Buletin of Animal Science, UGM (Supplement Edition),
December 2000 : 137-141
The effect of gossypol on the attachment of anaerobic rumen fungus Neocallimastix
frontalis strain RE1 to cellulose in culture
Ismartoyo *
* Department of Animal Science, Hasanuddin University, Makassar-Indonesia.
Abstract
An experiment was conducted to investigate the attachment of rumen fungus to cellulose in
the presence of gossypol in a consecutive batch culture system. This experiment was
performed by the radioactive labelling method described by Moniello et al (1996). The
results of this experiment indicated that total count of 14C-labelled fungus strain RE1
attached to cellulose in control treatment increased as times of incubation increased.
However, the presence of gossypol 0.1 mM decreased (p<0.05) the attachment of the rumen
fungus to cellulose. It was concluded that the presence of gossypol in culture affects the life
cycle of the rumen fungi possibly through binding of gossypol to attachment receptors on
the fungal surface.
Key words : Gossypol, rumen fungi, attachment, cellulose.
Pengaruh gossypol terhadap penempelan fungi rumen Neocallimastix frontalis strain
RE1 pada permukaan selulosa
Intisari
Penelitian ini dilakukan untuk menguji pengaruh gossypol terhadap penempelan fungi
rumen terhadap selulosa dalam sistem consecutive batch culture. Teknik penempelan
radioaktif (14C) pada fungi rumen dilakukan menurut metode sebagaimana telah dijelaskan
oleh Moniello et al (1996). Hasil penelitian ini menunjukkan bahwa penempelan fungi
rumen pada selulosa sangat dipengaruhi oleh konsentrasi gossypol dalam kultur.
Konsentrasi gossypol 0.1 mM menurunkan (p<0.05) jumlah fungi rumen yang melekat
pada selulosa. Kesimpulannya bahwa gossypol sangat menggangu siklus hidup fungi rumen
oleh karena terjadinya ikatan gossypol-protein dan atau gossypol-lipida kompleks pada
permukaan fungi rumen yang menghambat pelekatan dan degradasi rumen fungi terhadap
selulosa.
Kata kunci : Gossypol, fungi rumen, pelekatan, selulosa.
Introduction
It has been reported in other experiments (Ismartoyo et al, 1993; Ismartoyo et al, 1994) that
gossypol inhibits rumen microbial growth and fermentation by rumen bacteria and rumen
protozoa. A further experiment in vitro was conducted to investigate whether the presence
of gossypol may inhibit attachment by the rumen fungi. As has been previously described
by Trinci et al (1994) the life cycle of a rumen fungus species consists of a motile stage
(zoospore) and a vegetative stage (zoosporangia with attached rhizoids). Zoospores attach
to plant particles and produce rhizoids which degrade the plant particles enzymically and
produce a sporangium in which new zoospores are formed. The life cycle continues with
liberation of new zoospores. It is thought this the life cycle of the rumen fungus might be
influenced by the presence of gossypol.
It has been reported that anaerobic fungi are able to solubilize a high proportion of the
highly lignified plant material in laboratory culture (Joblin and Naylor, 1989), and a mixed
fungal population can extensively degrade plant material upon incubation in vitro (Akin et
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Buletin of Animal Science, UGM (Supplement Edition),
December 2000 : 137-141
al, 1983). The rumen fungi Neocallimastix sp. and Piromyces sp. were found to be more
active than Caecomyces sp. in degrading resistant plant tissues (Roger et al, 1993). The
anaerobic rumen fungus Neocallimastix frontalis strain RE, a strain previously isolated at
the Rowett Research Institute, Scotland, UK, and which is easy to grow and actively
cellulolytic (Stewart et al, 1995) was, therefore, chosen in this study to investigate the
effect of gossypol on attachment to and degradation of cellulose. This study was performed
by using the radioactive labelling method described by Moniello et al (1996).
Materials and methods
Preparation of nutrient media
A modified version of medium 2 (Hobson, 1969) was prepared under anaerobic conditions
using O2-free CO2 ( Bryant, 1972; Stewart and Bryant,1988.). The medium 2 (100 ml)
contained 15 ml mineral I, 15 ml mineral II (mineral I and mineral II were described in the
previous experiment), 30 ml clarified rumen fluid, 0.1 ml rezazurin, 0.4 g NaHCO3, 0.25 g
yeast extract, 1.0 g casitone, 0.1 g cysteine HCl and 40 ml distilled H2O. Cellulose (20 x 8
mm Whatman No.1 filter paper strips) was weighed accurately (30 mg) and transferred to
16 x 125 mm Hungate tubes. Nutrient medium (5 ml) was added to the Hungate tubes
under O2-free CO2 and autoclaved at 121oC for 15 min.
Preparation of buffer solution
A buffer solution was prepared for washing the cellulose residues after incubation. The
solution (500 ml) was prepared by mixing 75 ml mineral solution I, 75 ml mineral solution
II (these minerals solutions were as previously described) and 350 ml distilled H2O, and
boiling on a hot plate. Whilst cooling the solution was bubbled with CO2 before adding
cysteine HCl to a final concentration of 0.1% (w/v).
Preparation of gossypol solution
Gossypol (previously described by Risco et al, 1997; Ismartoyo, 1999) solution was
prepared by dissolving gossypol in dimethyl sulfoxide (DMSO). A stock solution of 5 mM
gossypol was prepared by diluting 5.2 mg gossypol in 2 ml DMSO. Two working gossypol
solutions (2.5 mM and 0.5 mM) were prepared by diluting the stock solution (0.5 ml and
0.1 ml) with DMSO (0.5 ml and 0.9 ml), respectively.
Radiolabelling of rumen fungus Neocallimastix frontalis strain RE1
A culture (0.5 ml) of rumen fungus Neocallimastix frontalis strain RE1 growing on
cellophane was inoculated into basal medium containing 0.2% (w/v) glucose and 0.05%
Difco agar and incubated at 38oC for 24 h. Then 0.5 ml of this culture was inoculated into
tubes containing basal medium 2 containing [U-14C] D-glucose (specific activity 6.8 mCi
mmol-1, Sigma) at 2.7 mg l-1 and 0.05 % (w/v) Difco agar, then incubated at 38oC for 48 h
under anaerobic condition using O2-free CO2. After 48 h of incubation the 14C-labelled
fungal biomass was washed three times using fresh modified medium 2 (5 ml), centrifuged
at 1065*g for 5 min and resuspended in 5 ml medium 2. The suspended fungal biomass
was shaken by hand before inoculation into culture tubes (below).
Incubation of cellulose with 14C-labelled fungus strain RE1
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Using sterilised syringes a suspension of 14C-labelled fungus strain RE1 (0.5 ml) and
DMSO with and without gossypol (100 l) were injected into each culture tube in each
treatment containing 5 ml modified basal medium 2 and a filter paper strip (20 x 8 mm, 30
mg). The gossypol concentrations used were 0.01 mM, 0.05 mM and 0.1 mM. The final
concentration of DMSO in culture was 2% (v/v). Four treatment were performed namely
(RE1 + DMSO solvent) = control with DMSO alone, (RE1 + 0.01 mM gp.) = DMSO +
0.01 mM gossypol, (RE1 + 0.05 mM gp.) = DMSO + 0.05 mM gossypol, and (RE1 + 0.1
mM gp.) = RE1 + 0.1 mM gossypol. The tubes were incubated at 38 oC for 6 h. The
incubation time of 6 h was chosen on the basis of previous studies (Moniello et al, 1996) on
the attachment of fungal zoospores and plantlets to cellulose which found that 5 - 6 h of
incubation at 39oC was the optimal time for the attachment of 14C-labelled fungal biomass
to cellulose.
Radioactive counting
After 6 h of incubation all tubes were washed twice using 2 ml buffer solution and the
supernatants were decanted. The cellulose residues were transferred to plastic containers to
which were added 3 ml scintillation fluid (Optiphase, FSA, Loughborough, UK). The
containers were sealed and the count per minute (CPM) of 14C-labelled fungi were counted
using a counter liquid scintillation counter (Tri-Carb, 460CD, Packard). Separate tests
showed that the presence of the cellulose did not affect the detection of radioactive counts
(A.J. Richardson, personal communication). Data of CPM 14C-labelled fungi were then
statistically analysed using completely random design (CRD) as described by Steel and
Torrie (1980).
Results
To establish the effect of gossypol on attachment to cellulose by rumen fungi, three
concentrations of gossypol (0.01, 0.05 and 0.1 mM) were studied for their effects on the
attachment of rumen fungus strain Neocallimastix frontalis to cellulose over 6 h of
incubation. The amounts of 14C-labelled fungus strain RE1 attached to cellulose at 6 h of
incubation is shown in Table1.
Table 1. The mean count per minute (CPM) of 14C-labelled fungus strain RE1 attached to cellulose in the
absence and in the presence of 0.01, 0.05 and 0.1 mM gossypol, (n = 3).
Treatment
T6
RE1 + DMSO
1862.5 b (691.8 - 2913.5)
RE1 + 0.01 mM gossypol
834.9 b (409.5 - 1418.0)
RE1 + 0.05 mM gossypol
276.6 ab (202.5 - 301,0)
RE1 + 0.1 mM gossypol
167.1 a (84.6 - 280.3)
RE1 = Rumen fungus strain Neocallimastix frontalis; DMSO = dimethyl sulfoxide; T6 = incubation at 6 h.
Means with different subscripts in the same column (T6) are significantly different (p<0.05, SED = 505).
Values in the brackets are the ranges of the CPM values of the 3 replicates.
Table 1 shows that gossypol (0.05 mM) severely depressed the attachment of rumen fungus
Neocallimastix frontalis strain RE1 to cellulose. However, the total counts of CPM varied
widely in each treatment. The reasons of the large variation of the CPM and the inhibition
of the attachment were uncertain. A separate investigation by A. J. Richardson (personal
communication) showed that the biomass of strain RE1 that attached to the filter paper
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Buletin of Animal Science, UGM (Supplement Edition),
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consisted of tangled clumps of zoosporangia, which attached via their rhizoids. Variations
in the radioactive counts probably reflected variations in the size of these clumps.
A time course experiment was performed to examine the effect of 0.1 mM gossypol on the
attachment of the rumen fungus strain RE1 to cellulose at 0, 3, 6 and 24 h of incubation.
The results of this experiment are shown in Table 2 and Figure 1.
Table 2. The mean counts per minute (CPM) of 14C-labelled fungus strain RE1 attached to cellulose at 0, 3, 6
and 24 h of incubation, (n = 3).
Treatment
To
T3
T6
T24
RE1 + DMSO
RE1 + 0.1 mM gossypol
SED
304.3 a
282.3 a
84.5; ns.
1016.3 b
350.7 a
219.3
1035.3 b
361.3 a
177.9
1220.0 b
303.7 a
203
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Figure 1. Count per minute of (CPM) of C-labelled fungus strain RE1 of a control (RE1 + DMSO)
compared to the gossypol treatment (RE1 + 0.1 mM gossypol) at 0, 3, 6, and 24 h incubations.
Table 2 shows that total counts of 14C-labelled fungus strain RE1 attached to cellulose in
control treatment (RE1 + DMSO) significantly increased (p<0.05) as times of incubation
increased. The presence of gossypol (0.1 mM) at T3, T6 and T24 significantly (p<0.05)
reduced the amounts of 14C-labelled fungus strain RE1 attached to cellulose in culture
(Figure 1). This study clearly demonstrated that attachment of rumen fungi to cellulose
was inhibited by the presence of gossypol (0.1 mM) in culture. There was no published
information on the effects of gossypol on the attachment of rumen fungi to cellulose. Using
the antinutrient compounds coumarin and the norlupinane alkaloid sparteine Moniello et al
(1996) reported inhibition of the attachment of the rumen fungus Neocallimastix frontallis
to cellulose, cellulose solubilization, and the proportion of lactate in the fermentation
products of the fungus in vitro. The result of the present study suggest that the cellulolysis
may also be reduced by the presence of antinutrient compounds including gossypol. The
effects of gossypol on the degradation of the cellulose are examined in the next experiment
in vitro.
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Conclusion
In conclusion the presence of gossypol in culture affects the life cylcle of rumen fungi
possibly through binding of gossypol to receptors on the fungal surface and the gossypolreceptors complex (probably gossypol-protein and/or gossypol-lipid complex) might
enveloping the cellulose which protect rumen fungi to attach cellulose.
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