Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Interactions between rumen amylolytic and lactate-utilizing bacteria in growth on starch

The growth and metabolism of the rumen amylolytic bacteria Streptococcus bovis, Butyrivibrio fibrisolvens and Bacteroides ruminicola, growing in pure cultures and co-cultures with the rumen lactilytic bacteria Megasphaera elsdenii and Veillonella alcalescens were followed. The interaction of amylolytic bacteria with V. alcalescens represents a simple food chain. The interaction with M. elsdenii is more complex, since there is a simultaneous competition for products of the starch degradation.     

Influence of the rumen anaerobic fungus Neocallimastix frontalis on the proteolytic activity of a defined mixture of rumen bacteria growing on a solid substrate

A grass + fishmeal ruminant feed was incubated for 7 d in a mineral salts medium with the non-proteolytic rumen bacteria Bacteroides succinogenes, Ruminococcus flavefaciens, Megasphaera elsdenii and proteolytic strains of Bacteroides ruminicola, Selenomonas ruminantium and Streptococcus bovis in the presence and absence of the anaerobic fungus Neocallitnastix frontalis . The fungus increased the dry matter digestion from 65·0 to 69·4%, and more than doubled the proteolytic activity of the culture filtrate. However, a greater difference was observed with the solid material, where the proteolytic activity increased from 0·71 to 6·89 mg ¹⁴C-casein hydrolysed/g/h, due mainly to EDTA-sensitive fungal protease.     

Antibiotic activity of an isocyanide metabolite of Trichoderma hamatum against rumen bacteria

A metabolite of Trichoderma hamatum, 3-(3-isocyanocyclopent-2-enylidene)propionic acid, was tested for its effects on growth of and carbohydrate metabolism in 11 strains of functionally important rumen bacteria. To standardize the biological activity of this unstable metabolite, a rapid, aerobic disc diffusion assay was developed using Escherichia coli ATCC 11775. In an anaerobic broth dilution assay using a medium lacking rumen fluid and containing a soluble carbohydrate, the minimum inhibitory concentration of the metabolite which completely inhibited growth of the rumen bacteria for 18 h at 39 degrees C was generally less than 10 micrograms X mL-1; however, the minimum inhibitory concentrations for Megasphaera elsdenii B159 and Streptococcus bovis Pe(1)8 were 10-25 and 25-64 micrograms X mL-1, respectively. In general, the Gram-negative strains were more sensitive than the Gram positive. The minimum inhibitory concentration for Bacteroides ruminicola 23 grown with glucose was 1 micrograms X mL-1; for B. ruminicola GA33 (glucose), B. succinogenes S85 (cellobiose), and Succinivibrio dextrinosolvens 24 (maltose), it was 2 microgram X mL-1. When added to a cellulose-containing rumen fluid medium, 1-4 micrograms X mL-1 of the metabolite delayed cellulose hydrolysis by B. succinogenes S85, Ruminococcus albus 7, and R. flavefaciens FD1 for up to 4 days, and 6-7 micrograms X mL-1 prevented hydrolysis for at least 1 month. In the presence of the metabolite, the proportion of acetate produced from soluble carbohydrate by the majority of strains increased, but with some strains net production of acetate decreased relative to production of other acidic fermentation products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.     

The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

A survey of peptidase activity in rumen bacteria.

Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ala2, Ala5, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala2, and a smaller number, including Bacteroides ruminicola, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Lachnospira multipara and Ruminobacter amylophilus, broke down Ala5. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba. ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba. ruminicola and Bu. fibrisolvens hydrolysed Ala5 to Ala3 and Ala2, with little ALa4 being produced, in a manner similar to rumen fluid. The activity of Ba. ruminicola against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala3-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu. fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArg-MNA is a diagnostic substrate, it was concluded that Ba. ruminicola was the most important single species in peptide breakdown in the rumen.     

Deoxyribonuclease activity in rumen bacteria

Deoxyribonuclease activity was surveyed in 22 strains belonging to 12 species of rumen bacteria, with lambda bacteriophage DNA as substrate. Activity was readily detected in broken cell preparations from 15 of these strains. Particularly high levels of activity were present in cells and culture supernatant of all 5 strains of Bacteroides succinogenes, and 2 out of 6 strains of Bacteroides ruminicola, examined.     

Interactions between rumen amylolytic and lactate-utilizing bacteria in growth on starch

The growth and metabolism of the rumen amylolytic bacteria Streptococcus bovis, Butyrivibrio fibrisolvens and Bacteroides ruminicola , growing in pure cultures and co-cultures with the rumen lactilytic bacteria Megasphera elsdenii and Veillonella alcalescens were followed. The interaction of amylolytic bacteria with V. alcalescens represents a simple food chain. The interaction with M. elsdenii is more complex, since there is a simultaneous competition for products of the starch degradation.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Comparison of Substrate Affinities Among Several Rumen Bacteria: a Possible Determinant of Rumen Bacterial Competition

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Streptococcus bovis, and Butyrivibrio fibrisolvens were grown in continuous culture. Estimates of substrate affinities were derived from Lineweaver-Burk plots of dilution rate versus substrate concentration. Each bacterium was grown on at least four of the six substrates: glucose, maltose, sucrose, cellobiose, xylose, and lactate. Wide variations in substrate affinities were seen among the substrates utilized by a species and among species for the same substrate. These wide differences indicate that substrate affinity may be a significant determinant of bacterial competition in the rumen where soluble substrate concentrations are often low. Growth of these bacteria in continuous culture did not always follow typical Michaelis-Menten kinetics. Inflated theoretical maximum growth rates and non-linear Lineweaver-Burk plots were sometimes seen. Maintenance energy expenditures and limitation of growth rate by factors other than substrate concentration (i.e., protein synthesis) are discussed as possible determinants of these deviations.     

Effects of Combinations of Substrates on Maximum Growth Rates of Several Rumen Bacteria

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Butyrivibrio fibrisolvens, and Streptococcus bovis were grown in media containing nonlimiting concentrations of glucose, sucrose, maltose, cellobiose, xylose and/or lactate. Each bacterium was grown with every substrate that it could ferment in every possible two-way combination. Only once did a combination of substrates result in a higher maximum growth rate than that observed with either substrate alone. Such stimulations of growth rate would be expected if specific factors unique to individual substrates (transport proteins and/or enzymes) were limiting. Since such synergisms were rare, it was concluded that more general factors limit maximum growth rates in these five bacteria.     

Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay

AIMS: To develop a real-time Taq nuclease assay (TNA) to enable the in vivo enumeration of Megasphaera elsdenii. METHODS AND RESULTS: Megasphaera elsdenii YE34 was phenotypically characteristic of the species and had 16S rDNA sequence similarity of 98% to previously described isolates. Calibration of the number of cells of M. elsdenii against the cycle threshold of fluorescent dye release gave a straight-line relationship with a correlation coefficient approximating unity. The specificity of the assay for M. elsdenii was confirmed by performing it against a panel of 24 heterogeneous, mainly ruminal bacteria. Megasphaera elsdenii was not detected in ruminal contents from a pasture-fed steer but was readily detected 2 and 50 h after the probiotic introduction of the bacterium into the rumen. CONCLUSIONS: Real-time TNA has provided a sensitive and specific means of enumerating the M. elsdenii population in rumen contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Megasphaera elsdenii is an important lactate-degrading ruminal bacterium that has been selected for probiotic use to prevent acidosis and enhance starch utilization in grain-fed cattle. The assay developed in this study provides a tool for determining the ability of probiotically-introduced M. elsdenii to establish useful populations in the rumen.     

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.     

Cyclic AMP in ruminal and other anaerobic bacteria

Abstract An examination of cAMP levels in predominant species of ruminal bacteria and other anaerobic bacteria was conducted. Cellular cAMP concentrations of glucose-grown cultures of Butyrivibrio fibrisolvens 49, Prevotella ruminicola D31d, Selenomonas ruminantium HD4 and D, Megasphaera elsdenii B159, Sreptococcus bovis JB1, Bacteroides thetaiotaomicron 5482, and Clostridium acetobutylicum ATCC 824 were determined at various times during growth by a competitive binding radioimmunoassay procedure. The results were compared to those for Escherichia coli NRRL B3704. The levels of cAMP ranged from undetectable for B. thetaiotaomicron to approximately 15 pmol/mg cell protein for P. ruminicola D31d. Varying the growth substrate in a manner previously shown to elicit regulatory response did not alter the level of cAMP in these organisms. In general, cAMP concentrations present in these organisms were much lower than the 6–25 pmol/mg cell protein observed for E. coli . The levels of cAMP in P. ruminicola were consistently higher than levels in other anaerobes, particularly during the early exponential and stationary phases of growth. Based on these data it seems unlikely that cAMP is involved in regulation of substrate catabolism in the anaerobic bacteria examined except in P. ruminicola where it may have an unknown regulatory function.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system.

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

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.