite origin. Then using enzymes that produce hydrogen as a byproduct. If it
the remaining cellulose is utilized by the symbionts. can be determined which enzymes are used to create
hydrogen, and which genes produce them, this process could
Many of cellulose degrading and lignin degrading
potentially be scaled up with bioreactors to generate
bacteria have been so far isolated and characterized in
hydrogen from woody biomass, in commercial quantities.
various regions across the world. This is our attempt to
Moreover the potential for the production of biofuel from
isolate potent celluolytic bacteria and screen its enzymatic
the fermentation of the simple sugars obtained from
activity from the gut of termites found in the Terai region of
lignocellulose decomposition in this way is tremendous. The
Nepal.
world's increasing demands for an alternative source of
Termites harbor a dense array of microbial community, energy have always urged scientists to turn to biofuel. Since
where hundreds of microbes are known to be found. The many of the developed countries in the world have already
microbes may be bacteria, fungi or protozoans. Each of started bioethanol production using cellulolytic bacteria
these microbes possesses a distinct source for their survival. obtained from many organisms including termites, it has
Their growth efficiency may vary based on the presence of become urgently necessary for a country like ours to exploit
specific substrates. So we used three different types of these organisms for biofuel production.
media for their isolation: Nutrient agar, Potato dextrose agar,
We isolated several cultures of bacteria from the
and M1 media. Nutrient agar was used for the isolation of

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 Subodh K. Upadhyaya et al.: Isolation and characterization of cellulolytic bacteria from gut of termite

termites' gut and grouped them based on their similarities,
dissimilarities and biochemical tests. All of the isolates were
gram negative, rods, except two isolates which were cocci.
All of them tested positive in catalase and oxidase test.
Hence, we suspected the isolated organisms to belong to
Genera Cellulomanas, Enterobacter and Citrobacter .
(Bergey’s manual of determinative biology, 7th edition).
We also carried out an enzymatic assay to test the
efficiency of the enzyme produced by the bacteria.
Absorbances of the enzyme samples from different bacterial
strains were measured by the help of the spectrophotometer.
Comparative analysis between absorbance readings of
different bacterial cultures, results of the biochemical tests
and congo red assay were helpful in grouping up of the
available cultures.
However these methods of characterization of the
bacteria based on conventional techniques could not enable
us to fully identify the bacteria at their strain's level. This
requires the need of molecular level characterization that can
overcome the flaws in the conventional step. Moreover these
molecular characterization techniques are easier than the
conventional methods. Thus the characterization of the
bacteria with the conventional methods should be used in
association with the modern molecular 16s rRNA
techniques.
ACKNOWLEDGEMENT
The authors gratefully acknowledge University Grants
Commission, Nepal for providing grant for this project and
the contributions of everyone involved this project.
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BIOGRAPHIES
Subodh Kumar Upadhyaya is Assistant Professor at Department of
Biotechnology, School of Science, Kathmandu University, Nepal.
Anuroop Manandhar and Hemanta Mainali, worked as Teaching
Assistant at Department of Biotechnology, School of Science, Kathmandu
University, Nepal.
Anaya R Pokhrel, Anurag Rijal, Barun Pradhan and Bhabuk Koirala,
were Undergraduate students at Department of Biotechnology, School of
Science, Kathmandu University, Nepal

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