Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

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.     

Use of adenosine 5'-triphosphate as an indicator of the microbiota biomass in rumen contents

A number of techniques were tested for their efficiency in extracting adenosine 5'-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H(2)SO(4), using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 mug of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in "cell-free" rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 mug of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

The uptake of bacteria and amino acids by Ophryoscolex caudatus, Diploplastron affine and some other rumen Entodiniomorphid protozoa

The rate of uptake of mixed rumen bacteria and free amino acids by washed suspensions of seven species of rumen ciliate protozoa has been followed. By assuming that the behaviour of these protozoa was the same under these conditions as during growth it was shown that Ophryoscolex caudatus could obtain the amino acids for growth by the engulfment of rumen bacteria. However, all the cellulolytic protozoa studied (Diploplastron affine, Diplodinium anacanthum, Diplodinium anisacanthum, Enoploplastron triloricatum, Eremoplastron bovis and Ostracodinium obtusum bilobum) were unable to obtain sufficient amino acids from either source to grow at even 25% of the maximum rate and it is postulated that they might utilize plant protein. O. caudatus grown in vitro did not engulf Klebsiella aerogenes or Escherichia coli but took up other bacteria and a rumen yeast at rates of up to 54000 organisms/protozoon/h from a population density of 10⁹/ml. When grown in vivo it was more selective and engulfed mixed rumen bacteria at only 10% of the rate obtained with protozoa grown in vitro. D. affine grown in vitro did not engulf Bacteroides ruminicola, Esch. coli, Kl. aerogenes or Proteus mirabilis but took up mixed rumen bacteria from a population of 10⁹/ml at a rate of 2200 bacteria/ protozoon/h.     

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

Abstract

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.     

Use of Adenosine 5′-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

A number of techniques were tested for their efficiency in extracting adenosine 5′-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H₂SO₄, using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 μg of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in “cell-free” rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 μg of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

Interactions between rumen bacteria and ciliate protozoa in their attachment to barley straw

The attachment of ¹⁴C-choline-labelled mixed rumen protozoa to barley straw in vitro was not significantly affected when bacteria prepared from rumen fluid were added to the incubation mixture. There was similarly little effect on protozoal attachment when the straw had already been colonized by a bacterial population for 24 h. In contrast, it was deduced from measurements of enzyme activities associated with straw that bacterial attachment was reduced if protozoa were present. Bacteria that had colonized the straw for 25 h beforehand were less susceptible to predation by protozoa.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.     

Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Accumulation of Reserve Carbohydrate by Rumen Protozoa and Bacteria in Competition for Glucose

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.     

Inhibition by 1,10-phenanthroline of the breakdown of peptides by rumen bacteria and protozoa

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii , had activities which were inhibited >95% by 1,10-phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10-phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10-phenanthroline, suggesting that protozoa too have a metallo-dipeptidase activity but that it is normally protected from inhibition by 1,10-phenanthroline. Breakdown of the pentapeptide, Ala₅, was also inhibited 27% by 1,10-phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10-phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro-organisms and may be a means of controlling ammonia production from peptides in the rumen.     

Protozoa involved in butyric rather than lactic fermentative pattern during latent acidosis in sheep

We used six ruminally cannulated Texel wethers to study the relative role of protozoa and lactate-metabolizing bacteria in ruminal fermentative patterns during an induced latent acidosis. The sheep were fed an alfalfa hay diet (H) and latent acidosis was induced, following a short transition period of one week, with a grain-rich acidotic diet (W, 60% wheat + 40% alfalfa hay). Ruminal pH, ruminal volatile fatty acids (VFA), lactate and NH3 concentrations, protozoa and lactate-utilizing bacterial counts, the relative proportions of three main bacteria implicated in lactate metabolism (a lactate-producing species, Streptococcus bovis, and two lactate-utilizing species, Selenomonas ruminantium, and Megasphaera elsdenii) using specific 16S-rRNA-targeting oligonucleotide probes, and lactate dehydrogenase (LDH) activity were determined for both diets. The pH parameters (mean, minimum, maximum, time and area under pH 6.0 and 5.5) measured with the W diet were indicative of a latent (i.e., subacute and maintained) acidosis. However, a butyric rather than lactic latent acidosis was observed in this study. Total ruminal lactate concentration remained at low levels with the acidotic diet (< 4 mmol x L(-1)), but changes were observed in VFA composition, which was oriented towards butyrate at the expense of acetate (P < 0.05), while propionate remained constant. In agreement with the low ruminal lactate concentration, no changes in the proportion of S. bovis 16S-rRNA were observed. The lactate-metabolizing bacterial population also remained fairly constant in number, proportion and activity. The increase in butyrate concentration was accompanied by a proliferation of entodiniomorphs (P < 0.01). These results suggest that the protozoa limited lactate accumulation and possibly also the decrease in pH during latent acidosis. Experiments with defaunated and faunated sheep could provide further evidence of the role of protozoa in the development of rumen latent acidosis.     

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.     

Correlation between microbial enzyme activities in the rumen fluid of sheep under different treatments

Five total mixed rations prepared from finger millet (Eleusine Coracana) straw as a roughage (48%) and mixed concentrate (52%), supplemented with a 1% isoacid mixture (i-C4, i-C5, C5 and phenylacetic acid in equal proportions) or oil (groundnut oil, 5% more than the control) or urea (5% more nitrogen than the control), and protein (groundnut cake, 5% more nitrogen than the control) were given in a Latin square experiment to sheep. Enzymatic activities were estimated for urease, cellulase, protease, amylase, and lipase in various fractions of rumen fluid on the one hand and rumen microbial biomass on the other hand. Rumen samples were taken 3-4 hours after feeding and mixed rumen bacteria were separated as a strained rumen fluid without protozoa (SRFWP), cell free rumen fluid (CFRF) and enzymes associated with the bacteria cell (EABC). Samples of SRFWP and EABC contained higher enzyme activities than CFRF. Depending on the type of enzymes in each fraction, some significant coefficient of determination (r2) was seen. These values showed very close cooperative action between proteolytic and amylolytic enzymes under the experimental conditions, or perhaps the presence of some species of bacteria with both activities. Lipolytic bacteria are completely specialized for lipase production only (P < 0.05). The results showed oil, isoacid and crude protein enhanced microbial production (P < 0.05) and this can change the pattern of enzymes in the rumen of sheep.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Formation of Lysine from, α, ε-Diaminopimelic Acid Contained in Rumen Bacterial Cell Walls by Rumen Ciliate Protozoa

Cell walls containing α,ε-diaminopimelate-l,7-¹⁴C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.

From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.     

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble ¹⁴C from ¹⁴C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoal population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter , while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and thzis effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

Factors Influencing Agnotobiotic Cultures of the Rumen Ciliate, Entodinium simplex

The nutrition of Entodinium simplex was studied, with foliage of bluegrass (Poa pratense), perennial ryegrass (Lolium perenne), and grains of wheat (Triticum vulgare) as substrates in agnotobiotic cultures. Entodinium grew poorly when the substrates were autoclaved; better growth was obtained when the substrates were sterilized with ethylene oxide vapor. The concentration of ethylene oxide and the amount of moisture influenced the sterility and nutritional adequacy of the treated substrate. Autolysates and hydrolysates of mixed rumen protozoa stimulated growth. Protozoal extracts did not replace factors destroyed by autoclaving. Clarified rumen fluid assisted the cultivation of entodinia from small inocula but was detrimental to established cultures. Success of cultures was influenced by the medium used to grow the inoculum as well as by the medium inoculated. The results indicated that the composition of the bacterial population influences the growth of E. simplex.