Effect of phenolic monomers on ruminal bacteria

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.     

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

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.     

Bacteriocins of ruminal bacteria

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strainE. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strainS. bovis II/I) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci. An erratum to this article is available at .     

Effects of phenolic monomers on growth of Acidothermus cellulolyticus

Previous studies on biological pretreatment of switchgrass by solid-state fermentation with Acidothermus cellulolyticus 11B have shown that inhibitory compounds prevent growth on untreated switchgrass. A. cellulolyticus was grown in liquid medium containing cellobiose with phenolic monomers added to determine if the phenolic compounds are one possible source of inhibition. Cinnamic acid derivatives (trans-p-coumaric, trans-ferulic, and hydrocinnamic acids), hydroxybenzoic acids (p-hydroxybenzoic, syringic, and vanillic acids), benzaldehydes (vanillin and p-hydroxybenzaldehyde), and condensed tannin monomers (catechin and epicatechin) were tested at levels up to 20 mM. All compounds exhibited a dose-response relationship and strongly inhibited growth at 20 mM. trans-p-Coumaric acid was found to be the strongest inhibitor of A. cellulolyticus growth, with a specific growth rate of 0.004 h(-1) at 1 mM (0.18 h(-1) without phenolic monomer). GC-MS and HPLC methods were used to confirm the presence of these phenolic compounds in switchgrass and measure the amounts extracted using different conditions. The amounts of phenolic compounds measured were found to be higher than the threshold for growth inhibition. Leaching with water at 55°C was inefficient at removing bound phenolics, whereas NaOH treatment improved efficiency. Phenolic compounds spiked into alkaline pretreated switchgrass were also found to inhibit growth of A. cellulolyticus in solid-state fermentation. However, addition of insoluble polyvinylpolypyrrolidone (PVPP) to switchgrass improved growth of A. cellulolyticus in liquid cultures, providing a possible approach for alleviating microbial inhibition due to phenolic compounds in lignocellulose.     

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.     

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)     

Superoxide dismutase in ruminal bacteria

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

Effect of heterotrophic ruminal bacteria on xylan metabolism by the anaerobic fungus Piromyces communis

Six rumen bacteria were cocultured with the rumen fungus Piromyces communis and the effects on xylanolysis determined. The rate and extent of xylan utilization was enhanced in cocultures with Prevotella ruminicola or Succinivibrio dextrinosolvens. The positive effects of Suc. dextrinosolvens and Prev. ruminicola on xylanolysis by P. communis correlated with effective cross-feeding by the bacteria on arabinose and xylose released from xylan. Xylanolysis was not enhanced in cocultures of P. communis with Streptococcus bovis, Veillonella parvula or Ruminococcus flavefaciens. A comparison of fermentation product profiles and of extracellular enzyme activities showed that whereas saccharolytic species and Butyrivibrio fibrisolvens were dominant in cocultures, P. communis dominated in the culture with R. flavefaciens. Extracellular xylanase and β-xylosidase activities were not increased by cocultivation of P. communis with any of the heterotrophic bacteria.     

Diet-dependent shifts in ruminal butyrate-producing bacteria

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.     

GATC-specific restriction-modification systems in ruminal bacteria

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.     

Glucose transport by mixed ruminal bacteria from a cow.

The glucose transport of mixed ruminal bacteria harvested from a holstein cow fed 5.0 kg of Italian ryegrass and 1.5 kg of flaked corn a day was investigated. The Eadie-Hofstee plot characterized two transport systems: a high-affinity, low-velocity system and a low-affinity, high-velocity system. The former system (K(m) = 16 microM; Vmax = 2.2 nmol/min/mg of protein) is considered dominant under this feeding condition based on the glucose concentration in the rumen (< 1 mM). In light of the facts that the protonophore SF6847 and the lipophilic triphenylmethyl phosphonium ion had no effect on the high-affinity system and an artificially generated proton gradient and electrical potential across the cell membrane did not increase glucose transport, a proton motive force is not be involved in the system. On the other hand, from the facts that chlorhexidine inhibited about 90% of the high-affinity system while iodoacetate showed no significant effect, and a high phosphoenolpyruvate-dependent phosphorylation of glucose was actually shown, the phosphoenolpyruvate-dependent phosphotransferase system is considered the main system in the high-affinity system. Moreover, as shown by the facts that harmaline inhibited about 30% of the high-affinity system and the artificially generated sodium gradient across the cell membrane significantly stimulated glucose transport, this system also includes sodium symport to some degree. The high-affinity system was sensitive to a decrease in pH (< 6.5) and was inhibited by the presence of sucrose, mannose, and fructose.     

Restriction and modification systems of ruminal bacteria

A high frequency of type II restriction endonuclease activities was detected inSelenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated fromS. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected inS. ruminantium strains as well as in several other species belonging to theSporomusa subbranch of low G+C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).     

Cellobiose transport by mixed ruminal bacteria from a Cow.

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 microM for the Km and 10 nmol/min/mg of protein for the Vmax. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher Vmax values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.     

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.