Abstract: Anaerobic fungi were isolated from the rumens of cattle, buffalo and goat. A total of 133 isolates were obtained. Three of these isolates namely Neocallimastix frontalis B9, Piromyces mae B6 and Orpinomyces joyonii C3 were studied for their cellulolytic activity by using 14C-cellulose produced by Acetobacter xylinum as the substrate. The effects of phenolic acids on the cellulolytic activity of a fungal isolate N. frontalis B9 from buffalo was also determined. The results showed that among the fungal species, N. frontalis B9 had the highest cellulolytic activity, followed by P. mae B6 and O. joyonii C3. The cellulolytic activities of N. frontalis isolates from buffalo, cattle and goat were not significantly different. Both ρ-coumaric and ferulic acids reduced significantly (p<0.05) the celluloytic activity of N. frontalis B9.
INTRODUCTION
Studies have shown that certain phenolic monomers have effects on the biomass and hydrolytic enzyme activities of rumen fungi (Paul et al., 2003) and depress cellulose degradation (Varel and Jung, 1986). Direct studies on the effects of phenolics on cell wall degradation are complicated by the large variety of polysaccharides present in plant cell walls. An alternative experimental approach is to use pure cellulose that can be obtained from a cellulose-synthesizing bacterium, Acetobacter xylinum, grown in glucose. The cellulose can be labelled with 14C by growing the bacterium in U-14C glucose medium. The use of 14C-labelled bacterial cellulose in digestibility studies provides a simple and versatile method to measure total cellulolytic activity. The digestion products can be easily monitored by measuring the release of radioactivity from the 14C-labelled cellulose.
The present study was conducted to assess the degradation rates of 14C-cellulose by various rumen fungi and to determine the effects of phenolic acids such as ρ-coumaric and ferulic acids on the degradation rate.
MATERIALS AND METHODS
Isolation and Maintenance of Anaerobic Rumen Fungi
Animals
Two male Kedah Kelantan cattle (Bos indicus), a male swamp buffalo
(Bubalus bubalis) and a goat (Capra hircus) were kept indoors
in single pens in the animal house at the Department of Animal Science, Universiti
Putra Malaysia. The animals had free access to drinking water and mineral blocks.
Each animal was fitted with a rumen cannula and fed once daily with guinea grass
(Panicum maximum) ad libitum.
The agar growth medium used to isolate rumen fungi was prepared following the method of Bauchop (1979). Fungal identifications were based on the classification of anaerobic gut fungi by Ho and Barr (1995). The pure cultures obtained were maintained in ball-milled filter paper medium or rice straw medium at 39°C and subcultured every three days to ensure that the fungal isolates maintained their cellulolytic activity.
Growth and Culture Maintenance of Acetobacter Xylinum
Acetobacter xylinum ATCC 23770 was kindly donated by Dr. G.L.R. Gordon from CSIRO, Prospect, New South Wales, Australia. The culture was maintained in Wheaton tubes containing 5 mL of Glucose-Phosphate-Peptone-Yeast (GPPY) medium (5 g of yeast extract, 5 g of peptone, 6.8 g KH2PO4 and 10 g glucose/L, pH 6.3). Subculturing was done monthly by transferring the whole bacterial pellicle which developed on the surface of the medium into a tube with fresh GPPY medium. The tubes were incubated loosely capped at 30°C.
Preparation of 14C-Cellulose from Acetobacter Xylinum
Pure 14C-cellulose was prepared from A. xylinum cultures
using the method described by Du Preez and Kistner (1986). One hundred and fifty
milliliter of GPPY medium was aseptically dispensed into disposable plastic
tissue culture flasks (180 mL) with a surface area of about 175 cm2 (Cat.
No. 1-56502, A/S Nunc, Kamstrup, DK 4000 Roskilde, Denmark) and then 1 mL of
filter-sterlised D-[U-14C]glucose (Amersham Biosciences: CFB96) was
aseptically dispensed into the flask. A single pellicle from a 5 mL culture
of A. xylinum that had been incubated for 7 to 10 days was immediately
transferred into the flask. The flask was incubated in a flat position, loosely
cap, at 30°C for 17 to 18 days.
Two batches of 14C-cellulose were prepared. For Batch 1, 6 μCi of D-[U]-14C-glucose was used for each flask, while for Batch 2, 38.5 μCi of D-[U-14C] glucose was used to increase the specific activity of 14C-cellulose.
Harvesting of 14C-Cellulose
After 17 to 18 days of incubation, the pellicles formed on the surface of
the medium in the flasks were removed and washed with distilled water until
the washings were clear. The pellicles were boiled in 2 M KOH for 20 min and
washed with distilled water until the washings were neutral. The pellicles were
then macerated in small quantities in a blender at high speed for 30 sec. The
14C-cellulose slurries were frozen overnight and freeze dried. The
freeze-dried 14C-cellulose was weighed and kept in a desiccator at
ambient temperature. The initial specific activity of the 14C-cellulose
used was determined after treatment with the commercial cellulase from Trichoderma
viride.
Counting of 14C-Cellulose
A 1 mL sample was added to 9 mL of water-miscible β-scintillant solution
(OptiPhase Hisafe 3; Wallac Oy, 20101 Turku, Finland) and the radioactivity
was counted by standard liquid scintillation procedures with correction for
counting efficiency by using an internal 14C standard. Daily count
of specific activity was determined. The amount of solubilized 14C
in each tube was calculated as dpm per mL. The counting of the radioactivity
of solubilized 14C-cellulose was done by using the liquid scintillation
counter (Beckman Instrument Inc. LS Analyzer).
Degradation of 14C-Cellulose by Three Rumen Fungal Species
Rumen fungal species namely Neocallimastix frontalis B9 and Piromyces
mae B6, both isolated from buffalo and Orpinomyces joyonii C3 isolated
from cattle were chosen for this study. The basal medium used was a pre-reduced
anaerobic culture medium without rumen fluid (Akin, 1980). It was modified to
contain 0.2% (v/v) ball-milled Batch 1 14C-cellulose.
The three fungal species used were first cultured in ball-milled filter paper medium for 3 days at 39°C and 0.5 mL of the fungal culture was inoculated into Hungate tubes containing 10 mL of 14C-cellulose medium. Three replicate tubes were made for each fungal species. The tubes were incubated at 39°C for 96 h. The release of radioactivity from the 14C-cellulose was determined by analysing 1 mL of the liquid culture withdrawn at 0 h (immediately following inoculation), 24, 48, 72 and 96 h of incubation. The contents of the tubes were mixed by a single inversion 1 h before the samples of culture liquid were withdrawn for analysis.
Degradation of 14C-Cellulose by N. frontalis from Buffalo,
Cattle and Goat
Neocallimastix frontalis B9 isolated from buffalo, N. frontalis
C20 from cattle and N. frontalis G8 from goat were compared in their
ability to digest 14C-cellulose. Degradation of 14C-cellulose
by N. frontalis isolates was determined according to the method previously
described. Batch 2 14C-cellulose was used.
Effects of ρ-Coumaric and Ferulic Acids on the Degradation of 14C-Cellulose
by N. frontalis from Buffalo
Neocallimastix frontalis B9 isolated from swamp buffalo was used
in this study. The method was as previously described, but culture medium contained
Batch 2 14C-cellulose and either ρ-coumaric or ferulic acids
at 0.1% concentration.
RESULTS
Rumen Fungal Isolates
A total of 133 isolates were obtained from the rumens of cattle and buffalo
and 115 isolates were identified as N. frontalis (Fig.
1), 15 isolates were Piromyces mae (Fig. 2) and
3 isolates were Orpinomyces joyonii (Fig. 3). A representative
isolate of each species was selected based on their good growth in straw and
ball-milled filter paper media and on their ability to maintain zoosporogenesis
and viability in the subcultures for the studies on the degradation of 14C-cellulose.
Cellulose Production from Glucose by Acetobacter xylinum
Static culture of A. xylinum formed a thick surface mat, called
a pellicle in which the embedded cells of this obligate aerobe had direct contact
with the liquid/air interface. The pellicle was a visible film of cellulose
covering the surface of the growth medium. It was colourless and opaque. It
was also jelly-like but strong and flexible. The specific activity of the 14C-cellulose
obtained was 1475.2 dpm mg-1 for Batch1 and 9394.8 dpm mg-1
for Batch 2.
Degradation of 14C-cellulose by N. frontalis B9, P.
mae B6 and O. joyonii C3
Percentage degradation of 14C-cellulose by N. frontalis B9
was significantly (p<0.05) higher than that of P. mae B6 or O.
joyonii C3 at 24, 48, 72 and 96 h (Table 1). Maximum percentage
degradation was observed at 72 h for all the fungal species. At this incubation
period, the radioactivity count for cultures grown with N. frontalis
B9, P. mae B6 and O. joyonii C3 were 2429, 1494 and 1105 dpm
mL-1, respectively (Fig. 4) and the culture medium
was clear due to the degradation of cellulose.
| Fig. 1: | Neocallimastix frontalis showing an exogenous sporangium (S) with a short, eggcup-shaped sporangiophore (SP) and a sporangium with a ruptured apical portion (arrow). Note a zoospore (Z) remaining in the sporangium. Bar = 20 µm |
| Fig. 2: | Piromyces mae showing an exogenous sporangium with a sporangiophore (SP) and a constriction at the base of the sporangiophore (arrows) and rhizoids (R). Bar = 20 µm |
| Fig. 3: | Orpinomyces joyonii showing rhizomycellium complex (R) with sporangia (S). Bar = 20 µm |
Degradation of 14C-Cellulose by N. frontalis Isolates
from Buffalo, Cattle and Goat
The isolates N. frontalis B9 from buffalo, N. frontalis C20
from cattle and N. frontalis G8 from goat were compared in their ability
to degrade 14C-cellulose. The percentage degradation of 14C-cellulose
were not significantly different among N. frontalis isolates at all
incubation times (Table 2).
14C degradation rate increased steadily with incubation time and the highest radioactivity counts were observed at 96 h for all the three isolates (Fig. 5). At 96 h of incubation, N. frontalis B9 from buffalo showed the highest percentage (83.5%) of 14C-cellulose degradation.
Since there were no significant differences in the degradation rates of 14C-cellulose among the N. frontalis isolates, N. frontalis B9 was selected for the experiment on the effect of phenolic acid on the degradation of 14C-cellulose.
Effects of ρ-Coumaric and Ferulic Acids on the Degradation of 14C-Cellulose
by N. frontalis B9
As shown in Table 3, both ρ-coumaric acid and ferulic
acid, at 0.1% concentration, had no significant effect on the 14C-cellulose
degrading activities of N. frontalis B9 at 24 h, but they significantly
(p<0.05) reduced the activities at 72 and 96 h when compared to the control.
| Table 1: | Percentage degradation of 14C-cellulose by N. frontalis B9, P. mae B6 and O. joyonii C3 |
| Values presented are means±SD of three replicates. a-cMeans within the same row with different superscripts are significantly different (p<0.05). The initial specific activity of the 14C-cellulose used was 1475 dpm mg-1 (Batch 1) | |
| Fig. 4: | Radioactivity counts of 14C (dpm mL-1) by N. frontalis B9, P. mae B6 and O. joyonii C3 |
| Fig. 5: | Radioactivity counts of 14C (dpm mL-1) by N. frontalis strains from buffalo, cattle and goat |
| Table 2: | Percentage of degradation of 14C-cellulose by N. frontalis isolates from buffalo, cattle and goat |
| Values presented are means±SD of three replicates. Means within the same row are not significantly different. The initial specific activity of the 14C-cellulose used was 9395 dpm mg-1 (Batch 2) | |
| Table 3: | Effects of ρ-coumaric and ferulic acids on the degradation of 14C-cellulose by N. frontalis B9 from buffalo |
| Values presented are means±SD of three replicates. a-bMeans within the same row with different superscripts are significantly different (p<0.05). The initial specific activity of the 14C-cellulose used was 9395 dpm mg-1 (Batch 2) | |
At 72 h, the degradation percentage of 14C-cellulose for the control culture was twice of that with the phenolic acids. During this period, N. frontalis B9 solubilized 80.7% of 14C-cellulose in the control culture, but only 40.6 and 34.7% in cultures with ρ-coumaric acid and ferulic acid, respectively. Degradation rate increased in the first 24 h for all cultures, but the rate began to decrease for cultures treated with phenolic monomers at further incubation periods. However, degradation of 14C-cellulose continued to increase for the control culture (Fig. 6).
| Fig. 6: | Effects of ρ-coumaric (PCA) and ferulic acids (FA) on the degradation of 14C-cellulose by N. frontalis B9 from buffalo |
DISCUSSION
The cellulose produced by A. xylinum is a native (Type 1) with a crytallinity index considerably higher than that of the much-used substrates, filter paper and Avicel (Hestrin and Schramm, 1954). It is of exceptionally high purity, free of lignin and hemicellulose. This property, together with the ease with which it can be radio-labelled to a desired specific activity, make it a highly suitable substrate for studies of cellulose degradation.
In the present study, 14C-labelled cellulose from A. xylinum was produced and used to determine the cellulolytic activities of three rumen fungal species, viz., N. frontalis B9, P. mae B6 and O. joyonii C3. The results showed that N. frontalis B9 exhibited the highest percentage degradation of 14C-cellulose (86.4%) followed by P. mae (53.2%) and O. joyonii (39.3%). It was also found that the cellulolytic activities of N. frontalis isolates from three different ruminant host species were not significantly different. Cellulolytic activity of O. joyonii was comparatively low probably due to the rudimentary rhizoid produced by O. joyonii limited the ability of the fungus to attach to cellulose particles prior to digestion of the substrate.
Orpin and Letcher (1979) first demonstrated the cellulolytic activity of N. frontalis when purified cellulose was degraded by the organism. Later, Pearce and Bauchop (1985) reported that most of the cellulose in the medium was digested by N. frontalis after six days of growth. Gordon (1985) found that about half of the cellulose and hemicellulose components in the media were lost from 4 to 5 day-old cultures of Neocallimastix and Piromyces spp.. In the present study, more than 80% of the pure cellulose was degraded by N. frontalis by 96 h of incubation.
Phenolic compounds occur naturally in plants, particularly as secondary metabolites and are generally involved in plant defense mechanisms. Ferulic and ρ-coumaric acids in forage cell walls may act as crosslinking agents between polysaccharides and lignin components (Scalbert et al., 1985). Akin and Rigsby (1985) studied the effects of three phenolic acids, ρ-coumaric acid, ferulic acid and sinapic acid on cellulolytic activity of anaerobic rumen fungi. They found that at relatively low concentration (0.1% w/v), these compounds decreased rumen fungal populations in vitro and the dry weight loss of plant cell walls (the substrate) was reduced by 25.5%.
In the present study, the addition of ρ-coumaric acid and ferulic acid in the media significantly (p<0.05) reduced the degradation of cellulose by N. frontalis B9. At 96 h, 83.7% of 14C-cellulose was solubilized in the control culture without the phenolic acids, but in cultures with ρ-coumaric acid or ferulic acid, only 51.9 and 50.5% of 14C-cellulose were degraded, respectively. The negative effects of these phenolic acids on cellulose degradation by N. frontalis B9 found in the present study agree with the findings of other workers. Jung and Sahlu (1986) reported that ρ-coumaric acid and ferulic acid reduced filter paper degradation by rumen fungi. The reduced degradation rate could be attributed to the inhibition of cellulolytic enzyme production as Paul et al. (2003) had observed that ρ-coumaric acid inhibited carboxymethylcellulase activity of Piromyces sp. It was also possible that phenolic monomers similar to those in plant cell walls inhibited the ability of rumen fungi to colonize and degrade the substrates (Akin and Rigsby, 1985). These results suggest that plant phenolics have a detrimental effect on the growth and cellulolytic activity of anaerobic rumen fungi.