Genomic architecture of three newly isolated unclassified Butyrivibrio species elucidate their potential role in the rumen ecosystem.

One cellulose-degrading strain CB08 and two xylan-degrading strains XB500-5 and X503 were isolated from buffalo rumen. All the strains were designated as putative novel species of Butyrivibrio based on phylogeny, phylogenomy, digital DNA-DNA hybridization, and average nucleotide identity with their closest type strains. The draft genome length of CB08 was ~3.54 Mb, while X503 and XB500-5 genome sizes were ~3.24 Mb and ~3.27 Mb, respectively. Only 68.28% of total orthologous clusters were shared among three genomes, and 40-44% of genes were identified as hypothetical proteins. The presence of genes encoding diverse carbohydrate-active enzymes (CAZymes) exhibited the lignocellulolytic potential of these strains. Further, the genome annotations revealed the metabolic pathways for monosaccharide fermentation to acetate, butyrate, lactate, ethanol, and hydrogen. The presence of genes for chemotaxis, antibiotic resistance, antimicrobial activity, synthesis of vitamins, and essential fatty acid suggested the versatile metabolic nature of these Butyrivibrio strains in the rumen environment.

Red and processed meat consumption within two different dietary patterns: Effect on the colon microbial community and volatile metabolites in pigs.

Pigs were fed either red and processed meat or chicken meat within either a prudent or a Western dietary pattern for four weeks (2 × 2 full factorial design). The colon microbial community and volatile organic compounds were assessed (either quantified or based on their presence). Results show that Lactobacilli were characteristic for the chicken × prudent dietary pattern treatment and Paraprevotella for the red and processed meat × prudent dietary pattern treatment. Enterobacteriaceae and Desulfovibrio were characteristic for the chicken × Western dietary pattern treatment and Butyrivibrio for the red and processed meat × Western dietary pattern treatment. Campylobacter was characteristic for chicken consumption and Clostridium XIVa for red and processed meat, irrespective of the dietary pattern. Ethyl valerate and 1-methylthio-propane were observed more frequently in pigs fed red and processed meat compared to chicken meat. The prevalence of 3-methylbutanal was >80% for pigs receiving a Western dietary pattern, whereas for pigs fed a prudent dietary pattern the prevalence was <35%. The concentration of butanoic acid was significantly higher when the prudent dietary pattern was given, compared to the Western dietary pattern, but no differences for other short chain fatty acids or protein fermentation products were observed.

Comparative Genomics of Rumen Butyrivibrio spp. Uncovers a Continuum of Polysaccharide-Degrading Capabilities.

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.IMPORTANCE Feeding a global population of 8 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Members of the genera Butyrivibrio and Pseudobutyrivibrio are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings have highlighted the immense enzymatic machinery of Butyrivibrio and Pseudobutyrivibrio species for the degradation of plant fiber, suggesting that these bacteria occupy similar niches but apply different degradation strategies in order to coexist in the competitive rumen environment.

Sharpea azabuensis: a ruminal bacterium that produces trans-11 intermediates from linoleic and linolenic acid.

To investigate the metabolism of 18:2n-6 and 18:3n-3 by pure cultures of Sharpea azabuensis, two different strains (RL 1 and ST18) were each incubated in the presence of 40 µg ml-1 18:2n-6 or 18:3n-3. Pure cultures of Butyrivibriofibrisolvens D1 and Butyrivibrio proteoclasticus P18 were included as control treatments. Similar to the metabolism of B. fibrisolvens, both S. azabuensis strains converted 18:2n-6 or 18:3n-3 to cis-9, trans-11 CLA or cis-9, trans-11, cis-15 CLnA, after which it was further reduced to trans-11 18:1 or trans-11, cis-15 18:2, respectively. B. proteoclasticus additionally reduced trans-11 18:1 to 18:0. Trans-11, cis-15 18:2 was also further metabolized by B. proteoclasticus, although trans-11 18:1 did not accumulate, and only minor amounts of 18:0 were formed. The time frame of 18:2n-6 and 18:3n-3 biohydrogenation by S. azabuensis was comparable with B. fibrisolvens, indicating that S. azabuensis and B. fibrisolvens might be alternative biohydrogenators of 18:2n-6 and 18:3n-3 in the rumen.

Butyrivibrio hungatei MB2003 Competes Effectively for Soluble Sugars Released by Butyrivibrio proteoclasticus B316T during Growth on Xylan or Pectin.

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316T were grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316T was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316T grown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316T when either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316T and MB2003 was assessed; B316T transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCE Feeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Butyrivibrio species are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber.

Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet.

High-grain feeding used in the animal production is known to affect the host rumen bacterial community, but our understanding of consequent changes in goats is limited. This study was therefore aimed to evaluate bacterial population dynamics during 20 days adaptation of 4 ruminally cannulated goats to the high-grain diet (grain: hay - ratio of 40:60). The dietary transition of goats from the forage to the high-grain-diet resulted in the significant decrease of rumen fluid pH, which was however still higher than value established for acute or subacute ruminal acidosis was not diagnosed in studied animals. DGGE analysis demonstrated distinct ruminal microbial populations in hay-fed and grain-fed animals, but the substantial animal-to-animal variation were detected. Quantitative PCR showed for grain-fed animals significantly higher number of bacteria belonging to Clostridium leptum group at 10 days after the incorporation of corn into the diet and significantly lower concentration of bacteria belonging to Actinobacteria phylum at the day 20 after dietary change. Taxonomic distribution analysed by NGS at day 20 revealed the similar prevalence of the phyla Firmicutes and Bacteroidetes in all goats, significantly higher presence of the unclassified genus of groups of Bacteroidales and Ruminococcaceae in grain-fed animals and significantly higher presence the genus Prevotella and Butyrivibrio in the forage-fed animals. The three different culture-independent methods used in this study show that high proportion of concentrate in goat diet does not induce any serious disturbance of their rumen ecosystem and indicate the good adaptive response of caprine ruminal bacteria to incorporation of corn into the diet.

Effect of adsorbants on in vitro biohydrogenation of 22:6n-3 by mixed cultures of rumen microorganisms.

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.

The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

AIMS: To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. METHODS AND RESULTS: Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific ß-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The ß-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of ß-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km  = 2·82 g l(-1) and Vmax  = 4·01 µmoles reducing sugar equivalents × mg(-1)  × min(-1) . CONCLUSION: Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Temporal dynamics of the metabolically active rumen bacteria colonizing fresh perennial ryegrass.

This study investigated successional colonization of fresh perennial ryegrass (PRG) by the rumen microbiota over time. Fresh PRG was incubated in sacco in the rumens of three Holstein × Friesian cows over a period of 8 h, with samples recovered at various times. The diversity of attached bacteria was assessed using 454 pyrosequencing of 16S rRNA (cDNA). Results showed that plant epiphytic communities either decreased to low relative abundances or disappeared following rumen incubation, and that temporal colonization of the PRG by the rumen bacteria was biphasic with primary (1 and 2 h) and secondary (4-8 h) events evident with the transition period being with 2-4 h. A decrease in sequence reads pertaining to Succinivibrio spp. and increases in Pseudobutyrivibrio, Roseburia and Ruminococcus spp. (the latter all order Clostridiales) were evident during secondary colonization. Irrespective of temporal changes, the continually high abundances of Butyrivibrio, Fibrobacter, Olsenella and Prevotella suggest that they play a major role in the degradation of the plant. It is clear that a temporal understanding of the functional roles of these microbiota within the rumen is now required to unravel the role of these bacteria in the ruminal degradation of fresh PRG.

Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.

The ruminant provides a powerful model for understanding the temporal dynamics of gastrointestinal microbial communities. Diet-induced milk fat depression (MFD) in the dairy cow is caused by rumen-derived bioactive fatty acids, and is commonly attributed to the changes in the microbial population. The aim of the present study was to determine the changes occurring in nine ruminal bacterial taxa with well-characterised functions, and abundance of total fungi, ciliate protozoa and bacteria during the induction of and recovery from MFD. Interactions between treatment and time were observed for ten of the twelve populations. The total number of both fungi and ciliate protozoa decreased rapidly (days 4 and 8, respectively) by more than 90% during the induction period and increased during the recovery period. The abundance of Streptococcus bovis (amylolytic) peaked at 350% of control levels on day 4 of induction and rapidly decreased during the recovery period. The abundance of Prevotella bryantii (amylolytic) decreased by 66% from day 8 to 20 of the induction period and increased to the control levels on day 12 of the recovery period. The abundance of Megasphaera elsdenii and Selenomonas ruminantium (lactate-utilising bacteria) increased progressively until day 12 of induction (>170%) and decreased during the recovery period. The abundance of Fibrobacter succinogenes (fibrolytic) decreased by 97% on day 4 of induction and increased progressively to an equal extent during the recovery period, although smaller changes were observed for other fibrolytic bacteria. The abundance of the Butyrivibrio fibrisolvens/Pseudobutyrivibrio group decreased progressively during the induction period and increased during the recovery period, whereas the abundance of Butyrivibrio hungatei was not affected by treatment. Responsive taxa were modified rapidly, with the majority of changes occurring within 8 d and their time course was similar to the time course of the induction of MFD, demonstrating a strong correlation between changes in ruminal microbial populations and MFD.

Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows.

This study investigated the effect of Capsicum oleoresin in granular form (CAP) on nutrient digestibility, immune responses, oxidative stress markers, blood chemistry, rumen fermentation, rumen bacterial populations, and productivity of lactating dairy cows. Eight multiparous Holstein cows, including 3 ruminally cannulated, were used in a replicated 4×4 Latin square design experiment. Experimental periods were 25 d in duration, including a 14-d adaptation and an 11-d data collection and sampling period. Treatments included control (no CAP) and daily supplementation of 250, 500, or 1,000 mg of CAP/cow. Dry matter intake was not affected by CAP (average 27.0±0.64 kg/d), but milk yield tended to quadratically increase with CAP supplementation (50.3 to 51.9±0.86 kg/d). Capsicum oleoresin quadratically increased energy-corrected milk yield, but had no effect on milk fat concentration. Rumen fermentation variables, apparent total-tract digestibility of nutrients, and N excretion in feces and urine were not affected by CAP. Blood serum ß-hydroxybutyrate was quadratically increased by CAP, whereas the concentration of nonesterified fatty acids was similar among treatments. Rumen populations of Bacteroidales, Prevotella, and Roseburia decreased and Butyrivibrio increased quadratically with CAP supplementation. T cell phenotypes were not affected by treatment. Mean fluorescence intensity for phagocytic activity of neutrophils tended to be quadratically increased by CAP. Numbers of neutrophils and eosinophils and the ratio of neutrophils to lymphocytes in peripheral blood linearly increased with increasing CAP. Oxidative stress markers were not affected by CAP. Overall, in the conditions of this experiment, CAP did not affect feed intake, rumen fermentation, nutrient digestibility, T cell phenotypes, and oxidative stress markers. However, energy-corrected milk yield was quadratically increased by CAP, possibly as a result of enhanced mobilization of body fat reserves. In addition, CAP increased neutrophil activity and immune cells related to acute phase immune response.

Effects of incremental amounts of fish oil on trans fatty acids and Butyrivibrio bacteria in continuous culture fermenters.

Previous studies have shown that adding fish oil (FO) to ruminant animal diets increased vaccenic acid (VA; t11 C18:1) accumulation in the rumen. Therefore, the objective of this study was to evaluate the effect of dietary FO amounts on selected strains of rumen bacteria involved in biohydrogenation. A single-flow continuous culture system consisting of four fermenters was used in a 4 × 4 Latin square design with four 9 days consecutive periods. Treatment diets were as follows: (i) control diet (53:47 forage to concentrate; CON), (ii) control plus FO at 0.5% (DM basis; FOL), (iii) control plus FO at 2% (DM basis; FOM) and (iv) control plus FO at 3.5% (DM basis; FOH). Fermenters were fed treatment diets three times daily at 120 g/day. Samples were collected from each fermenter on day 9 of each period at 1.5, 3 and 6 h post-morning feeding and then composited into one sample per fermenter. Increasing dietary FO amounts resulted in a linear decrease in acetate and isobutyrate concentrations and a linear decrease in acetate-to-propionate ratio. Propionate, butyrate, valerate and isovalerate concentrations were not affected by FO supplementation. Concentrations of C18:0 in fermenters linearly decreased, while concentrations of t10 C18:1 and VA linearly increased as dietary FO amounts increased. The concentrations of c9t11 and t10c12 conjugated linoleic acid were not affected by FO supplementation. The DNA abundance for Butyrivibrio fibrisolvens, Butyrivibrio vaccenic acid subgroup, Butyrivibrio stearic acid subgroup and Butyrivibrio proteoclasticus linearly decreased as dietary FO amounts increased. In conclusion, FO effects on trans fatty acid accumulation in the rumen may be explained in part by FO influence on Butyrivibrio group.

Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus.

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fiber-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains-a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fiber-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings.

Glycerol inhibition of ruminal lipolysis in vitro.

Supplemental glycerol inhibits rumen lipolysis, a prerequisite for rumen biohydrogenation, which is responsible for the saturation of dietary fatty acids consumed by ruminant animals. Feeding excess glycerol, however, adversely affects dry matter digestibility. To more clearly define the effect of supplemental glycerol on rumen lipolysis, mixed populations of ruminal bacteria were incubated with 6 or 20% glycerol (vol/vol). After 48-h anaerobic incubation of mixed culture rumen fluid, rates of free fatty acid production (nmol/mL per h) for the 6 and 20% glycerol-supplemented samples were decreased by 80 and 86%, respectively, compared with rates from nonsupplemented control cultures (12.4±1.0; mean ± SE). Conversely, assay of the prominent ruminal lipase-producing bacteria Anaerovibrio lipolyticus 5S, Butyrivibrio fibrisolvens 49, and Propionibacterium species avidum and acnes revealed no effect of 2 or 10% (vol/vol) added glycerol on lipolytic activity by these organisms. Supplementing glycerol at 6% on a vol/vol basis, equivalent to supplementing glycerol at approximately 8 to 15% of diet dry matter, effectively reduced lipolysis. However, the mechanism of glycerol inhibition of ruminal lipolysis remains to be demonstrated.

Ricinoleic acid inhibits methanogenesis and fatty acid biohydrogenation in ruminal digesta from sheep and in bacterial cultures.

Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for CLA synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from 4 sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9,cis-12-18:2) or with a combination of LA and RA or LA and castor oil (LA, RA, and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA rumenic acid (cis-9,trans-11-18:2) accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. Butyrivibrio proteoclasticus but not B. fibrisolvens metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were decreased (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo studies are now required to confirm the potential of these additives.

Production of conjugated linoleic acid (CLA) by Bifidobacterium breve LMC520 and its compatibility with CLA-producing rumen bacteria.

This study was performed to characterize the ability of an active Bifidobacterium strain to produce conjugated linoleic acid (CLA) and to test its possible utilization as a probiotic compatible to the ruminal condition. Bifidobacterium breve LMC520 can actively convert linoleic acid (LA) to cis-9,trans-11-CLA, which is a major isomer derived from microbial conversion. LMC520 showed reasonable tolerance under acidic conditions (pH 2.5 with 1% pepsin) and in the presence of oxgall (0-3%). The growth and CLA production of LMC520 were tested under ruminal conditions and compared with those of Butyrivibrio fibrisolvens A38, which is a major CLA producer in the rumen as an intermediate in the biohydrogenation (BH) process. LMC520 converted 15% of LA to CLA under ruminal conditions, which was 2 times higher activity than that of A38, and there was no decline in CLA level during prolonged incubation of 48 h. The BH activity of LMC520 was comparable to that of A38. When LMC520 was cocultured with A38, even with slight decrease of CLA due to high BH activity by A38, but the level of CLA was maintained by the high CLA-producing activity of LMC520. This comparative study shows the potential of this strain to be applied as a functional probiotic not only for humans but also for ruminants as well as to increase CLA production.

The glycobiome of the rumen bacterium Butyrivibrio proteoclasticus B316(T) highlights adaptation to a polysaccharide-rich environment.

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4 Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens.

BACKGROUND: Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA. RESULTS: Linoleic acid (LA; cis-9, cis-12-18:2) and alpha-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans-11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n-3)) and docosahexaenoic acid (DHA; 22:6(n-3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%. CONCLUSIONS: It was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Metabolism of conjugated linoleic acids and 18 : 1 fatty acids by ruminal bacteria: products and mechanisms.

Cultures of ruminal bacteria known to metabolize unsaturated fatty acids were grown in medium containing 50 microg ml(-1) of geometric and positional isomers of conjugated linoleic acid (CLA) or 18 : 1 fatty acids and 37.4 % deuterium oxide to investigate the mechanisms responsible for fatty acid metabolism. Butyrivibrio fibrisolvens JW11 converted cis-9,trans-11-18 : 2 and trans-9,trans-11-18 : 2 to trans-11-18 : 1 as the main product, labelled at C-9, and metabolized trans-10,cis-12-18 : 2 to trans-10-18 : 1, labelled at C-13, and smaller amounts of trans-12-18 : 1 and cis-12-18 : 1. Butyrivibrio proteoclasticus P-18 did not grow in the presence of cis-9,trans-11-18 : 2 or trans-10,cis-12-18 : 2, but grew in medium containing trans-9,trans-11-18 : 2, forming 18 : 0. Propionibacterium acnes, a ruminal species that isomerizes linoleic acid to trans-10,cis-12-18 : 2, did not metabolize CLA isomers further. B. fibrisolvens metabolized small amounts of trans-10-18 : 1, trans-11-18 : 1 and cis-9-18 : 1, but the products formed were not detected. B. proteoclasticus, on the other hand, carried out substantial conversion of 18 : 1 substrates to 18 : 0. P. acnes hydrated cis-9-18 : 1 and trans-11-18 : 1 to 10-OH-18 : 0, which was further oxidized to yield 10-O-18 : 0. The deuterium enrichment in the intermediates formed during incubations with 9,11 geometric isomers of CLA was about half that of the products from trans-10,cis-12 CLA and 18 : 1 isomers, suggesting that the reduction of 9,11 geometric isomers CLA by ruminal bacteria occurs via different mechanisms compared with the metabolism of other unsaturated fatty acids.

Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping.

BACKGROUND: Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures. METHODOLOGY/PRINCIPAL FINDINGS: We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential (1)H-NMR profiling and two-dimensional (2D) (1)H, (13)C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D (1)H, (13)C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects. CONCLUSIONS: RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.