Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

p-Coumaroyl and feruloyl arabinoxylans from plant cell walls as substrates for ruminal bacteria.

Growth of the ruminal bacteria Ruminococcus flavefaciens FD1, Selenomonas ruminantium HD4, and Butyrivibrio fibrisolvens 49 was limited by ester-linked feruloyl and p-coumaroyl groups. The limitation of growth on phenolic acid-carbohydrate complexes varied with individual bacteria and appeared to be influenced by ability to hydrolyze carbohydrate linkages.     

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.     

Esterase activities in Butyrivibrio fibrisolvens strains.

Thirty strains of Butyrivibrio fibrisolvens isolated in diverse geographical locations were examined for esterase activity by using naphthyl esters of acetate, butyrate, caprylate, laurate, and palmitate. All strains possessed some esterase activity, and high levels of activity were observed with strains 49, H17c, S2, AcTF2, and LM8/1B. Esterase activity also was detected in other ruminal bacteria (Bacteroides ruminicola, Selenomonas ruminantium, Ruminobacter amylophilus, and Streptococcus bovis). For all B. fibrisolvens strains tested, naphthyl fatty acid esterase activity paralleled culture growth and was predominantly cell associated. With strains 49, CF4c, and S2, the activity was retained by protoplasts made from whole cells. Esterase activity was detected with all strains when grown on glucose, and some strains showed higher activity levels when grown on other substrates (larchwood xylan or citrus pectin). When nitrophenyl esters of fatty acids were used to measure esterase activity, generally four- to sevenfold-higher activity levels were detected, and with a number of strains substantial levels were found in the culture fluid. Cultures of these strains (H17c, NOR37, D1, and D30g) contained xylanase and acetyl xylan esterase activities, neither of which was associated to any great extent with the cells. Acetyl xylan esterase has not been previously detected in ruminal bacteria and may be important to overall digestion of forage by these organisms.     

Esterase activities in Butyrivibrio fibrisolvens strains

Thirty strains of Butyrivibrio fibrisolvens isolated in diverse geographical locations were examined for esterase activity by using naphthyl esters of acetate, butyrate, caprylate, laurate, and palmitate. All strains possessed some esterase activity, and high levels of activity were observed with strains 49, H17c, S2, AcTF2, and LM8/1B. Esterase activity also was detected in other ruminal bacteria (Bacteroides ruminicola, Selenomonas ruminantium, Ruminobacter amylophilus, and Streptococcus bovis). For all B. fibrisolvens strains tested, naphthyl fatty acid esterase activity paralleled culture growth and was predominantly cell associated. With strains 49, CF4c, and S2, the activity was retained by protoplasts made from whole cells. Esterase activity was detected with all strains when grown on glucose, and some strains showed higher activity levels when grown on other substrates (larchwood xylan or citrus pectin). When nitrophenyl esters of fatty acids were used to measure esterase activity, generally four- to sevenfold-higher activity levels were detected, and with a number of strains substantial levels were found in the culture fluid. Cultures of these strains (H17c, NOR37, D1, and D30g) contained xylanase and acetyl xylan esterase activities, neither of which was associated to any great extent with the cells. Acetyl xylan esterase has not been previously detected in ruminal bacteria and may be important to overall digestion of forage by these organisms.     

Carbon Dioxide Requirement of Various Species of Rumen Bacteria

The carbon dioxide requirement of 32 strains of rumen bacteria, representing 11 different species, was studied in detail. Increasing concentrations of CO(2) were added as NaHCO(3) to a specially prepared CO(2)-free medium which was tubed and inoculated under nitrogen. Prior depletion of CO(2) in the inoculum was found to affect the level of requirement; however, the complexity and buffering capacity of the medium did not appear to be involved. An absolute requirement for CO(2) was observed for eight strains of Bacteroides ruminicola, three strains of Bacteroides succinogenes, four strains of Ruminococcus flavefaciens, two strains of Lachnospira multiparus, one strain of Succinimonas amylolytica, and two strains of Butyrivibrio fibrisolvens. Inconsistent growth responses were obtained in CO(2)-free media with one strain each of B. fibrisolvens, Ruminococcus albus, and Selenomonas ruminantium. Growth of six additional strains of B. fibrisolvens, and single strains of Eubacterium ruminantium and Succinivibrio dextrinosolvens was markedly increased or stimulated by increasing concentrations of CO(2). Peptostreptococcus elsdenii B159 was the only organism tested which appeared to have no requirement, either absolute or partial, for CO(2). Higher concentrations of CO(2) were required for the initiation of growth, as well as for optimal growth, by those species which produce succinic acid as one of their primary end products.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes.

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

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.     

Some rumen bacteria degrading fructan

Degradation of fructan obtained from timothy ( Phleum pratense L.) by the following six species of bacteria isolated from sheep rumen was studied: Streptococcus bovis, Bacteroides ruminicola, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Treponema bryantii and Treponema saccharophilum. The enzymatic activity of the bacteria was analysed by TLC. The highest activity was found in whole cells and in the strains B. fibrisolvens No. 3 and T. saccharophilum S.     

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.     

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.     

Amylolytic activity of selected species of ruminal bacteria.

A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amylolytic activity of selected species of ruminal bacteria

A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of heavy metals and other trace elements on the fermentative activity of the rumen microflora and growth of functionally important rumen bacteria

The inhibitory effects of high concentrations of essential and non-essential trace elements were tested on the rumen microflora using the rate of fermentation in vitro as the assay. The elements (and the concentration causing 50% inhibition) in decreasing order of toxicity were Hg2+ (20 microgram/ml), Cu2+ (21 microgram/ml), Cr6+ (70 microgram/ml), Se4+ (73 microgram/ml), Ni2+ (160 microgram/ml), Cd2+ (175 microgram/ml), As3+ (304 microgram/ml) and As5+ (1610 microgram/ml). The elements tested that were either weak or noninhibitory at concentrations greater than 400 microgram/ml included Zn2+, Cr2+, Fe2+, Mn2+, Pb2+, and Co2+. Methylmercury was as inhibitory as mercuric chloride to the fermentation. When the inhibitory effect of Cd2+ was tested on separated bacterial and protozoal fractions, it was more inhibitory to the bacteria. The inhibitory effects of trace elements were also determined for a number of axenic cultures of rumen bacteria. The bacteria which most frequently exhibited the greatest sensitivity were Bacteroides succinogenses, Ruminococcus albus, Bacteroides amylophilus, and Eubacterium ruminantium. Those often exhibiting intermediate sensitivities included Butyrivibrio fibrisolvens, Selenomonas ruminantium, and Megasphera elsdenii, while Streptococcus bovis was very refractory to all elements tested. Rumen fluid provided a modest protective effect for the bacteria.