Succinogenes S85 Research Articles

Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.

Dynamic genome-based metabolic modeling of the predominant cellulolytic rumen bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is a cellulolytic bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the automatic reconstruction of metabolic model workspace. The reconstruction was based on genome annotation, five template-based orthology methods, gap filling, and manual curation. The metabolic network of F. succinogenes S85 comprises 1,565 reactions with 77% linked to 1,317 genes, 1,586 unique metabolites, and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for the computation of elementary flux modes. A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the root mean squared error of 19%. The resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step toward the integration of omics microbial information into predictive models of rumen metabolism. IMPORTANCE F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model of rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.

Polyclonal antibodies inhibit growth of key cellulolytic rumen bacterial species.

Antibodies targeting specific bacterial species could allow for modification of the rumen microbial population to enhance rumen fermentation. However, there is limited knowledge of targeted antibody effects on rumen bacteria. Therefore, our objective was to develop efficacious polyclonal antibodies to inhibit the growth of targeted cellulolytic bacteria from the rumen. Egg-derived, polyclonal antibodies were developed against pure cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium for each of the three targeted species. Antibody efficacy was determined via inoculation time (0 h and 4 h) and dose response. Antibody doses included: 0 (CON), 1.3 × 10-4 (LO), 0.013 (MD), and 1.3 (HI) mg antibody per ml of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density and total acetate concentration after a 52 h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P < 0.05) in live bacterial cells during the mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P < 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total acetate accumulation after 52 h incubation, indicating that antibodies have less of an inhibitory effect on non-target strains. Addition of anti-FS85 to non-cellulolytic strains did not affect (P ≥ 0.89) OD, substrate disappearance, or total VFA concentrations, providing further evidence of specificity against fiber-degrading bacteria. Western blotting with anti-FS85 indicated selective binding to F. succinogenes S85 proteins. Identification by LC-MS/MS of 8 selected protein spots indicated 7 were outer membrane proteins. Overall, polyclonal antibodies were more efficacious at inhibiting the growth of targeted cellulolytic bacteria than non-targeted bacteria. Validated polyclonal antibodies could serve as an effective approach to modify rumen bacterial populations.

207 Polyclonal Antibodies Inhibit Growth of Key Cellulolytic Rumen Bacterial Species

Abstract The objective was to develop efficacious polyclonal antibodies to inhibit growth of targeted cellulolytic rumen bacterial species. Many rumen microorganisms have overlapping functions, so inhibiting the activity of targeted bacterial species may help elucidate their dynamic contributions to rumen fermentation. Egg-derived, polyclonal antibodies were developed against whole-cell cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium of pure cultures of the 3 targeted species. Antibody efficacy was determined via an inoculation time (0 and 4 h) and dose response. Antibody doses included: 0 (CON), 0.013 (LO), 1.3 (MD), 13 (HI) mg antibody per 10 mL of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P &amp;lt; 0.01) final optical density and total volatile fatty acid (VFA) concentration after a 52-h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P &amp;lt; 0.05) of live bacterial cells during mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P &amp;lt; 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total VFA accumulation after 52 h incubation. Overall, polyclonal antibodies were more efficacious at inhibiting growth of targeted cellulolytic bacteria than non-targeted bacteria. Specific polyclonal antibodies can be used to further characterize functional contributions of bacterial species to rumen fermentation.

Effects of yeast and yeast cell wall polysaccharides supplementation on beef cattle growth performance, rumen microbial populations and lipopolysaccharides production

This experiment was conducted to investigate the effects of live yeast and yeast cell wall polysaccharides on growth performance, rumen function and plasma lipopolysaccharides (LPS) content and immunity parameters of beef cattle. Forty Qinchuan cattle were randomly assigned to one of four treatments with 10 replicates in each treatment. The dietary treatments were: control diet (CTR), CTR supplemented with 1 g live yeast (2×1010 live cell g−1 per cattle per day (YST1), CTR supplemented with 2 g live yeast per cattle per day (YST2) and CTR supplemented with 20 g of yeast cell wall polysaccharides (30.0%≤β-glucan≤35.0%, and 28.0%≤mannanoligosaccharide≤32.0%) per cattle per day (YCW). The average daily gain was higher (P=0.023) and feed conversion ratio was lower (P=0.042) for the YST2 than the CTR. The digestibility of neutral detergent fiber (P=0.039) and acid detergent fiber (P=0.016) were higher in yeast supplemented groups. The acetic acid:propionic acid of the YST2 was lower compared with the CTR (P=0.033). Plasma LPS (P=0.032), acute phase protein haptoglobin (P=0.033), plasma amyloid A (P=0.015) and histamine (P=0.038) were lower in the YST2 compared with the CTR. The copies of fibrolytic microbial populations such as Fibrobacter succinogenes S85, Ruminococcus albus 7 and Ruminococcus flavefaciens FD-1 of the YST2 were higher (P<0.001), while the copies of typical lactate producing bacteria Streptococcus bovis JB1 was lower (P<0.001) compared with the CTR. Little differences were observed between the CTR, YST1 and YCW in growth performance, ruminal fermentation characteristics, microbial populations, immunity indices and total tract nutrient digestibility. It is concluded that the YST2 could promote fibrolytic microbial populations, decrease starch-utilizing bacteria, reduce LPS production in the rumen and LPS absorption into plasma and decrease inflammatory parameters, which can lead to an improvement in growth performance in beef cattle.

Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels.

Alcoholic fermentation of thermochemical and biological hydrolysates derived from Miscanthus biomass by Clostridium acetobutylicum ATCC 824

This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biological saccharification of Miscanthus giganteus (MG) for generation of fermentable saccharides and its subsequent fermentation into solvents i.e. acetone, ethanol and butanol (ABE) using Clostridium acetobutylicum ATCC 824. Saccharide hydrolysates were derived from MG by thermochemical (water, acid and alkali at 130 °C) and biological saccharification (Fibrobacter succinogenes S85) processes and were subjected to batch fermentation for 120 h using C. acetobutylicum ATCC 824. At the end of fermentation of thermochemically-derived hydrolysates, 742 g m−3 of saccharides from water treatment, 9572 g m−3 of saccharides from acid treatment and 4054 g m−3 of saccharides from alkali treatment were fermented and yielded 0.045, 0.0069 and 0.01 g g−1 of total solvents, respectively. Similarly, at the end of fermentation of biological hydrolysate (using F. succinogenes), 2504 g m−3 of saccharides was fermented and yielded 0.091 g g−1 of total solvents. The highest yield of total solvents was achieved by water (thermochemical) and biological saccharification of MG using C. acetobutylicum. Whereas, acid and alkali-treated hydrolysates showed lower yields of solvents presumably due to production of inhibitory compounds during saccharification. Compared to thermochemical saccharification, biological saccharification using F. succinogenes is a promising approach since it yielded the highest amount of solvents whilst being eco-friendly. Our future studies will focus on optimisation of biological saccharification (using F. succinogenes) and sequential co-culture fermentation (using C. acetobutylicum). The development of alternative consolidated bioprocessing approach using biological saccharification will contribute towards making lignocellulosic biofuels a reality.

A global analysis of gene expression in Fibrobacter succinogenes S85 grown on cellulose and soluble sugars at different growth rates

BackgroundCellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium.ResultsSeveral patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose.ConclusionsThis work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.

FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota

Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

Biochanin A improves fibre fermentation by cellulolytic bacteria.

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity. When bovine microbial rumen cell suspensions (n=3) were incubated (24h, 39°C) with ground hay, cellulolytic bacteria proliferated, short-chain fatty acids were produced and pH declined. BCA (30μgml-1 ) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n=3) were incubated (48h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent. These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition. BCA could serve as a feed additive to improve cellulosis when cattle are consuming high-fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization invivo.

Diauxic growth of Fibrobacter succinogenes S85 on cellobiose and lactose.

Fibrobacter succinogenes rapidly colonizes the preruminant calf rumen and becomes a dominant cellulolytic bacterium in the rumen after weaning. Although F. succinogenes actively degrades cellulose in the rumen, it seems that there is no or little of its substrate, cellulose, in the rumen of preweaned calves. We thus evaluated the ability of F. succinogenes to utilize lactose, a main sugar of milk, with or without the presence of cellobiose. We grew F. succinogenes S85 on media containing 2.5% lactose combined with 0%-0.2% cellobiose or a medium with 0.2% cellobiose but without lactose. The generation times on the 0.2% cellobiose medium and the 2.5% lactose medium were 1.9 and 16.2 h, respectively. The bacterium showed rapid growth on cellobiose and diauxic growth on the lactose media containing 0.05%-0.2% cellobiose. Moreover, the production of β-galactosidase was low in the presence of 0.1%-0.2% cellobiose. Since the β-galactosidase contained a signal peptide and a Por secretion system C-terminal sorting domain, we speculate that the β-galactosidase would be secreted from the bacterial cells by the Por secretion system. Our data indicate the possibility that F. succinogenes could colonize preruminant calf rumen, consuming the lactose present in cow milk.

Generation and Characterization of Acid Tolerant Fibrobacter succinogenes S85

Microorganisms are key components for plant biomass breakdown within rumen environments. Fibrobacter succinogenes have been identified as being active and dominant cellulolytic members of the rumen. In this study, F. succinogenes type strain S85 was adapted for steady state growth in continuous culture at pH 5.75 and confirmed to grow in the range of pH 5.60–5.65, which is lower than has been reported previously. Wild type and acid tolerant strains digested corn stover with equal efficiency in batch culture at low pH. RNA-seq analysis revealed 268 and 829 genes were differentially expressed at pH 6.10 and 5.65 compared to pH 6.70, respectively. Resequencing analysis identified seven single nucleotide polymorphisms (SNPs) in the sufD, yidE, xylE, rlmM, mscL and dosC genes of acid tolerant strains. Due to the absence of a F. succinogenes genetic system, homologues in Escherichia coli were mutated and complemented and the resulting strains were assayed for acid survival. Complementation with wild-type or acid tolerant F. succinogenes sufD restored E. coli wild-type levels of acid tolerance, suggesting a possible role in acid homeostasis. Recent genetic engineering developments need to be adapted and applied in F. succinogenes to further our understanding of this bacterium.

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85.

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.

Biogas production from brewery spent grain enhanced by bioaugmentation with hydrolytic anaerobic bacteria

Lignocellulosic substrates are widely available but not easily applied in biogas production due to their poor anaerobic degradation. The effect of bioaugmentation by anaerobic hydrolytic bacteria on biogas production was determined by the biochemical methane potential assay. Microbial biomass from full scale upflow anaerobic sludge blanket reactor treating brewery wastewater was a source of active microorganisms and brewery spent grain a model lignocellulosic substrate. Ruminococcus flavefaciens 007C, Pseudobutyrivibrio xylanivorans Mz5T, Fibrobacter succinogenes S85 and Clostridium cellulovorans as pure and mixed cultures were used to enhance the lignocellulose degradation and elevate the biogas production. P. xylanivorans Mz5T was the most successful in elevating methane production (+17.8%), followed by the coculture of P. xylanivorans Mz5T and F. succinogenes S85 (+6.9%) and the coculture of C. cellulovorans and F. succinogenes S85 (+4.9%). Changes in microbial community structure were detected by fingerprinting techniques.

Monitoring of gene expression in Fibrobacter succinogenes S85 under the co-culture with non-fibrolytic ruminal bacteria.

Fibrobacter succinogenes is one of the most pivotal fibrolytic bacterial species in the rumen. In a previous study, we confirmed enhancement of fiber digestion in a co-culture of F. succinogenes S85 with non-fibrolytic ruminal strains R-25 and/or Selenomonas ruminantium S137. In the present study, mRNA expression level of selected functional genes in the genome of F. succinogenes S85 was monitored by real-time RT-PCR. Growth profile of F. succinogenes S85 was similar in both the monoculture and co-cultures with non-fibrolytics. However, expression of 16S rRNA gene of F. succinogenes S85 in the co-culture was higher (P < 0.01) than that of the monoculture. This finding suggests that metabolic activity of F. succinogenes S85 was enhanced by coexistence with strains R-25 and/or S. ruminantium S137. The mRNA expression of fumarate reductase and glycoside hydrolase genes was up-regulated (P < 0.01) when F. succinogenes S85 was co-cultured with non-fibrolytics. These results indicate the enhancement of succinate production and fiber hydrolysis by F. succinogenes S85 in co-cultures of S. ruminantium and R-25 strains.

Magnesium requirement of some of the principal rumen cellulolytic bacteria

Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (P<0.001). The requirement for R. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH3-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.

English

This study determined the effects of aromatic amino acids (AA) and phenyl acids (phenylacetic acid and phenylpropionic acid) on fermentation of xylan by selected rumen bacteria. The ruminal bacteria were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia (no AA); ammonia plus a complete mixture of 20 AA commonly found in protein (CAA); ammonia plus complete AA mixture minus aromatic AA (MAR); ammonia plus phenyl acids (no AA+PA); ammonia plus complete AA mixture without aromatic AA plus phenyl acids (MAR+PA). There was varied growth response, with Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, and Butyrivibrio fibrisolvens JW11 not being able to grow. The supply of CAA mixture significantly (P&lt;0.05) increased acetate production in all species, in comparison with no AA treatment, by 54, 30, 97 and 33% in Prevotella bryantii B14, Ruminococcus albus SY3, R. flavefaciens 17 and Pseudobutyrivibrio xylanivorans JK170 cultures, respectively. The addition of phenyl acids significantly (P&lt;0.05) increased acetate yields by 34, 8 and 5% in P. bryantii B14, R. albus SY3 and P. xylanivorans JK170 cultures, respectively. Sugar utilization by all the species significantly (P&lt;0.05) increased by 42 and 77% when CAA mixture was provided. The deletion of aromatic AA from the complete AA mixture caused a significant (P&lt;0.05) reduction in sugar utilization in P. bryantii B14 and P. xylanivorans JK170 by 25 and 10%, respectively. Phenyl acids addition increased sugar utilization by 4.8 and 5.1% in R. albus SY3 and R. flavefaciens 17, respectively. The supply of CAA mixture significantly (P&lt;0.05) increased microbial yield in P. bryantii B14, R. albus SY3, R. flavefaciens 17 and P. xylanivorans JK170 species. It is concluded that supplementation of complete mixture of AA is beneficial in stimulating bacterial growth in the rumen. &nbsp; Key words: Aromatic amino acids, phenylacetic acid, phenylpropionic acid, rumen bacteria, xylan.

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.

Involvement of recently cultured group U2 bacterium in ruminal fiber digestion revealed by coculture withFibrobacter succinogenesS85

In a previous study, we reported the ecological significance of uncultured bacterial group U2 in the rumen. In this study, the involvement of a recently cultured group U2 bacterium, strain R-25, in fiber digestion was tested in coculture with the fibrolytic bacterium Fibrobacter succinogenes S85. Dry matter (DM) digestion, growth and metabolites were examined in culture using rice straw as the carbon source. Although strain R-25 did not digest rice straw in monoculture, coculture of strain R-25 and F.succinogenes S85 showed enhanced DM digestion compared with that for F.succinogenes S85 monoculture (36.9±0.6% vs. 32.8±1.3%, P<0.05). Growth of strain R-25 and production of the main metabolites, d-lactate (strain R-25) and succinate (F.succinogenes S85), were enhanced in the coculture. Enzyme assay showed increased activities of carboxymethylcellulase and xylanase in coculture of strain R-25 and F.succinogenes S85. Triculture including strain R-25, F.succinogenes S85 and Selenomonas ruminantium S137 showed a further increase in DM digestion (41.8±0.8%, P<0.05) with a concomitant increase in propionate, produced from the conversion of d-lactate and succinate. These results suggest that the positive interaction between strains R-25 and F.succinogenes S85 causes increased rice straw digestion.

Screening of a Xylanase Clone from a Fosmid Library of Rumen Microbiota in Hu Sheep

The glycosyl hydrolase family 11, which is responsible for carbohydrate metabolism, was identified in the open reading frame (ORF) 6 of a xylanase positive clone from a fosmid library of rumen microbiota of Hu sheep. A BLASTP search of GenBank revealed that ORF6 encoded a 355-amino acid putative endoxylanase, having 61% similarity (e−73) to endo-1,4-β-xylanase of Fibrobacter succinogenes S85 (YP_003250510.1). Predicted with the SWISS-MODEL, there were two separate β-sandwich clusters linked with a high serine containing linker in ORF6. The N-terminal β-sandwich is a novel endoxylanase of the glycosyl hydrolase family 11 with a specific activity of 1150.00 U/mg. The optimal pH and temperature for this enzyme were shown to be pH 5.0 and 50°C, respectively. The C-terminal helped increase the stability of the xylanase but decreased the activity to some degree. The C-terminal β-sandwich could bind avicel, but no conserved domain could be found. It may be a novel carbohydrate-binding module.