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.     

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.     

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.     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentose transport by the ruminal bacterium Butyrivibrio fibrisolvens

Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose sugars. Washed cells of strain D1 accumulated radiolabelled xylose ( K _m= 1.5 μ M) and arabinose ( K _m= 0.2 μ M) when the organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.     

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.     

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens.

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic transformation of the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis by electroporation

Electroporation methods for introduction of plasmid DNA into the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis were developed. Electroporation of the strictly anaerobic B. fibrisolvens was carried out in an anaerobic glovebox with a buffer of 10% (v/v) glycerol and 1 mM MgCl₂ in distilled water. Streptococcus bovis electroporation could be carried out aerobically with a buffer of 10% (v/v) glycerol in distilled water. The Escherichia coli/Bacillus subtilis shuttle vector pBS42 could be transformed into B. fibrisolvens strain H17c, selecting for chloramphenicol resistance. The Streptococcus sanguis/E. coli shuttle vector pVA838 could replicate and express erythromycin resistance in Strep. bovis. Both vectors were stable in each organism in the absence of antibiotic selection. While the efficiency was low (<10²/μg DNA), the results demonstrate a means to introduce cloned genes into these organisms.     

Oxaloacetate Synthesis in Butyrivibrio fibrisolvens

Phosphoenolpyruvate carboxykinase (adenosine 5′-triphosphate) was the only enzyme capable of carboxylating pyruvate or phosphoenolpyruvate that could be demonstrated in sonicated cells or cell-free extracts of a group 1 butyrivibrio.     

Oxaloacetate Synthesis in Butyrivibrio fibrisolvens

[This corrects the article on p. 1247 in vol. 37.].     

The role of carbonate in the metabolism of glucose by Butyrivibrio fibrisolvens

The amount of Na2CO3 added to semi-synthetic medium determined the length of the lag phase, the growth rate and the dry weight of three strains of Butyrivibrio fibrisolvens (WV1, NOR37, B835). With increasing CO3(2-) concentration the molar growth yield of bacteria, from glucosewas increased and, of the fermentation products, formate increased more than the other acids. CO3(2-)-limited cultures of strain WV1 (Group 2 Butyrivibrio) and strain NOR37 (Troup 1 Butyrivibrio) incorporated 14CO3(2-) into lactate and formate. In NOR37, lactate and formate had equal specific activities; in WV1, the formate specific activity was twice that of lactate. Strain WV1 had an active pyruvate synthase and an energy-dependent exchange between CO3(2-) and formate was demonstrated. In strain WV1 butyrate was produced mainly from glucose.     

Degradation and utilization of xylan by the ruminal bacteria Butyrivibrio fibrisolvens and Selenomonas ruminantium.

The cross-feeding of xyland hydrolysis products between the xylanolytic bacterium Butyrivibrio fibrisolvens H17c and the xylooligosaccharide-fermenting bacterium Selenomonas ruminantium GA192 was investigated. Cultures were grown anaerobically in complex medium containing oat spelt xylan, and the digestion of xylan and the generation and subsequent utilization of xylooligosaccharide intermediates were monitored over time. Monocultures of B. fibrisolvens rapidly degraded oat spelt xylan, and a pool of extracellular degradation intermediates composed of low-molecular-weight xylooligosaccharides (xylobiose through xylopentaose and larger, unidentified oligomers) accumulated in these cultures. The ability of S. ruminantium to utilize the products of xylanolysis by B. fibrisolvens was demonstrated by its ability to grow on xylan that had first been digested by the extracellular xylanolytic enzymes of B. fibrisolvens. Although enzymatic hydrolysis converted the xylan to soluble products, this alone was not sufficient to assure complete utilization by S. ruminantium, and considerable quantities of oligosaccharides remained following growth. Stable xylan-utilizing cocultures of S. ruminantium and B. fibrisolvens were established, and the utilization of xylan was monitored. Despite the presence of an oligosaccharide-fermenting organism, accumulations of acid-alcohol soluble products were still noted; however, the composition of carbohydrates present in these cultures differed from that seen when B. fibrisolvens was cultivated alone. Residual carbohydrates present at various times during growth were of higher average degree of polymerization in cocultures than in cultures of B. fibrisolvens alone. Structural characterization of these residual products may help define the limitations on the assimilation of xylooligosaccharides by ruminal bacteria.     

Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10(-6). The 10(-8) and 10(-9) dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited Clostridium sticklandii SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to Butyrivibrio fibrisolvens B835. Strain JL5 inhibited B. fibrisolvens 49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of B. fibrisolvens 49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of C. sticklandii SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and C. sticklandii SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains.

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

High-frequency transfer of a naturally occurring chromosomal tetracycline resistance element in the ruminal anaerobe Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens strains resistant to tetracycline were isolated from the bovine rumen. Two of three Tcr B. fibrisolvens tested were able to donate tetracycline resistance at frequencies ranging from 10(-7) to 10(-1) per donor cell in anaerobic filter matings to a rifampin-resistant mutant of the type strain of B.fibrisolvens, 2221R. The recipient strain 2221R exhibited rapid autoaggregation, which might be a factor in the high transfer rates observed. Tcr transconjugants of B. fibrisolvens 2221R were also capable of further transferring tetracycline resistance to a fusidic acid-resistant mutant, 2221F. Comparison of genomic DNAs by pulsed-field gel electrophoresis demonstrated altered band profiles in transconjugants, consistent with the acquisition of a large mobile chromosomal element. The transferable elements from the two B. fibrisolvens donors 1.23 and 1.230 (TnB123 and TnB1230, respectively) showed the same preferred insertion site in the B. fibrisolvens 2221R chromosome and are likely to be similar, or identical, elements. Hybridization experiments showed no close relationship between TnB1230 and int-xis regions from Tn916 or Tn5253. Although DNA from the B. fibrisolvens donor strains hybridized with probes carrying tet(M) or tet(O) sequences, transconjugants were found to have acquired a distinct band that hybridized only weakly with these probes, suggesting that a second, distantly related Tcr determinant had been transferred.     

Characterization of the Lipids of Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens strain D-1 was grown on a lipid-free chemically defined medium. The lipids were extracted with chloroform-methanol and separated into nonpolar and polar fractions by silicic acid column chromatography. Further separations were made by preparative thin-layer chromatography. The lipid fractions were identified by specific staining reactions and R(F) values, by phosphorus and nitrogen determinations, by chromatography of hydrolysis products, and by the use of infrared spectroscopy. The major nonpolar lipid was free fatty acid. Four major polar lipids were identified: phosphatidylethanolamine, phosphatidyl glycerol, lipoaminoacid, and glycolipid. The lipoaminoacid contained alanine, leucine, and isoleucine. The glycolipid contained galactose. The major fatty acids identified were C16:0 and C18:1. The significance of the presence of lipoaminoacid is discussed.