Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum.

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

The influence of ruminal concentrations of O2 and CO2 on fermentative metabolism of the rumen entodiniomorphid ciliateEudiplodinium maggii

The effects of ruminal concentrations of CO₂ and O₂ on glucose-stimulated and endogenous fermentation of the rumen ciliateEudiplodinium maggii were investigated. The principal metabolic products were butyrate, acetate, lactate, propionate, H₂, and CO₂.¹³C NMR spectroscopy revealed glycerol to be an important, but previously unidentified, fermentation product of this organism. Glucose uptake and metabolite formation rates were influenced by the headspace composition during protozoal incubations. Glucose uptake was most rapid in the presence of low O₂ in N₂ (1–3 µM O₂ dissolved in the protozoal suspension). Pathways located in the hydrogenosomes were O₂ sensitive, and low O₂ concentrations resulted in lowered acetate, H₂, and CO₂ formation. The presence of high CO₂ (65% gaseous headspace by volume) resulted in elevated acetate and butyrate formation; fumarate and propionate were similarly found to accumulate at higher concentration than previously detected in the supernatants. Results suggest that under conditions similar to those prevailing in the rumen (i.e., high CO₂),Eu. maggii produces higher levels of important ruminal volatile fatty acids, and thus its relative contribution to rumen metabolism may have been underestimated.     

Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium) showed that both organisms have an efficient O2-scavenging capability (apparent Km values 2.3 and 0.3 microM, respectively). Reversible inhibition of H2 production increased almost linearly with increasing O2 up to 1.5 microM; higher levels of O2 gave irreversible inhibition. In situ determinations of H2, CH4, O2 and CO2 in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O2 was present before feeding at 1-1.5 microM and decreased to undetectable levels (less than 0.25 microM) within 25 min after feeding. A transient increase in O2 concentration after feeding occurred only in defaunated animals and resulted in suppression of CH4 and CO2 production. The presence of washed holotrich protozoa decreases the O2 sensitivity of CH4 production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri. It is concluded that holotrich protozoa play a role in ruminal O2 utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H2 utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O2 levels, and are themselves regulated by the low ambient O2 concentrations they experience in the rumen.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Oxygen consumption by ruminal microorganisms: protozoal and bacterial contributions

The relative contributions to O2 consumption made by the protozoal and bacterial populations present within the rumen were determined by using an open-type oxygen electrode system. Measurements indicated that two separate microbial populations contributed approximately equally to ruminal O2 consumption over the O2 concentration range experienced in situ (0.25 to 1.0 microM). The populations were observed to consume O2 under liquid-phase O2 concentrations of up to 7 microM, above which point rapid inactivation of O2 utilization was observed. Km values for the mixed population of bacteria and protozoa were 0.36 +/- 0.17 and 3.2 +/- 0.4 microM at concentrations of less than 1.6 and greater than 1.6 microM, respectively. O2 affinity values obtained for both the protozoal and bacterial populations were similar. O2 affinities of the isolated entodiniomorphid ciliates Polyplastron multivesiculatum and Eudiplodinium maggii showed O2 inhibition thresholds of 10 and 5, respectively, and apparent half-saturation constants (Km values) of 1.7 and 5.2 microM O2, respectively. Corresponding Vmax values were 7.8 microM O2 per min per 10(5) organisms for P. multivesiculatum and 3.6 microM O2 per min per 10(5) organisms for E. maggii. Mass spectroscopic analysis detected average rates of H2 production of 12.0 and 3.7 microM H2 per min per 10(5) organisms for P. multivesiculatum and E. maggii, respectively. Trace levels of dissolved O2 (less than 0.25 microM) stimulated the H2 production rate of E. maggii eightfold but inhibited that of P. multivesiculatum by 18%.     

Oxygen consumption by ruminal microorganisms: protozoal and bacterial contributions.

The relative contributions to O2 consumption made by the protozoal and bacterial populations present within the rumen were determined by using an open-type oxygen electrode system. Measurements indicated that two separate microbial populations contributed approximately equally to ruminal O2 consumption over the O2 concentration range experienced in situ (0.25 to 1.0 microM). The populations were observed to consume O2 under liquid-phase O2 concentrations of up to 7 microM, above which point rapid inactivation of O2 utilization was observed. Km values for the mixed population of bacteria and protozoa were 0.36 +/- 0.17 and 3.2 +/- 0.4 microM at concentrations of less than 1.6 and greater than 1.6 microM, respectively. O2 affinity values obtained for both the protozoal and bacterial populations were similar. O2 affinities of the isolated entodiniomorphid ciliates Polyplastron multivesiculatum and Eudiplodinium maggii showed O2 inhibition thresholds of 10 and 5, respectively, and apparent half-saturation constants (Km values) of 1.7 and 5.2 microM O2, respectively. Corresponding Vmax values were 7.8 microM O2 per min per 10(5) organisms for P. multivesiculatum and 3.6 microM O2 per min per 10(5) organisms for E. maggii. Mass spectroscopic analysis detected average rates of H2 production of 12.0 and 3.7 microM H2 per min per 10(5) organisms for P. multivesiculatum and E. maggii, respectively. Trace levels of dissolved O2 (less than 0.25 microM) stimulated the H2 production rate of E. maggii eightfold but inhibited that of P. multivesiculatum by 18%.     

Changes of fermentation pathways of fecal microbial communities associated with a drug treatment that increases dietary starch in the human colon.

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-13C]glucose and 12CO2 or with unlabeled glucose and 13CO2, the distribution of 13C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO2. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO2 reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.     

The effect of Aspergillus oryzae fermentation extract on the growth of fungi and ciliate protozoa in the rumen

When added to the diet of sheep, 2 g/d, Aspergillus oryzae fermentation extract (AO) stimulated total and cellulolytic bacterial numbers in rumen fluid by 34 and 90% respectively. AO had no effect on the numbers of protozoa or fungal zoospores. AO did not affect hydrogen production by the rumen fungi Neocallimastix frontalis (RE1), N. patriciarum (CX) or Piromonas communis (P) in pure culture or protozoal activity in vitro , estimated from the rate of breakdown of [¹⁴C] leucine-labelled Selenomonas ruminantium. It was concluded that increases in ruminal fibre digestion observed previously in animals fed AO, were most likely due to a stimulation of bacteria rather than eukaryotes in the rumen microbial population.     

Hereditary spherocytosis of man. Altered binding of cytoskeletal components to the erythrocyte membrane

The endogenous respiration of the rumen ciliate Dasytricha ruminantium maintained under an O2 tension of 2kPa (approximately 0.02 atm) was partially inhibited by KCN (40% inhibition) and NaN3 (58% inhibition). The organisms lack cytochromes, and sensitivity of respiration to KCN, NaN3, chloroquine and quercetin suggest that the operation of flavoprotein-iron-sulphur-mediated electron transport. As in Tritrichomonas foetus, hydrogenosomal respiration can be stimulated by the addition of CoA in the presence of 0.025% Triton X-100; stimulation by ADP was not detected. Stimulation of pyruvate-supported O2 uptake by Pi suggests that acetate is produced via acetyl phosphate.     

Rumen Microbial Ecology in Mule Deer

Mule deer rumen microbial populations from animals in the natural habitat in Utah and from captive deer fed various rations were studied. The microorganisms were characterized on the basis of morphology and Gram reaction. Rumen samples contained 13 identifiable types of bacteria and one genus of ciliate protozoa (Entodinium). Highest rumen bacterial populations were produced on rations containing barley. No differences in proportions of ruminal bacteria in the various morphological groups could be detected when animals were fed either natural browse plants or alfalfa hay. The total numbers of bacteria were similar for animals feeding on controlled diets of browse or hay and those in the natural habitat. Numbers of some bacterial types were directly related to ciliate protozoal numbers, whereas others were inversely related. Highest rumen ciliate protozoal populations were observed on rations containing barley. No differences in protozoal populations were noted between diets containing only browse or hay. Seasonal variations were noted in ciliate protozoal numbers from deer feeding in the natural habitat. The total number of ciliate protozoa decreased in the fall and winter and remained low until spring. There were indications that salt in the deer diet favorably affected rumen ciliate protozoa. Rather than revealing direct deer management applications, this study serves to stimulate and illuminate new approaches to research in range and wildlife nutrition.     

Ability of Acidaminococcus fermentans to oxidize trans-aconitate and decrease the accumulation of tricarballylate, a toxic end product of ruminal fermentation.

Mixed ruminal bacteria convert trans-aconitate to tricarballylate, a tricarboxylic acid which chelates blood divalent cations and decreases their availability (J. B. Russell and P. J. Van Soest, Appl. Environ. Microbiol. 47:155-159, 1984). Decreases in blood magnesium in turn cause a potentially fatal disease known as grass tetany. trans-Aconitate was stoichiometrically reduced to tricarballylate by Selenomonas ruminantium, a common ruminal bacterium in grass-fed ruminants (J. B. Russell, Appl. Environ. Microbiol. 49:120-126, 1985). When mixed ruminal bacteria were enriched with trans-aconitate, a trans-aconitate-oxidizing bacterium was also isolated (G. M. Cook, F. A. Rainey, G. Chen, E. Stackebrandt, and J. B. Russell, Int. J. Syst. Bacteriol. 44:576-578, 1994). The trans-aconitate-oxidizing bacterium was identified as Acidaminococcus fermentans, and it converted trans-aconitate to acetate, a nontoxic end product of ruminal fermentation. When S. ruminantium and A. fermentans were cocultured with trans-aconitate and glucose, tricarballylate never accumulated and all the trans-aconitate was converted to acetate. Continuous-culture studies (dilution rate, 0.1 h-1) likewise indicated that A. fermentans could outcompete S. ruminantium for trans-aconitate. When mixed ruminal bacteria were incubated in vitro with 10 mM trans-aconitate for 24 h, 45% of the trans-aconitate was converted to tricarballylate. Tricarballylate production decreased 50% if even small amounts of A. fermentans were added to the incubation mixes (0.01 mg of protein per mg of mixed bacterial protein). When A. fermentans (2 g of bacterial protein) was added directly to the rumen, the subsequent conversion of trans-aconitate to tricarballylate decreased 50%, but this effect did not persist for more than 18 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of Yucca shidigera extract on ruminal ammonia concentrations and ruminal microorganisms.

An extract of the desert plant Yucca shidigera was assessed for its possible benefit in ruminal fermentation. The extract bound ammonia in aqueous solution when concentrations of ammonia were low (up to 0.4 mM) and when the extract was added at a high concentration to the sample (20%, vol/vol). The apparent ammonia-binding capability was retained after autoclaving and was decreased slightly following dialysis. Acid-precipitated extract was inactive. No evidence of substantial ammonia binding was found at higher ammonia concentrations (up to 30 mM). When Y. shidigera extract (1%, vol/vol) was added to strained rumen fluid in vitro, a small (6%) but significant (P < 0.05) decrease in ammonia concentration occurred, apparently because of decreased proteolysis. Inclusion of Y. shidigera extract (1%, vol/vol) in the growth medium of the rumen bacterium Streptococcus bovis ES1 extended its lag phase, while growth of Butyrivibrio fibrisolvens SH13 was abolished. The growth of Prevotella (Bacteroides) ruminicola B(1)4 was stimulated, and that of Selenomonas ruminantium Z108 was unaffected. Protozoal activity, as measured by the breakdown of 14C-leucine-labelled S. ruminantium in rumen fluid incubated in vitro, was abolished by the addition of 1% extract. The antimicrobial activities were unaffected by precipitating tannins with polyvinylpyrrolidone, but a butanol extract, containing the saponin fraction, retained its antibacterial and antiprotozoal effects. Saponins from other sources were less effective against protozoa than Y. shidigera saponins. Y. shidigera extract, therefore, appears unlikely to influence ammonia concentration in the rumen directly, but its saponins have antimicrobial properties, particularly in suppressing ciliate protozoa, which may prove beneficial to ruminal fermentation and may lead indirectly to lower ruminal ammonia concentrations.     

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.     

Evaluation of a Method of Measuring Fermentation Rates and Net Growth of Rumen Microorganisms

The method of el-Shazly and Hungate for measuring gas production in rumen contents was slightly modified and used throughout this investigation. The variation in the fermentation rates due to samples collected separately from a sheep fed on hay was less than 2%. When samples obtained through a stomach tube were compared with samples collected through the rumen fistula, the variation was about 3%. The rates of gas and acid production were approximately similar in samples obtained from the rumen at the same time when no sodium bicarbonate was added. During in vitro incubation of whole ruminal contents, there was a highly significant correlation between the net growth rate values (obtained by using fermentation capacity as an index) and the change in concentration of viable rumen bacteria or total ciliate protozoa.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Studies on the effects of selenium on rumen microbial fermentation in vitro

The effects of selenium (Se) on ruminant microbial fermentation were investigated in vitro using rumen microflora collected from a rumen-fistulated dairy cow. First, the effects ofl-selenomethionine (SeMet; at 0.2 or 2 ppm Se) in the presence or absence of wheat bran (WB, 500 mg per incubation flask) were evaluated. Second, the effects of several forms of Se (elemental Se: 50 ppm Se; sodium selenite: 2 ppm Se; SeMet: 2 ppm Se) were compared. Results showed that the amounts of short-chain fatty acids (SCFAs) tended to be increased by SeMet treatment, whereas SeMet in the presence of WB transiently suppressed fermentation. The addition of SeMet tended to increase the production of acetate while reducing the production of butyrate with and without WB supplementation. Among the different Se compounds tested, the amounts of SCFAs were greater with SeMet treatment, which yielded a higher proportion of acetate compared to other treatments. Selenite did not influence the total SCFAs concentrations; however, it increased the relative proportion of butyrate at the expense of acetate. Elemental Se did not significantly affect fermentation. Higher bacterial Se concentrations were observed for selenite than for SeMet. It was concluded that Se supplementation can influence rumen microbial fermentation and that Se compounds differ in this regard.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.