Synergistic fibrolysis in the rumen by cellulolytic Ruminococcus flavefaciens and non-cellulolytic Selenomonas ruminantium: Evidence in defined cultures

To investigate the rumen bacterial interaction between cellulolytic Ruminococcus flavefaciens and non‐cellulolytic Selenomonas ruminantium, fiber digestibility and fermentation products were determined in defined cultures consisting of these two species. Avicel, orchardgrass hay, rice straw and alfalfa hay were used as substrates for 72 h incubation to monitor digestibility, volatile fatty acids, succinate, lactate and bacterial number. In monoculture, R. flavefaciens digested the fiber sources at 21–32%, while S. ruminantium strains did not. When R. flavefaciens was cocultured with one of three different strains (GA192, S137 and S150) of S. ruminantium, fiber digestion exceeded the value recorded by R. flavefaciens alone. In particular, cocultures with S. ruminantium S137 showed significantly higher digestibility for all the fiber sources than R. flavefaciens alone (P < 0.05). Propionate production and growth of S. ruminantium was notable in all cocultures but not in monocultures. Succinate was accumulated in monoculture of R. flavefaciens, while the accumulation was not observed in cocultures. These results indicate that R. flavefaciens provides fiber hydrolysis products to S. ruminantium as growth substrates. In addition, S. ruminantium could activate R. flavefaciens by rapidly consuming the products. Such cross‐feeding between cellulolytic and non‐cellulolytic bacteria could enhance fiber digestion, although the extent of the enhancement may depend on strain combinations.     

Molecular cloning, nucleotide sequence and characteristics of a xylanase gene (xynA) from Ruminococcus albus 7

A 2.6-kb DNA fragment encoding a xylanase gene ( xyn A) was cloned from the rumen hemicellulolytic bacterium Ruminococcus albus 7. The deduced primary structure of the protein (XynA) was divided into a signal peptide region and 3 domains. Domain A was identified as a family 11 (G) catalytic domain, but one amino acid residue was replaced by another in an active site signature 1 of family 11. Domain B is a stabilizing domain for the catalytic domains of families 10 and 11. Deletion of domain B reduced stability of the xylanase at high temperature and at high and low pH. Domain B may be useful for protein engineering of xylanase. Domain C has sequence similarity to deacetylases and NodB proteins.     

Microbial population in the rumen of swamp buffalo (Bubalus bubalis) as influenced by coconut oil and mangosteen peel supplementation

Four, rumen fistulated swamp buffalo bulls were used to study microbial populations in the rumen when supplemented with coconut oil and mangosteen peel. Animals were randomly assigned to a 4 × 4 Latin square design. Four treatments were un‐supplemented (Control), supplementation with coconut oil at 50 g/kg (CO5), supplementation with mangosteen peel at 30 g/kg (MP3) and supplementation with CO5 and MP3 (COM), of total DM intake. Animals received concentrate at 10 g/kg of BW, and rice straw was given ad libitum. Abundance of total bacteria was increased by CO5 supplementation, whereas populations of protozoa and Fibrobacter succinogenes were reduced by CO5 and COM supplementation. Dietary supplementation did not affect methanogen, Ruminococcus flavefaciens or Ruminococcus albus abundances. Dietary treatments changed denaturing gradient gel electrophoresis (DGGE) band patterns of methanogens and protozoa when compared with the control group, especially when supplemented with MP3. Supplementation of COM resulted in the greatest difference in pattern of DGGE bands for total bacteria compared with the control. Coconut oil and mangosteen peel supplementation resulted in changing of rumen microbial abundances and communities; however, combination of them could be more benefit to improve rumen fermentation of swamp buffalo fed on rice straw.     

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes . Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes . DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis , Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii , B. fibrisolvens , Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes -like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.     

Partial characterization of structure and function of a xylanase gene from the rumen hemicellulolytic bacterium Eubacterium ruminantium

A gene encoding for xylanase activity in the rumen hemicellulolytic bacterium Eubacterium ruminantium was cloned into pBR322 in Escherichia coli (E. coli ). The primary clone had a 5.7 kb insert produced by Eco RI partial digestion. Subcloning followed by sequencing allowed for the discovery that this enzyme has a glycosyl‐hydrolase family 10 catalytic domain with a family 9 carbohydrate binding module at C‐terminus and a region partially homologous to a family 22 carbohydrate binding module at N‐terminus. Cloned xylanase is specifically active against xylan and oligoxyloside to produce xylobiose and xylotriose, showing optimal pH and temperature at 7.0 and 50°C, respectively. Molecular size of the xylanase (91 kDa) was confirmed by zymogram analysis of the E. coli clone, which agreed with the predicted size from the DNA sequence. Functions of the two modules at C‐ and N‐termini were evaluated by using xylanase variants with and without the respective module and the C‐terminal module was found to be functional in binding to acid‐swollen cellulose and insoluble oat‐spelt xylan, whereas the N‐terminal module was inactive for binding them.     

Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali‐treated rice straw

Colonization patterns of representative rumen bacteria were compared between untreated rice straw (UTS) and sodium hydroxide‐treated rice straw (SHTS). UTS and SHTS were incubated in the rumen of sheep for 10 min, 1, 2, 6, 12, 24, 48 and 96 h using the nylon bag method. The population sizes of 13 representative bacterial species or groups were quantified by real‐time PCR. The total bacterial population size (abundance) was similar in both UTS and SHTS. Fibrobacter succinogenes showed a higher population size compared to other fibrolytic species and was detected at a higher level in SHTS (3.7%) than in UTS (2.6%). Ruminococcus albus and Ruminococcus flavefaciens were also detected at higher levels in SHTS (0.15% and 0.29%) than in UTS (0.03% and 0.18%). Population sizes of non‐fibrolytic species, such as Selenomonas ruminantium, Anaerovibrio lipolytica and Succinivibrio dextrinosolvens were higher in UTS than in SHTS. Coefficient of determination (r²) on population changes between bacterial species or groups were higher in UTS than in SHTS, suggesting the necessity of stronger bacterial interactions for UTS digestion. Therefore, not only colonization of fibrolytic species, but also synergistic interactions between different bacterial species may be key to the ruminal digestion of rice straw.     

Ecological characterization of three different phylogenetic groups belonging to the cellulolytic bacterial species Fibrobacter succinogenes in the rumen

To study the group‐dependent ecology of Fibrobacter succinogenes in the rumen, real‐time polymerase chain reaction assays for two phylogenetic groups (groups 2 and 3) of F. succinogenes were newly established and applied to rumen samples. Both the assays targeting the bacterial 16S rDNA were sensitive and accurate, showing wide quantifiable ranges (10⁴?10⁹ and 10²?10⁹ copies of 16S rDNA) and high recoveries of known amounts of added DNA (96.9 and 98.0%). The quantity of group 1 was confirmed to be numerable by subtracting assay values of groups 2 and 3 from that of F. succinogenes species (groups 1–3). By using the developed assays and the above subtractive calculation, the quantities of all three groups were evaluated in solid and liquid fractions of the rumen content and also on hay stems. In the solid fraction, groups 1 and 2 were abundantly present, compared with group 3 (P < 0.05). On untreated hay stems, group 1 was dominant throughout 48 h. In addition, group 1 showed growth even on the cellulase‐treated hay stems, unlike the other two groups. These results suggest that F. succinogenes group 1 greatly contributes to rumen fiber digestion, even for less degradable materials.     

Seasonal differences in rumen bacterial flora of wild Hokkaido sika deer and partial characterization of an unknown bacterial group possibly involved in fiber digestion in winter

Rumen digesta was obtained from wild Hokkaido sika deer to compare bacterial flora between summer and winter. Bacterial flora was characterized with molecular‐based approaches and enrichment cultivation. Bacteroidetes was shown as a major phylum followed by Firmicutes, with similar proportions in both seasons. However, two phylogenetically unique groups in Bacteroidetes were found in each season: unknown group A in winter and unknown group B in summer. The ruminal abundance of unknown group A was the highest followed by Ruminococcus flavefaciens in winter. Moreover, the abundance of these two was higher in winter than in summer. In contrast, the abundance of unknown group B was higher in summer than in winter. In addition, this group showed the highest abundance in summer among the bacteria quantified. Unknown group A was successfully enriched by cultivating with oak bark and sterilized rumen fluid, particularly that from deer. Bacteria of this group were distributed in association with the solid rather than the liquid rumen fraction, and were detected as small cocci. Accordingly, unknown group A is assumed to be involved in degradation of fibrous materials. These results suggest that wild Hokkaido sika deer develop a rumen bacterial flora in response to changes in dietary conditions.     

Use of bean husk as an easily digestible fiber source for activating the fibrolytic rumen bacterium Fibrobacter succinogenes and rice straw digestion

A series of in sacco and in vitro studies were carried out to evaluate bean husks for activation of fibrolytic rumen bacteria and rice straw digestion. First, lablab bean husk, chickpea husk and rice straw were suspended in the rumen of sheep to analyze the bacterial consortium developed on each fiber source. Known members of fiber‐associating bacteria were found on both lablab bean husk and rice straw, but some of these bacteria were lacking on chickpea husk. Second, a pure culture study was carried out using six strains of Fibrobacter succinogenes. Both husks stimulated the growth of all tested strains, including a strain that did not grow on rice straw. The strain OS128 that showed the highest growth on rice straw displayed even higher growth on lablab bean husk without a time lag. Finally, two‐step incubations were carried out to determine whether prior incubation of rumen fluid with husks stimulates subsequent rice straw digestion. Higher digestibility of rice straw was recorded in the second‐round incubation following the first incubation with bean husks. These results suggest that the tested bean husks improve the digestion of rice straw by activating fibrolytic F. succinogenes and other associated bacteria.     

Assessment of pregnancy‐associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera

Pregnancy‐associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)‐PAG systems: RIA‐1 (antiserum raised against bovine PAG67kDa; AS#497), RIA‐2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA‐3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA‐2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA‐2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA‐3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA‐1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG‐RIA systems to measure PAG concentration in swamp buffalo samples.     

Contribution of different rumen microbial groups to gas,short‐chain fatty acid and ammonium production from different diets—an approach in an in vitro fermentation system

In this study, the relative contribution of different microbial groups to ruminal metabolism was investigated for different diets. The rumen microbial cultures included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi and were established by physical and chemical methods. Gas production, short‐chain fatty acid (SCFA) and ammonium production were measured at 24 hr in in vitro incubations using the Hohenheim gas test (HGT) procedure. Seven donor animal diets with different concentrate‐to‐roughage ratios (C:R: 10:90, 30:70, 50:50, 70:30, 70:30BC (BC = NaHCO₃), 90:10 and 90:10BC) and five HGT diets (C:R: 10:90, 30:70, 50:50, 70:30 and 90:10) were formulated. Incubations in the HGT were always based on inoculum from sheep diets with the respective C:R ratio. Gas and ammonium production increased (p < 0.001) as a result of a gradual increase in concentrate proportion of the diets. In general, SCFA production followed the same trend. Whole rumen fluid and bacteria + fungi produced approximately 50% higher gas volume than protozoa and fungi + protozoa fractions, whereas gas production with bacteria + protozoa was at an intermediate level. Coculture of protozoa either with bacteria or with fungi produced more ammonium. Populations without bacteria were characterized by a particularly high acetate/propionate ratio. Although an interaction between microbial group and diet was observed for several variables, no clear direction could be established. Manipulating rumen fluid by selectively suppressing specific rumen microbial groups may be a helpful tool in elucidating their role in nutrient degradation and turnover in vitro.     

Pediococcus acidilactici isolated from the rumen of lambs with rumen acidosis, 16S rRNA identification and sensibility to monensin and lasalocid

A lactic-acid producing bacterium was isolated from the rumen of lambs with rumen acidosis. The cells were Gram-positive, nonmotile, nonsporing, catalase negative spherical, 1.5-2.0 μm in diameter, and occur in pairs and tetrads. Analysis of 16S ribosomal RNA indicated that the rumen bacterium was a strain of Pediococcus acidilactici with 99% of nucleotide homology. This bacterium was sensible to monensin and lasalocid at the unique dose tested of 300 ppm. The concentration of lactic acid and DM degradation decreased (P < 0.05) when monensin or lasalocid were added to the culture media after 24, 48 and 72 h of incubation. In contrast, total VFA concentration and pH were higher (P < 0.05) in the culture media added with the ionophores. Up to now S. bovis is considered the main ruminal bacterium related with rumen acidosis, but the importance of P. acidilactici should be also reconsidered in experimental studies focused on the control rumen acidosis.     

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10–14, 15–22, and 23–30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, “Biosynthesis of secondary metabolites” was significantly higher in the fluid fraction than in the solid fraction. “Two-component system” in the middle stage was significantly lower and “Purine metabolism” in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as “Biosynthesis of secondary metabolites” and “Sugar metabolism.” Moreover, the solid fraction was correlated with “Purine metabolism” and “Biosynthesis of secondary metabolism”. These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.