Crystal Structures of Bacterial Pectin Methylesterases Pme8A and PmeC2 from Rumen Butyrivibrio.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed ß-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

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.

Effect of an inulin-type fructan from Platycodon grandiflorum on the intestinal microbiota in rats exposed to PM2.5.

In this study, an inulin-type fructan (PGPI-1-a) was isolated from the roots of Platycodon grandiflorum. PGPI-1-a was composed of (2 â†’ 1)-linked ß-D-fructofuranose (Fruf) and a terminal α-d-glucopyranose (Glcp) with a molecular weight of 12.1 kDa. PM2.5 exposure has brought a great threat to human health in recent years. Therefore, this study explored the effect of PGPI-1-a on the intestinal microbial community structure of rats exposed to PM2.5 using the animal model of PM2.5 inhalation exposure. The results showed that PGPI-1-a could regulate the intestinal microbiota by partly restoring the perturbed levels of Peptoniphilaceae_[G-2] and Lachnospiraceae_[G-2] caused by PM2.5 exposure. In addition, the relative abundance of Butyrivibrio, a butyric acid-producing genera, significantly increased after PGPI-1-a intervention. These results indicated that PGPI-1-a could improve the imbalance of intestinal microbiota due to PM2.5 exposure to a certain extent.

A specific structure and high richness characterize intestinal microbiota of HIV-exposed seronegative individuals.

Intestinal microbiota facilitates food breakdown for energy metabolism and influences the immune response, maintaining mucosal homeostasis. Overall, HIV infection is associated with intestinal dysbiosis and immune activation, which has been related to seroconversion in HIV-exposed individuals. However, it is unclear whether microbiota dysbiosis is the cause or the effect of immune alterations and disease progression or if it could modulate the risk of acquiring the HIV infection. We characterize the intestinal microbiota and determine its association with immune regulation in HIV-exposed seronegative individuals (HESN), HIV-infected progressors (HIV+), and healthy control (HC) subjects. For this, feces and blood were collected. The microbiota composition of HESN showed a significantly higher alpha (p = 0.040) and beta diversity (p = 0.006) compared to HC, but no differences were found compared to HIV+. A lower Treg percentage was observed in HESN (1.77%) than HC (2.98%) and HIV+ (4.02%), with enrichment of the genus Butyrivibrio (p = 0.029) being characteristic of this profile. Moreover, we found that Megasphaera (p = 0.017) and Victivallis (p = 0.0029) also are enriched in the microbiota composition in HESN compared to HC and HIV+ subjects. Interestingly, an increase in Succinivibrio and Prevotella, and a reduction in Bacteroides genus, which is typical of HIV-infected individuals, were observed in both HESN and HIV+, compared to HC. Thus, HESNs have a microbiota profile, similar to that observed in HIV+, most likely because HESN are cohabiting with their HIV+ partners.

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.

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening.

To increase intramuscular fat accumulation, Japanese Black cattle are commonly fed a high-grain diet from 10 to 30 months of age although it can result in the abnormal accumulation of organic acids in the rumen. We explored the effect of long-term high-concentrate diet feeding on ruminal pH and fermentation, and its effect on the rumen bacterial community in Japanese Black beef cattle during a 20-month fattening period. Nine castrated and fistulated Japanese Black beef cattle were housed with free access to food and water throughout the study period (10-30 months of age). The fattening stages included Early, Middle, and Late stages (10-14, 15-22, and 23-30 months of age, respectively). Cattle were fed high-concentrate diets for the experimental cattle during fattening. The body weight of the cattle was 439 ± 7.6, 561 ± 11.6, and 712 ± 18.5 kg (mean ± SE) during the Early, Middle, and Late stages, respectively. Ruminal pH was measured continuously during the final 7 days of each stage, and rumen fluid and blood samples were collected on day 4 (fourth day during the final 7 days of the pH measurements). The 24-h mean ruminal pH during the Late stage was significantly lower than that during the Early stage. Total volatile fatty acid (VFA) during the Late stage was significantly lower than during the Early and Middle stages, but no changes were noted in individual VFA components. The lactic acid concentration during the Late stage was significantly higher than that during the Early and Middle stages. The bacterial richness indices decreased significantly during the Late stage in accordance with the 24-h mean ruminal pH. Among the 35 bacterial operational taxonomic units (OTUs) shared by all samples, the relative abundances of OTU8 (Family Ruminococcaceae) and OTU26 (Genus Butyrivibrio) were positively correlated with the 24-h mean ruminal pH. Total VFA concentration was negatively correlated with OTU167 (Genus Intestinimonas), and lactic acid concentration was correlated positively with OTU167 and OTU238 (Family Lachnospiraceae). These results suggested that long-term high-grain diet feeding gradually lowers ruminal pH and total VFA production during the Late fattening stage. However, the ruminal bacterial community adapted to feeding management and the lower pH during the Late stage by preserving their diversity or altering their richness, composition, and function, to enhance lactic acid production in Japanese Black beef cattle.

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.

Relative abundance of Megamonas hypermegale and Butyrivibrio species decreased in the intestine and its possible association with the T cell aberration by metabolite alteration in patients with Behcet's disease (210 characters).

OBJECTIVES: We have previously demonstrated that the phylum Actinobacteria, the family Lactobacillaceae, and the genus Bifidobacterium increased in relative abundance of gut microbiota in patients with Behcet's disease (BD). The phylum Firmicutes and the class Clostridia were predominant in the feces of normal individuals. The class Clostridia includes short-chain fatty acid-producing bacteria, important for the balance between regulatory T cells and helper T type 17 (Th17) cells. It is possible that the bacterial compositional alteration causes low intestinal short-chain fatty acid concentrations, leading to skewed immune functions in patients with BD. METHODS: To test the hypothesis, we examined species composition and gene functions from the 16S rRNA data by utilizing PICRUSt software. RESULTS: We have shown that relative abundance of Eggerthella lenta, Acidaminococcus species, Lactobacillus mucosae, Bifidobacterium bifidum, Lactobacillus iners, Streptococcus species, and Lactobacillus salivarius increased significantly in patients with BD. Relative abundance of Megamonas hypermegale, Butyrivibrio species, Streptococcus infantis, and Filifactor species increased significantly in normal individuals compared with BD patients. In the functional annotation analysis by PICRUSt, we found prevalent gene functions of the pentose phosphate pathway and the inosine monophosphate biosynthesis in patients with BD. The data suggested that BD gut microbes altered nucleic acid and fatty acid synthesis. CONCLUSIONS: These compositional and functional alterations of gut microbes may accompany unfavorable molecular exchanges between intestinal immunocompetent cells and gut microbes, and these interactions may have an association with the immune aberration in patients with BD.

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.

Effects of co-inoculating rice straw with ruminal microbiota and anaerobic sludge: digestion performance and spatial distribution of microbial communities.

Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch experiments. The CH4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88-212.88 and 24.51-29.08 U/mL in co-inoculated samples, respectively, and were significantly different compared to the results for single inoculum (p < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase observed in samples with a higher RM ratio. The microbial compositions were clearly altered after digestion. Fibrobacter, Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane production. Syntrophy involving propionate oxidizers with associated methanogens occurred in the liquid phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.

Immunogenic inhibition of prominent ruminal bacteria as a means to reduce lipolysis and biohydrogenation activity in vitro.

Lipolysis and biohydrogenation in ruminal animals promote the accumulation of saturated fatty acids in their meat and milk. Antibodies were generated against key ruminal lipase contributors Anaerovibrio lipolyticus, Butyrivibrio fibrisolvens, Propionibacterium avidum and acnes. An anti-Pseudomonas lipase antibody was generated to determine if an antibody against a purified protein would be more effective. Each bacterium was cultured and assayed without or with increasing levels of each antibody. Butyrivibrio fibrisolvens H17C also participates in biohydrogenation and therefore the antibody was tested to determine if it could effectively reduce biohydrogenation. Butyrivibrio fibrisolvens was assayed without and with the anti-B. fibrisolvens antibody and linoleic or α-linolenic acid. All antibodies were effective at reducing lipolysis with the anti-Pseudomonas lipase averaging a 78% reduction. The anti-B. fibrisolvens showed a tendency for a reduction (P=0.0713) in biohydrogenation products of α-linolenic acid. Results demonstrate that lipolysis and biohydrogenation can be immunologically inhibited in vitro.

The Effect of Probiotics, Antibiotics, and Antipyretic Analgesics on Gut Microbiota Modification

Human gut microbial community is playing a critical role in human health and associated with different human disease. In parallel, probiotics, antibiotics, and antipyretic analgesics (AAs) were developed to improve human health or cure human diseases. We therefore examined how probiotics, antibiotics, and AAs influence to the gut microbiota. Three independent case/control studies were designed from the cross-sectional cohort data of 1,463 healthy Koreans. The composition of the gut microbiota in each case and control group was determined via 16S ribosomal RNA Illumina next-generation sequencing. The correlation between microbial taxa and the consumption of each drug was tested using zero-inflated Gaussian mixture models, with covariate adjustment of age, sex, and body mass index (BMI). Probiotics, antibiotics, and AAs consumption yielded the significant differences in the gut microbiota, represented the lower abundance of Megasphaera in probiotics, the higher abundance of Fusobacteria in antibiotics, and the higher abundance of Butyrivibrio and Verrucomicrobia in AAs, compared to each control group. The reduction of Erysipelotrichaceae family was common in three drugs consumption.

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.

Biohydrogenation of 22:6n-3 by Butyrivibrio proteoclasticus P18.

BACKGROUND: Rumen microbes metabolize 22:6n-3. However, pathways of 22:6n-3 biohydrogenation and ruminal microbes involved in this process are not known. In this study, we examine the ability of the well-known rumen biohydrogenating bacteria, Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18, to hydrogenate 22:6n-3. RESULTS: Butyrivibrio fibrisolvens D1 failed to hydrogenate 22:6n-3 (0.5 to 32 µg/mL) in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Growth of B. fibrisolvens was delayed at the higher 22:6n-3 concentrations; however, total volatile fatty acid production was not affected. Butyrivibrio proteoclasticus P18 hydrogenated 22:6n-3 in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Biohydrogenation only started when volatile fatty acid production or growth of B. proteoclasticus P18 had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n-3. The amount of 22:6n-3 hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n-3 and 22:0. Formation of neither 22:0 nor 22:6 conjugated fatty acids was observed during 22:6n-3 metabolism. Extensive metabolism was observed at lower initial concentrations of 22:6n-3 (5, 10 and 20 µg/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 µg/mL) inhibited its metabolism. Stearic acid formation (18:0) from 18:2n-6 by B. proteoclasticus P18 was retarded, but not completely inhibited, in the presence of 22:6n-3 and this effect was dependent on 22:6n-3 concentration. CONCLUSIONS: For the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n-3. The gradual appearance of intermediates indicates that biohydrogenation of 22:6n-3 by B. proteoclasticus P18 occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated 22 carbon fatty acids. During the simultaneous presence of 18:2n-6 and 22:6n-3, B. proteoclasticus P18 initiated 22:6n-3 metabolism before converting 18:1 isomers into 18:0.

Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers.

This study investigated the effects of ensiled mulberry leaves (EML) and sun-dried mulberry fruit pomace (SMFP) on the ruminal bacterial and archaeal community composition of finishing steers. Corn grain- and cotton meal-based concentrate was partially replaced with EML or SMFP. The diets had similar crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy. Following the feeding trial, the steers were slaughtered and ruminal liquid samples were collected to study the ruminal microbiome. Extraction of DNA, amplification of the V4 region of the 16S rRNA gene, and Illumina MiSeq pyrosequencing were performed for each sample. Following sequence de-noising, chimera checking, and quality trimming, an average of 209,610 sequences were generated per sample. Quantitative real-time PCR was performed to examine the selected bacterial species in the rumen. Our results showed that the predominant phyla were Bacteroidetes (43.90%), Firmicutes (39.06%), Proteobacteria (4.31%), and Tenericutes (2.04%), and the predominant genera included Prevotella (13.82%), Ruminococcus (2.51%), Butyrivibrio (2.38%), and Succiniclasticum (2.26%). Compared to the control group, EML and SMFP groups had a higher abundance of total bacteria (p < 0.001); however, the bacterial community composition was similar among the three groups. At the phylum level, there were no significant differences in Firmicutes (p = 0.7932), Bacteroidetes (p = 0.2330), Tenericutes (p = 0.2811), or Proteobacteria (p = 0.0680) levels among the three groups; however, Fibrobacteres decreased in EML (p = 0.0431). At the genus level, there were no differences in Prevotella (p = 0.4280), Ruminococcus (p = 0.2639), Butyrivibrio (p = 0.4433), or Succiniclasticum (p = 0.0431) levels among the groups. Additionally, the dietary treatments had no significant effects on the archaeal community composition in the rumen. Therefore, EML and SMFP supplementation had no significant effects on the ruminal bacterial or archaeal community composition of finishing steers.

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.