Termites are responsible for much of the degradation of wood and other
cellulose materials in the Tropics and sub-Tropics (Peralta et al.,
2004). While some wood are resistant to their attack, others are not (Nakayama
et al., 2000; Peralta et al., 2004). Cellulose form the
principal food of wood-eating termites. Termites` gut microflora have
been reported to play vital role in carbon nutrition and energy derived
from digestion of cellulose (Breznak and Brune, 1994). The lower termites
possess intestinal protozoa which help them in the digestion of wood,
while the higher termite which lack intestinal protozoa possess bacteria
in their hindguts.
The hindgut of termites are colonized by diverse group of bacteria such
as Bacillus, Micrococcus, Streptococcus, Bacteroides,
Streptomyces, Staphylococcus and various Enterobacteriaceae
(Amund et al., 1986; Femi-Ola et al., 2001).
Cellulose hydrolysis is accomplished with the aid of cellulase enzyme
complex which is made up of three classes of enzymes namely exoglucanase,
endoglucanase and β glucosidase (Beguin, 1990). The cellulolytic
organisms in the hindgut of termites are known to produce extracellular
cellulases necessary for the hydrolysis of cellulose. Certain extractives
from some woods have been reported to have termicidal properties (Duryea
et al., 1999; Femi-Ola et al., 2007; Nakayama et al.,
2000; Neya et al., 2004). However, there is paucity of information
on the effect of these extractives on the microflora in the hindgut of
the termites. Thus, this study investigated the effect of aqueous extracts
of some woods on the growth and cellulase production in some strains of
cellulolytic Bacillus subtilis.
MATERIALS AND METHODS
Culture: Bacillus subtilis (NCIB 3610) was obtained from
the Department of Microbiology, Obafemi Awolowo University, Ile-Ife. The
study was conducted during the wet season. Bacillus subtilis (BS5)
was isolated from the hindgut of wood-eating termites Amitermes evuncifer
(Silvestri) (Femi-Ola et al., 2001). Bacterial suspension was prepared
in normal saline from 24 h-old slant culture. The suspension was diluted
to give an extinction of 0.20. The total bacterial count in the suspension
was approximately 54x109 organisms mL-1.
Wood materials: Wood chips of Milicia excelsa (Welw) C.C.
Berg, Mansonia altissima Chev, Khaya grandifoliola C.Dc.,
Brachystegia eurycoma Harms and Terminalia superba Engl
and Diels were collected from Bashiri Sawmill in Ado-Ekiti, Ekiti-State,
Preparation of extracts: Twenty grams of the wood shaving of each
wood sample were suspended in 100 mL of distilled water in 250 mL flask.
The mixtures were held for 90 min in a water bath at 80°C; after which
they were allowed to stand for 3 days. Each extract was filtered through
sterile Millipore filter (0.22 μm) into a sterile flask. Dilution
of each extract was prepared in sterile citrate phosphate buffer (pH 6.5)
to give a final concentration of 25, 50 and 100 mg mL-1.
In vitro test of effect of wood extract: The effect of
wood extracts on cellulase production and cellulolytic bacteria were determined
by culturing the test organisms in carboxymethylcellulose medium containing
in g L-1:
K2HPO4, 0.2; KH2PO4, 0.5;
CaCl2.2H2O, 0.02; NaNO3, 2.0; NaCl, 0.5;
MgSO4.7H2O, 0.2; MnSO4.H2O,
0.02; FeSO4, 0.02; yeast extract 0.5; Carboxymethylcellulose
(CMC High viscosity BDH UK) 10.0. The basal medium was autoclaved at 121°C
for 15 mins while CMC was autoclaved separately and added to the basal
medium to give a final concentration of 1% (w/v). The medium (25 mL) was
supplemented with 1 mL of wood extract and then inoculated with 0.5 mL
suspension of the test organisms. Inoculated flasks were incubated at
35°C for 36 h. Growth was determined by measuring the Optical Density
(OD) at 470 nm. Uninoculated medium served as control in each concentration,
protein content was determined by Lowry et al. (1951). Cellulolytic
activity was assayed for by the copper arsenomolybdate colour reagent
method of Somogyi (1952).
Determination of cellulolytic activity: Cultures were harvested
by centrifugation at 5000 rpm for 20 min. Culture supernatants were used
for the assay of the extracellular enzyme. To 0.5 mL of 1% CMC in 0.1
M citrate phosphate buffer (pH 6.5) was added 0.5 mL of crude enzyme preparation.
The reaction mixture was incubated at 35°C for 1 h in a water bath.
The reaction mixture was terminated by heating at 100C° for 10 min,
while the cellulase activity was determined by the method of Somogyi (1952).
One unit of cellulase activity was expressed as the amount of enzyme required
to liberate 1 μg of glucose per minute.
Statistical analysis: Analysis of variance and Schefee pair wise
multiple comparison using SPSS version 11.0 software were carried on data
obtained from different experimental results.
RESULTS AND DISCUSSION
Addition of the wood extract significantly affected the growth of the
Bacillus strains (p<0.05).The Scheffe pair wise multiple comparison
showed that there were significant differences in the growth of the Bacillus
strains when the wood extracts was absent (control) and when added (Table
1). The inhibitory effect of the aqueous extract of B. eurycoma
was stronger than the other wood types. The concentration of the wood
extracts also had effect on the growth of test organisms; however the
differences were not significant (p>0.05).
There was significant difference in the amount of protein released by
the test organism in the presence and absence of wood extracts (p<0.05).
There was no significant difference between additions of aqueous extract
of T. superba and control (Table 2). Statistical
||Effect of wood extracts on growth of two strains of
|Averages within a column followed by the same letter(s)
are not significantly different as gauged by Schefee pair wise multiple
||Effect of wood extracts on cellulolytic activity of
Bacillus subtilis BS5 isolated from hindgut of Amitermes
|Averages within a column followed by the same letter(s)
are not significantly different, as gauged by Schefee pair wise multiple
analysis also showed that there was a significant difference (p<0.05)
in protein content on addition of different concentration of wood extracts.
Scheffe test showed that there were significant differences (p<0.05)
between the means of control and wood extracts with concentration 50 and
100 mg mL-1. There was however no significant difference (p>0.05)
in specific activity of cellulase produced by the different B. subtilis
The study has shown that aqueous extract of the woods inhibited the growth
and cellulase production in the strains of Bacillus subtilis
significantly. Although the growth and the cellulolytic activity of the
organisms were affected by the concentration of the wood extracts, the
differences were not significant. The celluloytic activity of the two
strains of B. subtilis was least affected by aqueous extract of
T. superba. The aqueous extract of B. eurycoma most effective,
as it inhibited the growth and cellulolytic activity of the Bacillus
strains at all concentrations employed.
The inhibition might be due to the chemical composition of the woods.
Extractives in form of resin, hormones and fatty acids have been reported
to account for 3±5% of softwood and 5±3% of heartwoods (Illston
et al., 1981; Neya et al., 2004). These substances and some
others have been suggested to contribute to the resistance of some woods
to termite infestation (Nakayama et al., 2000; Neya et al.,
2004; Peralta et al., 2004).
Amylase and protease inhibitors have been demonstrated in extracts
of kolanut (Cola nitida and C. acuminata) against the protease
and amylase of the kolanut weevil (Sophrorhinus insperatus Faust)
(Adedire and Balogun, 1992; Adedire, 1994). Extractives in the wood that
is antimicrobial and inhibitory to cellulase activity may be responsible
for the protection of some woods against termite attack.
Aqueous extract of some of the woods inhibited growth and cellulase production
in Bacillus subtilis strains. High concentration of wood extracts
(100 mg mL-1) of Milicia excelsa, Mansonia altissima,
Khaya grandifoliola and Brachystegia eurycoma were effective
in inhibiting cellulolytic activity in B. subtilis BS5 and B.
subtilis NCIB3610, respectively. Among the wood species, B.
eurycoma had the strongest inhibitory effects on the growth and cellulolytic
activity of B. subtilis BS5, isolated from the hindgut of A.
evuncifer. Extractives in wood contribute immensely to their protection
against termite attack. Aqueous extract of T. superba did not have
any inhibitory effect on cellulase production by the strains of B.