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Research Article
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Digestion of Rice Straw and Oil Palm Fronds by Microflora
from Rumen and Termite Bacteria, in vitro |
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M. Ramin,
A.R. Alimon,
J.M. Panandam,
K. Sijam,
A. Javanmard
and
N. Abdullah
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ABSTRACT
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The digestion and Volatile Fatty Acid (VFA) production
from rice straw and oil palm fronds by cellulolytic bacteria isolated
from the termite Coptotermes curvignathus were investigated. The
bacteria were Acinetobacter strain Raminalimon, Enterobacter
aerogenes strain Razmin C, Enterobacter cloacae strain Razmin
B, Bacillus cereus strain Razmin A and Chryseobacterium kwangyangense
strain Cb. Acinetobacter strain Raminalimon is an aerobic bacterium,
while the other species are facultative anaerobes. There were significant
differences (p<0.05) among the bacteria for Dry Matter (DM) lost and
acetic acid production from rice straw and Acinetobacter strain
Raminalimon showed the highest activity. The facultative bacteria
C. kwangyangense strain Cb (cfu mL-1 231x10-6,
OD: 0.5), E. cloacae (cfu mL-1 68x10-7, OD:
0.5) and E. aerogenes (cfu mL-1 33x10-7,
OD: 0.5) were used for digestion study with the rumen fluid microflora.
The in vitro gas production technique was applied for the comparative
study and the parameters measured were pH, gas (volume), dry matter lost,
acetic acid, propionic acid and butyric acid concentrations. pH was not
significantly (p<0.05) different among the five treatments. The bacterium
C. kwangyangense strain Cb showed the highest activity (p<0.05)
for DM lost, acetic acid, propionic acid and butyric acid production from
rice straw when compared to the other bacterial activities. There was
no significance (p<0.05) difference between the three bacteria for
the dry matter lost of oil palm fronds but the production of Volatile
Fatty Acids (VFA) was significantly (p<0.05) high in the treatment
which was inoculated with C. kwangyangense strain Cb. The Gen Bank
NCBI/EMBL accession numbers for the bacterial strains are EU332791, EU305608,
EU305609, EU294508 and EU169201.
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INTRODUCTION Termites are important in the
degradation of plant matter which includes cellulose, hemicellulose, lignin
and flavonoids (Harazono et al., 2003). The intestinal tracts of
termites comprise of one or several dilated hindgut compartments, which
harbor the bulk of the intestinal microbiota and were initially considered
as fermentation chambers analogous to the rumen of sheep and cattle (Brune
and Friedrich, 2000). The gut microorganisms of termites consist of protozoa,
bacteria, spirochetes and fungi. The most important metabolic activities
traditionally attributed to the gut microbiota are, first, hydrolysis
of cellulose and hemicellulose, second, fermentation of the depolymerization
products to short-chain fatty acids, which are then resorbed by the host
and third, intestinal nitrogen cycling and dinitrogen fixation (Breznak
and Brune, 1994; Brune, 1998). The term cellulase traditionally includes
two types of enzymes, exoglucanase that hydrolyses cellulose from its
non reducing or reducing ends and endo-ß-1,4-glucanase that hydrolyses
along the glucan chain of cellulose fibres randomly. In termites, it has
been proposed that endo-ß-1, 4-glucanase plays a primary role in cellulose
digestion (Konig, 2005). Carboxymethylcellulose (CMC), which measures
endo-ß-1, 4-glucanase activity is one of the most popular artificial substrates
for measuring cellulose activity because of its high solubility in water.
Thus carboxymethyl-cellulose has been preferentially used in most studies
of cellulose digestion in termites (Tokuda et al., 2005). An in
vitro gas production technique has been recently used to study activities
of rumen microflora on various substrates. A feedstuff is incubated with
buffered rumen fluid and the gases as well as the end product of fermentation
are indicators of fermentation kinetics (Rymer et al., 2005). Agriculture
by-products such as Oil Palm Frond (OPF) and others have been shown to
be available roughage feeds for ruminant animals (Khamseekhiew et al.,
2002), however, their high fiber contents are the main factors limiting
their digestibility and intake by ruminants. The objective of this study
was to conduct an in vitro study to measure the effects of the
cellulolytic bacteria isolated from the termite gut on the digestion of
a fiber substrate and to compare the in vitro digestibility of
rice straw and oil palm fronds by rumen microbial flora and termite bacteria.
MATERIALS AND METHODS Isolates
were prepared in the Microbiology Laboratory of Universiti Putra Malaysia
(UPM) and after preparation they were brought to the Animal Nutrition
Laboratory, UPM for in vitro studies.
Termite bacteria: Five bacterial species were previously isolated
from the gut of the termite Coptotermes curvignathus. The bacterial
species were identified as Acinetobacter strain Raminalimon (EU332791),
Enterobacter aerogenes strain Razmin C (EU305608), Bacillus
cereus Strain Razmin A (EU294508), Enterobacter cloacae strain
Razmin B (EU305609) and Chryseobacterium kwangyangense strain Cb
(EU169201).
In vitro digestion of rice straw by bacterial species from
the termite gut: Each bacterial species was inoculated in 10 mL Nutrient
Broth (NB) and the Optical Density (OD) was adjusted to 0.5. Then it was
mixed with 20 mL buffer medium (Getachew et al., 2005) the total
volume was 30 mL. Approximately 200 mg rice straw was ground to a maximum
particle size of 1 mm and were added to each syringe. The cultures in
triplicate were incubated at 39°C for 24 h. T1 with nutrient
broth without bacteria (Control), T2 with Acinetobacter,
T3 with Enterobacter aerogenes, T4 with
Enterobacter cloacae, T5 with Bacillus cereus and
T6 with Chryseobacterium Kwangyangense strain Cb and
T7 was a mix culture of all the bacteria.
In vitro true dry matter digestibility (IVTDMD): All conditions
were same as described before with some differences; a blank treatment
was added without any substrates (rice straw or oil palm frond) for subtraction
with treatments for the appearance of the particles
Table 1: |
Experimental design showing
treatments |
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Thirty five milliliter buffered rumen fluid was mixed with
each treatment (5 mL), Blank: Thirty five milliliter buffered
rumen + 5 mL nutrient broth, NB: Nutrient Broth
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inside the rumen fluid. The amount of substrates was approximately
500 mg. The buffer was flushed with CO2 to remove the oxygen
in the medium and the total medium in each syringe was 40 mL. The substrates
were oven dried for overnight at 60°C, ground through 1 mm screen sieve
and stored for the in vitro studies. Two in vitro studies
were performed, one for rice straw and another for oil palm frond.
Rumen fluid collection: Rumen fluid was collected before morning
feeding from Kedah-Kelantan (KK) cattle of about 4 years of age with an
average body weight of ±300 kg, fitted with a ruminal cannulae. The rumen
content was collected into 1 L capacity pre-warmed water flask. The cattle
were fed twice per day during the experiment, once at 09:00 h and again
at 16:30 h. Drinking water was freely available to the animal throughout
the experiment.
Treatments: The treatment for blank consisted of 30 mL buffer
(Getachew et al., 2005), 5 mL rumen fluid and 5 mL Nutrient Broth
(NB) without any substrate. Five treatments consisted of 30 mL buffer,
5 mL rumen fluid, NB and distilled water and were inoculated with 5 mL
of one of the bacteria. Approximately 500 mg rice straw was added to each
syringe. The last treatment was as the blank except that it had distilled
water instead of NB (Table 1).
Dry Matter (DM) lost, VFA production and gas production
(after 24 h) was calculated for blank and then subtracted from all treatments.
True dry matter digestibility (TDMD): After 24 h of incubation
the in vitro true dry matter digestibility was determined by refluxing
the residue of each treatment into 600 mL beakers with 70 mL Neutral-Detergent
Solution (NDS) for 1 h (Blummel et al., 1997). True digestibility
was calculated as the weight of substrate incubated minus the weight of
the residue after NDS treatment. The dry matter lost of each treatment
was subtracted with the blank.
VFA production: Volatile Fatty Acids from each treatment were
analyzed by the method of Minato and Kudo referred to by Jetana et
al. (1996).
Gas production and pH determination: Volumes of the gas produced
were read after 24 h of incubation. The total amount of the gas of each
treatment was subtracted with the blank. The pH of each treatment immediately
after incubation was determined using pH meter.
Statistical analyses: The mean of the parameter measured: pH,
dry matter lost, VFAs and gas production, were analyzed by analysis of
variance (ANOVA) using the Statistical Analysis System Institute software
(SAS, 1997). The differences between the treatment means were tested using
the Least Significant Difference (LSD) method.
RESULTS AND
DISCUSSION More details about some of the bacterial
isolates are given in Ramin et al. (2008). All data presented in
Table 2 should be subtracted with the blank; the amount
of dry matter lost in the blank relates to insoluble fraction of rice
straw. There were significant (p<0.05) differences among the treatments.
The amount of Dry Matter (DM) lost and acetic acid concentrations were
higher for the treatments with bacterial isolates, including Acinetobacter
(Table 2). Acinetobacter is an aerobic bacterium.
There was no significant difference between Enterobacter aerogenes,
Enterobacter cloacae and Chryseobacterium kwangyangense strain
Cb for dry matter lost and concentration of acetic acid. The concentrations
of the other volatile fatty acids were not so high. The mixed culture
did not significantly show a high effect on rice straw digestion. For
all treatments the production of gas was not so high. The second and third
in vitro studies were conducted to determine the best bacteria
from termites with rumen microflora for rice straw and oil palm fronds
digestion. For in vitro studies with the aid of rumen microflora
an anaerobic condition is necessary so the facultative bacteria with the
highest effect from the previous experiment were selected; the bacteria
were: E. aerogenes, (cfu mL-1 33x10-7, OD:
0.5) E. cloacae (cfu mL-1 68x10-7, OD: 0.5)
and C. kwangyangense strain Cb (cfu mL-1 231x10-6,
OD: 0.5).
The activity of the bacterial inoculate in each treatment
with rumen fluid was determined. The bacterium Chryseobacterium kwangyangense
strain Cb significantly (p<0.05) showed the best effect for DM lost,
acetic acid, propionic acid and butyric acid concentrations when compared
to the other treatments (Table 3).
In all treatments, the pH did not differ significantly except for the
treatment inoculated with Enterobacter aerogenes, which had a negligible
difference. The amount of gas production did not differ significantly
(p<0.05)
Table 2: |
Mean of the dry matter lost
and acetic acid concentrations of termite gut bacteria |
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a,b,c: Means in
the same column with different subscripts are different significantly
(p<0.05), Each treatment had three replicates, Substrate: Rice
straw, Control: Nutrient broth |
among the treatments, of which the gas production was lowest
for the treatment with distilled water. The concentration of volatile
fatty acids was higher (p<0.05) in the treatment inoculated with Chryseobacterium
kwangyangense strain Cb. In order to compare the effect of the bacteria
on another fiber content substrate, Oil Palm Fronds (OFP) were selected
as a substrate due to their high fiber contents. There was no significance
difference between the three bacterial species when compared for the digestion
of oil palm fronds (Table 4), but there were significance
(p<0.05) differences when the bacterial treatments were compared with
Nutrient Broth (NB) and distilled water. In the latter case the three
bacterial species showed the higher activity on dry matter lost. There
was no significance difference for pH between the treatments. However,
the high pH recorded seems to suggest that the condition were conducive
to cellulolytic bacteria fermentation; it could also be due to the low
VFA production from the highly indigestible OPF-based substrates (Khamseekhiew
et al., 2002). The production of gas was lowest for the treatment
with distilled water and it was significantly (p<0.05) higher for the
treatments which were inoculated with C. kwangyangense and E.
cloacae (Table 4). The production of Volatile Fatty
Acids especially acetic acid was significantly higher for the treatment
which included C. kwangyangense strain Cb (Table
4).
The ability of Termite bacteria to digest lignocellulose compounds has
been published. Acinetobacters, Bacillus cereus and Enterobacters
have been reported to be able to degrade 34-62% cellulose and 14-32% hemicellulose
and in addition Enterobacters are also able to degrade 18-39% lignin
(Konig, 2005), Acinetobacters are classified under the aerobic
group with the capability to degrade caffeic acid and syringic acid (lignin
monomers) and Enterobacters are grouped among bacteria with the
capability of modifying lignin monomers and other aromatic compounds (Kuhnigk
et al., 1994; Varma et al., 1994). Enterobacters
are also able to assimilate different phenolic compounds considered as
Table 3: |
Mean of the dry matter lost,
pH, gas production, acetic acid, propionic acid and butyric
acid concentrations of the treatments |
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a, b, c: Means
in the same column with different subscripts are different significantly
(p<0.05), Three replicates for each treatment, Bac A: E.
aerogenes, Bac B: E. cloacae, Bac C: C. Kwangyangense,
NB: Nutrient broth, Substrate: Rice straw |
Table 4: |
Mean of the dry matter lost,
pH, gas production, acetic acid, propionic and butyric acid
production of the treatments |
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a, b, c: Means
in the same column with different subscripts are different significantly
(p<0.05), Three replicates for each treatment, Bac A: E.
aerogenes, Bac B: E. cloacae, Bac C: C. kwangyangense,
NB: Nutrient broth, Substrate: Oil palm fronds |
lignin related simple monomers (Deschamps et al.,
1980). Borji et al. (2003) has isolated and identified some bacteria
capable of degrading straw lignin and polysaccharides and they were identified
as Bacillus sp., Enterobacter sp. and Ocrobacterium.
The bacterium from the Enterobacters family had a faster growth
rate than the other two species. Schafer et al. (1996) has identified
some bacteria from Enterobacteriaceae and Acinetobacters
family with the ability to degrade hemicellulose compounds. The first
investigations for isolation of facultative anaerobes come from Adams
and Boopathy (2005), who in their study identified some facultative anaerobes
as Enterobacter families from the termite Coptotermes formosanus.
Purwadaria et al. (2003) have investigated the fiber hydrolytic
enzyme activity from termite gut extract as poultry feed applications
and they demonstrated that the extract which comes from the microorganisms
inside termite gut were more appropriate in digesting feedstuffs with
high lignocellulose (fiber) such as rice bran. Volatile Fatty Acids (VFA)
are the chief end-products of cellulose digestion in the rumen, in the
hindgut of termites and acetic acid is the most common volatile fatty
acid produced (Mcfarlane and Alli, 1985). Odelson and Breznak (1983) have
also demonstrated that acetate dominated the extracellular pool of volatile
fatty acids in the hindgut fluid of Reticulitermes flavipes, Zootermopsis
angusticollis and Incisitermes schwarzi, where it occurred
at concentrations of 57.9 to 80.9 mM. The production of acetate is also
reported by Droge et al. (2006) from Spirochaeta coccoides
sp. nov from the hindgut of the termite Neotermes castaneus. There
are few reports on the isolation and identification of Chryseobacterium
species from insect gut specially termites. Organisms tentatively
described as Flavobacterium had only once been isolated from insect
intestinal tracts which were described by Eutick et al. (1978)
and the cellulolytic activity was not reported. Dugas et al. (2001)
is the second person who had identified a Chryseobacterium species
from the gut of the American cockroach fed with high fiber content diet
(30% crude fiber by weight). Some species of this bacterium are able to
degrade feather as described by Brandelli and Riffel (2005). David Landry
has patented (US20020072104A1) a pure glycosidase enzyme which was obtained
from the genus Chryseobacterium. These days feed additives or Direct
Fed Microbial (DFM) are among the most important supplements for ruminants
(Yang et al., 2004), examples are Saccharomyces cerevisiae
(Lynch and Martin, 2002) and Enterococcus faecium (Ghorbani et
al., 2002). It can be concluded that all bacteria were able to digest
rice straw and produce Volatile Fatty Acids (VFA) especially acetate.
Termite bacteria showed an improvement on rice straw and oil palm frond
digestion and production of VFA which proves their ability to digest fiber
materials. The bacterium Chryseobacterium kwangyangense strain
Cb showed the best effect on the dry matter lost, concentration of acetic
acid when compared to the other bacterial species used in the treatments.
Further studies need to be conducted to determine the potential of microorganisms,
especially bacteria from termite gut, for industrial applications such
as feed additives for poultry or ruminants diet. ACKNOWLEDGMENTS
We thank Mr. Mohammad Zawawi and Mr. Ali Nargeskhani for
their technical supports in this study.
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