Distribution and Evolution of the Xylanase Genes xynA and xynB and Their Homologues in Strains of Butyrivibrio fibrisolvens

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.     

Influence of Forage Phenolics on Ruminal Fibrolytic Bacteria and In Vitro Fiber Degradation

In vitro cultures of ruminal microorganisms were used to determine the effect of cinnamic acid and vanillin on the digestibility of cellulose and xylan. Cinnamic acid and vanillin depressed in vitro dry matter disappearance of cellulose 14 and 49%, respectively, when rumen fluid was the inoculum. The number of viable Bacteroides succinogenes cells, the predominant cellulolytic organism, was threefold higher for fermentations which contained vanillin than for control fermentations. When xylan replaced cellulose as the substrate, a 14% decrease in the digestibility of xylan was observed with vanillin added; however, the number of viable xylanolytic bacteria cultured from the batch fermentation was 10-fold greater than that of control fermentations. The doubling time of B. succinogenes was increased from 2.32 to 2.58 h when vanillin was added to cellobiose medium, and absorbance was one-half that of controls after 18 h. The growth rate of Ruminococcus albus and Ruminococcus flavefaciens was inhibited more by p-coumaric acid than by vanillin, although no reduction of final absorbance was observed in their growth cycles. Vanillin, and to a lesser extent cinnamic acid, appeared to prevent the attachment of B. succinogenes cells to cellulose particles, but did not affect dissociation of cells from the particles. B. succinogenes, R. albus, R. flavefaciens, and Butyrivibrio fibrisolvens all modified the parent monomers cinnamic acid, p-coumaric acid, ferulic acid, and vanillin, with B. fibrisolvens causing the most extensive modification. These results suggest that phenolic monomers can inhibit digestibility of cellulose and xylan, possibly by influencing attachment of the fibrolytic microorganisms to fiber particles. The reduced bacterial attachment to structural carbohydrates in the presence of vanillin may generate more free-floating fibrolytic organisms, thus giving a deceptively higher viable count.     

Digestion of Barley, Maize, and Wheat by Selected Species of Ruminal Bacteria

Differences in the digestion of barley, maize, and wheat by three major ruminal starch-digesting bacterial species, Streptococcus bovis 26, Ruminobacter amylophilus 50, and Butyrivibrio fibrisolvens A38, were characterized. The rate of starch digestion in all cereal species was greater for S. bovis 26 than for R. amylophilus 50 or B. fibrisolvens A38. Starch digestion by S. bovis 26 was greater in wheat than in barley or maize, whereas starch digestion by R. amylophilus 50 was greater in barley than in maize or wheat. B. fibrisolvens A38 digested the starch in barley and maize to a similar extent but was virtually unable to digest the starch in wheat. The higher ammonia concentration in cultures of B. fibrisolvens A38 when grown on wheat than when grown on barley or maize suggests that B. fibrisolvens A38 utilized wheat protein rather than starch. Scanning electron microscopy revealed that B. fibrisolvens A38 initially colonized cell wall material, while S. bovis 26 randomly colonized the endosperm and R. amylophilus 50 preferentially colonized starch granules. There was subsequent colonization but only superficial digestion of wheat starch granules by B. fibrisolvens A38. Variation in the association between starch and protein within the endosperm of cereal grains contributes to the differential effectiveness with which amylolytic species can utilize cereal starch.     

Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures. Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R. albus 8. Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R. albus when xylan prepared from oat spelts was used as a carbohydrate source. These results show that the xylanolytic potential of R. albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid.     

Fermentation of xylans byButyrivibrio fibrisolvens andThermoanaerobacter strain B6A: Utilization of uronic acids and xylanolytic activites

Butyrivibrio fibrisolvens andThermoanaerobacter strain B6A are xylanolytic anaerobes isolated from rumen and geothermal sources respectively. Both organisms fermented larchwood xylan, oatspelt xylan, or 4-O-methylglucuronoaxylan, extensively utilizing both the monosaccharide (glucose, xylose, arabinose) and uronic acid components. Citrus pectin or polygalacturonate also supported growth of both organisms, but onlyB. fibrisolvens was able to use the monomers glucuronate or galacturonate as the sole added energy source. Strain B6A was able to utilize these two uronic acids when glucose, xylose, arabinose, or oatspelt xylan was also provided as a second energy source. Xylanase, xylosidase, and arabinofuranosidase activities were found to be produced by strain B6A, but the levels and distribution (cell bound vs. culture fluid) were influenced by growth substrate. The highest levels were observed with growth on xylans when xylanase activity was mainly extracellular, but the other two activities were mostly cell bound. Apparently,Thermoanaerobacter strain B6A, but notB. fibrisolvens, requires xylan degradation products generated by these three activities to provide energy sources to utilize the uronic acid components on xylans.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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.     

Development of competitive PCR for detection of Butyrivibrio fibrisolvens in the rumen

Competitive PCR method was developed for the detection and enumeration ofButyrivibrio fibrisolvens. Sequences of 16S rDNA were obtained from our isolates (serving as a source of data for primer design) and were distinguished into nine different groups of butyrivibria. Specific primers for two distinct groups were designed with the help of BioEdit program. These primers were tested with DNA of 20 strains of ruminalB. fibrisolvens isolates. Annealing temperature 58. °C showed a little specificity but a better selectivity was found after raising it up to 65 °C. A group 1 competitive fragment of 16S rDNA of different length was constructed using restriction cutting withMspl followed by ligation; the size of the resulting fragment was cut down by 75 bp. The fragment worked in the presence of the original 16S rDNA fragment ofB. fibrisolvens JK 609.     

Xylanolytic Activity of Clostridium acetobutylicum

Of 20 strains of Clostridium spp. screened, 17 hydrolyzed larch wood xylan. Two strains of Clostridium acetobutylicum, NRRL B527 and ATCC 824, hydrolyzed xylan but failed to grow on solid media with larch xylan as the sole carbon source; however, strain ATCC 824 was subsequently found to grow on xylan under specified conditions in a chemostat. These two strains possessed cellulolytic activity and were therefore selected for further studies. In cellobiose-limited continuous cultures, strain NRRL B527 produced maximum xylanase activity at pH 5.2. Strain ATCC 824 produced higher xylanase, xylopyranosidase, and arabinofuranosidase activities in chemostat culture with xylose than with any other soluble carbon source as the limiting nutrient. The activities of these enzymes were markedly reduced when the cells were grown in the presence of excess glucose. The xylanase showed maximum activity at pH 5.8 to 6.0 and 65°C. The enzyme was stable on the alkaline side of pH 5.2 but was unstable below this pH value. The extracellular xylanolytic activity from strain ATCC 824 hydrolyzed 12% of the larch wood xylan during a 24-h incubation period, yielding xylose, xylobiose, and xylotriose as the major hydrolysis products. Strain ATCC 824, after being induced to grow in batch culture in xylan medium supplemented with a low concentration of xylose, failed to grow reproducibly in unsupplemented xylan medium. A mutant obtained by mutagenesis with ethyl methanesulfonate was able to grow reproducibly in batch culture on xylan. Both the parent strain and the mutant were able to grow with xylan as the sole source of carbohydrate in continuous culture with the pH maintained at either 5.2 or 6.0. Under these conditions, the cells utilized approximately 50% of the xylan.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Evaluation of native potential probiotic bacteria using an in vitro ruminal fermentation system

Rumen microbiota provides an important source of protein to grazing animals and produces volatile fatty acids (VFA), the main energy source for ruminants generated by fibre fermentation. Probiotics can be used to modulate rumen fermentation, and native microbiota is a source of potentially useful microorganisms. In this work, ruminal bacterial strains were isolated and subsequently identified, and their potential to modify fermentation patterns with wheat straw, microcrystalline cellulose and oat xylan as substrates was assessed by in vitro gas production and VFA fermentation patterns. Four of the isolates were identified as Pseudobutyrivibrio ruminis and two corresponded to new members of the Lachnospiraceae family. The addition of one P. ruminis (strain 50C) and one Lachnospiraceae (strain 21C) to the fermentation system which used wheat straw as the substrate significantly increased total VFA concentration without altering the total gas produced in one case and showed a decrease in total gas production in the other. All bacterial strains induced higher butyric acid concentrations with the three substrates (up to 31 mM in the case of Lachnospiraceae 21C incubated with oat xylan and 25 mM in microcrystalline cellulose fermenters to which P. ruminis 50C had been added) compared to the control, which had concentrations of <1 mM. Analysis of the fermentation products suggested that the addition of probiotics to the fermentation system had the potential to induce metabolic shifts that would result in better energy yields. These results show that native bacteria have promising features as fermentation modulators, thereby justifying further research to assess their use as probiotics for ruminants.     

Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteria

The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans Δ11 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis Δ9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria.     

Production of hemicellulases by three fungi pathogenic to sugar cane

Three fungal pathogens, Ceratocystis paradoxa (CP), Cephalosporium sacchari (CS), and Marasmius sacchari (MS) were screened for the production of hemicellulose-degrading enzymes (hemicellulases) by induction on bagasse hemicellulose B, and on a commercial preparation of hemicellulose (crude xylan). All three pathogen initially grew poorly on hemicellulose B and “crude xylan” as carbon source. Profuse growth was induced, however, by using mixtures of hemicellulose B and sucrose in the culture media for CP and CS until the organisms were capable of growing on media containing only hemicellulose B. These isolates were classified as CS₁ and CP₁. Profuse growth occurred when CS and CP were grown on carboxymethylcellulose (CMC) and also when these cultures were transferred to media containing only hemicellulose B. These isolates were classified as CS₂ and CP₂. When the above four isolates were grown on hemicellulose B as carbon source in submerged liquid culture, only CP₁ did not produce any extra-cellular hemicellulase(s), and CP₂ produced the highest yield of enzyme. CS₂ and CP₂ also produced extra-cellular CM-cellulase(s). The CP₂-culture isolate was selected for the study of conditions for the optimal production of extra-cellular hemicellulase(s). A preliminary study of the action of enzymes from CS and CP isolated on hemicellulose is reported.     

Induction of xylan-degrading enzymes inButyrivibrio fibrisolvens

The formation of xylanolytic enzymes byButyrivibrio fibrisolvens NCFB 2249 was induced by xylan, xylo-oligosaccharides, and xylobiose. Inhibition of RNA or protein synthesis prevented inducetion, and enzyme formation occured only when anaerobiosis was maintained. The rate of enzyme inducetion by xylan was affected by pH and inducer concentration, and highest levels of activity occurred when the initial pH and xylan concentration were pH 6.5–7 and 2 mg/ml respectively. The ability of the cells to respond to the inducer was reduced in slowly growing cells, although cells that were grown at dilution rates that appertain in the rumen ecosystem responded rapidly to the inducer.Butyrivibrio fibrisolvens also exhibited diauxic characteristics of carbohydrate utilization, and in consequence enzyme induction and xylanolysis were delayed until readily metabolized sugars (e. g., glucose, arabinose) had been consumed.     

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.     

A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO₂/C₂H₂ ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by ¹⁵N₂ incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.     

Simple method for determination of patulin production by Penicillium griseofulvum Dierckx.

The growth of several cellulolytic species of ruminal bacteria was measured in media containing either cellobiose or cellulose as the energy source and with or without added 3-phenylpropanoic acid (PPA). With Ruminoccoccus albus 7 and 8, the addition of PPA greatly enhanced the rate of cellulose utilization but had little effect on the rate of growth when cellobiose was the energy source. Comparative rates of growth obtained on either cellobiose or cellulose for Ruminococcus flavefaciens FD1 or C94 and Butyrivibrio fibrisolvens 12, 49, or A38 were similar regardless of the PPA content of the growth medium.     

Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C_18:2 c9-c12 (linoleic acid [LA]) to C_18:2 c9-t11 (rumenic acid [RA]) and then RA to C_18:1 t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C_18:0 (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (−30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (+107% and +56.1%, respectively; P < 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.The fatty acid profile of the meat and milk of ruminants is strongly affected by diet (2, 15). When ingested, the dietary polyunsaturated fatty acids (PUFA) undergo a process known as biohydrogenation (BH) carried out by ruminal microorganisms (20). During the BH of C_18:2(n-6) (linoleic acid [LA]) and C_18:3(n-3) (linolenic acid [LNA]) a number of C_18:1 and C_18:2 isomers are formed (6). The last step in the BH process leads to the formation of C_18:0 (stearic acid [SA]). Among the intermediate products formed during this process, the isomer C_18:2 c9t11 (rumenic acid [RA]) is active in preventing cancer in mammals (17). Only a small amount of the RA found in meat and milk originates during BH. It is produced to a larger extent in muscle and mammary glands from the desaturation of C_18:1 t11 (vaccenic acid [VA], another intermediate of ruminal BH) by the action of Δ⁹-desaturase enzyme (41, 43).Ruminal BH is carried out mostly by bacteria belonging to the Butyrivibrio genus (38). Butyrivibrio fibrisolvens has the capacity to convert LA to RA and RA to VA, while Butyrivibrio proteoclasticus (previously classified as Clostridium proteoclasticum [35]) hydrogenates VA to SA (38, 39). According to Or-Rashid et al. (37), ruminal protozoa also play a role in BH by converting LA to RA. However, this issue is still controversial, as Devillard et al. (11) have reported that protozoa do not have the capability of hydrogenating LA. The proportion of BH intermediates in the rumen can vary depending on changes in ruminal microbial populations (7, 51). Changes in ruminal fatty acid profiles are also reflected in intramuscular fatty acid composition (48, 52).Tannins are phenolic compounds that are widespread in plants. When ingested by ruminants in large amounts, tannins can reduce the activity and the proliferation of ruminal microorganisms (34). Tannins from Lotus corniculatus (33) or from Acacia spp. (12) reduce the proliferation of B. proteoclasticus B316^T and B. proteoclasticus P18, respectively. Durmic et al. (12) reported that VA increased and SA decreased when extracts from Acacia iteaphylla, which contains condensed tannins (1), were incubated in vitro with sheep ruminal fluid inoculated with B. fibrisolvens JW11 and B. proteoclasticus P18 strains. In two recent in vitro studies, the inclusion of tannins in fermentor systems containing bovine ruminal fluid inhibited the conversion of VA to SA, while no effect was detected on RA production (21, 47). These results have been also confirmed in vivo in the rumen of sheep fed a diet with 4.0% dry matter (DM) quebracho tannin (48). However, to date there is no in vivo study focusing on the effects of dietary tannins on the proliferation of the microorganisms involved in ruminal BH.We assessed whether dietary tannins may affect the BH pathway via changes in bacterial and protozoal ruminal populations. We gave particular emphasis to B. fibrisolvens and B. proteoclasticus. We also assayed the production of conjugated linoleic acids (CLAs) by linoleic acid isomerase (LA-I) enzyme.     

Broad Diversity and Newly Cultured Bacterial Isolates from Enrichment of Pig Feces on Complex Polysaccharides

One of the fascinating functions of mammalian intestinal microbiota is fermentation of plant cell wall components. Eight-week continuous culture enrichments of pig feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 575 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented in the bacterial isolates: Firmicutes (242), Bacteroidetes (185), Proteobacteria (65), Fusobacteria (55), Actinobacteria (23), and Synergistetes (5). The majority of the bacterial isolates had ≥97 % similarity to cultured bacteria with sequences in the RDP, but 179 isolates represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families of Lachnospiraceae, Enterococcaceae, Staphylococcaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. Many of the Firmicutes and Bacteroidetes isolates were identified as species that possess enzymes that ferment plant cell wall components, and the rest likely support these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 30 % of the isolates not represented in culture, there are new opportunities to study genomic and metabolic capacities of these members of the complex intestinal microbiota.