Effect of rice bran fermented with Ligilactobacillus equi on in vitro fermentation profile and microbial population.

This study was conducted to assess the effects of fermented rice bran (FRB) with Ligilactobacillus equi on ruminal fermentation using an in vitro system. Oat hay, corn starch, and wheat bran were used as substrate for control. Ten percent of wheat bran was replaced with rice bran (RB), rice bran fermented with distilled water, and rice bran fermented with L. equi for T1, T2, and T3, respectively. The experimental diets were mixed with buffered rumen fluid from wethers under nitrogen gas and incubated for 24 h at 39°C. The fermentation profile and microbial population were analyzed after the incubations. The results revealed that the RB and FRB (with or without L. equi) significantly reduced the gas, methane (CH4), and CH4 per dry matter digested (p < 0.001). Total short-chain fatty acid was also reduced in T1 and T2 in comparison with the control (p < 0.001). Propionate proportion was increased while butyrate proportion was reduced in response to treatment addition in cultures (p < 0.001). Anaerobic fungi and Fibrobacter succinogenes abundance were decreased in treatments (p < 0.001). Overall, CH4 production in vitro can be reduced by RB and FRB supplementation as a result of the reduction of fiber-degrading microorganisms and a decrease in gas production.

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.

The Phylogenomic Diversity of Herbivore-Associated Fibrobacter spp. Is Correlated to Lignocellulose-Degrading Potential.

Members of the genus Fibrobacter are cellulose-degrading bacteria and common constituents of the gastrointestinal microbiota of herbivores. Although considerable phylogenetic diversity is observed among members of this group, few functional differences explaining the distinct ecological distributions of specific phylotypes have been described. In this study, we sequenced and performed a comparative analysis of whole genomes from 38 novel Fibrobacter strains against the type strains for the two formally described Fibrobacter species F. succinogenes strain S85 and F. intestinalis strain NR9. Significant differences in the number of genes encoding carbohydrate-active enzyme families involved in plant cell wall polysaccharide degradation were observed among Fibrobacter phylotypes. F. succinogenes genomes were consistently enriched in genes encoding carbohydrate-active enzymes compared to those of F. intestinalis strains. Moreover, genomes of F. succinogenes phylotypes that are dominant in the rumen had significantly more genes annotated to major families involved in hemicellulose degradation (e.g., CE6, GH10, and GH43) than did the genomes of F. succinogenes phylotypes typically observed in the lower gut of large hindgut-fermenting herbivores such as horses. Genes encoding a putative urease were also identified in 12 of the Fibrobacter genomes, which were primarily isolated from hindgut-fermenting hosts. Screening for growth on urea as the sole source of nitrogen provided strong evidence that the urease was active in these strains. These results represent the strongest evidence reported to date for specific functional differences contributing to the ecology of Fibrobacter spp. in the herbivore gut.IMPORTANCE The herbivore gut microbiome is incredibly diverse, and a functional understanding of this diversity is needed to more reliably manipulate this community for specific gain, such as increased production in ruminant livestock. Microbial degraders of plant cell wall polysaccharides in the herbivore gut, particularly Fibrobacter spp., are of fundamental importance to their hosts for digestion of a diet consisting primarily of recalcitrant plant fibers. Considerable phylogenetic diversity exists among members of the genus Fibrobacter, but much of this diversity remains cryptic. Here, we used comparative genomics, applied to a diverse collection of recently isolated Fibrobacter strains, to identify a robust association between carbohydrate-active enzyme gene content and the Fibrobacter phylogeny. Our results provide the strongest evidence reported to date for functional differences among Fibrobacter phylotypes associated with either the rumen or the hindgut and emphasize the general significance of carbohydrate-active enzymes in the evolution of fiber-degrading bacteria.

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 ß-galactosidase was low in the presence of 0.1%-0.2% cellobiose. Since the ß-galactosidase contained a signal peptide and a Por secretion system C-terminal sorting domain, we speculate that the ß-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.

Changes in fibre-adherent and fluid-associated microbial communities and fermentation profiles in the rumen of cattle fed diets differing in hay quality and concentrate amount.

The rumen microbiota enable important metabolic functions to the host cattle. Feeding of starch-rich concentrate feedstuffs to cattle has been demonstrated to increase the risk of metabolic disorders and to significantly alter the rumen microbiome. Thus, alternative feeding strategies like the use of high-quality hay, rich in sugars, as an alternative energy source need to be explored. The aim of this study was to investigate changes in rumen microbial abundances in the liquid and solid-associated fraction of cattle fed two hay qualities differing in sugar content with graded amounts of starchy concentrate feeds using Illumina MiSeq sequencing and quantitative polymerase chain reaction. Operational taxonomic units clustered separately between the liquid and the solid-associated fraction. Phyla in the liquid fraction were identified as mainly Firmicutes, Proteobacteria and Bacteroidetes, whereas main phyla of the fibre-associated fraction were Bacteroidetes, Fibrobacteres and Firmicutes. Significant alterations in the rumen bacterial communities at all taxonomic levels as a result of changing the hay quality and concentrate proportions were observed. Several intermicrobial correlations were found. Genera Ruminobacter and Fibrobacter were significantly suppressed by feeding sugar-rich hay, whereas others such as Selenomonas and Prevotella proliferated. This study extends the knowledge about diet-induced changes in ruminal microbiome of cattle.

Dose-response effects of dietary pequi oil on fermentation characteristics and microbial population using a rumen simulation technique (Rusitec).

The effect of increasing the concentration of commercial pequi (Caryocar brasiliense) oil on fermentation characteristics and abundance of methanogens and fibrolityc bacteria was evaluated using the rumen simulation technique (Rusitec). In vitro incubation was performed over 15 days using a basal diet consisting of ryegrass, maize silage and concentrate in equal proportions. Treatments consisted of control diet (no pequi oil inclusion, 0 g/kg DM), pequi dose 1 (45 g/kg DM), and pequi dose 2 (91 g/kg DM). After a 7 day adaptation period, samples for fermentation parameters (total gas, methane, and VFA production) were taken on a daily basis. Quantitative real time PCR (q-PCR) was used to evaluate the abundance of the main rumen cellulolytic bacteria, as well as abundance of methanogens. Supplementation with pequi oil did not reduce overall methane production (P = 0.97), however a tendency (P = 0.06) to decrease proportion of methane in overall microbial gas was observed. Increasing addition of pequi oil was associated with a linear decrease (P < 0.01) in dry matter disappearance of maize silage. The abundance of total methanogens was unchanged by the addition of pequi oil, but numbers of those belonging to Methanomassiliicoccaceae decreased in liquid-associated microbes (LAM) samples (P < 0.01) and solid-associated microbes (SAM) samples (P = 0.09) respectively, while Methanobrevibacter spp. increased (P < 0.01) only in SAM samples. Fibrobacter succinogenes decreased (P < 0.01) in both LAM and SAM samples when substrates were supplemented with pequi oil. In conclusion, pequi oil was ineffective in mitigating methane emissions and had some adverse effects on digestibility and selected fibrolytic bacteria.

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.

Effects of corn silage and grass silage in ruminant rations on diurnal changes of microbial populations in the rumen of dairy cows.

Here, we examined diurnal changes in the ruminal microbial community and fermentation characteristics of dairy cows fed total mixed rations containing either corn silage (CS) or grass silage (GS) as forage. The rations, which consisted of 52% concentrate and 48% GS or CS, were offered for ad libitum intake over 20 days to three ruminal-fistulated lactating Jersey cows during three consecutive feeding periods. Feed intake, ruminal pH, concentrations of short chain fatty acids and ammonia in rumen liquid, as well as abundance change in the microbial populations in liquid and solid fractions, were monitored in 4-h intervals on days 18 and 20. The abundance of total bacteria and Fibrobacter succinogenes increased in solids in cows fed CS instead of GS, and that of protozoa increased in both solid and liquid fractions. Feeding GS favored numbers of F. succinogenes and Selenomonas ruminantium in the liquid fraction as well as the numbers of Ruminobacter amylophilus, Prevotella bryantii and ruminococci in both fractions. Minor effects of silage were detected on populations of methanogens. Despite quantitative changes in the composition of the microbial community, fermentation characteristics were less affected by forage source. These results suggest a functional adaptability of the ruminal microbiota to total mixed rations containing either GS or CS as the source of forage. Diurnal changes in microbial populations were primarily affected by feed intake and differed between species and fractions, with fewer temporal fluctuations evident in the solid than in the liquid fraction. Interactions between forage source and sampling time were of minor importance to most of the microbial species examined. Thus, diurnal changes of microbial populations and fermentative activity were less affected by the two silages.

Shifts in Rumen Fermentation and Microbiota Are Associated with Dissolved Ruminal Hydrogen Concentrations in Lactating Dairy Cows Fed Different Types of Carbohydrates.

BACKGROUND: Different carbohydrates ingested greatly influence rumen fermentation and microbiota and gaseous methane emissions. Dissolved hydrogen concentration is related to rumen fermentation and methane production. OBJECTIVES: We tested the hypothesis that carbohydrates ingested greatly alter the rumen environment in dairy cows, and that dissolved hydrogen concentration is associated with these changes in rumen fermentation and microbiota. METHODS: Twenty-eight lactating Chinese Holstein dairy cows [aged 4-5 y, body weight 480 ± 37 kg (mean ± SD)] were used in a randomized complete block design to investigate effects of 4 diets differing in forage content (45% compared with 35%) and source (rice straw compared with a mixture of rice straw and corn silage) on feed intake, rumen fermentation, and microbial populations. RESULTS: Feed intake (10.7-12.6 kg/d) and fiber degradation (0.584-0.692) greatly differed (P ≤ 0.05) between cows fed the 4 diets, leading to large differences (P ≤ 0.05) in gaseous methane yield (27.2-37.3 g/kg organic matter digested), dissolved hydrogen (0.258-1.64 µmol/L), rumen fermentation products, and microbiota. Ruminal dissolved hydrogen was negatively correlated (r < -0.40; P < 0.05) with molar proportion of acetate, numbers of fungi, abundance of Fibrobacter succinogenes, and methane yield, but positively correlated (r > 0.40; P < 0.05) with molar proportions of propionate and n-butyrate, numbers of methanogens, and abundance of Selenomonas ruminantium and Prevotella spp. Ruminal dissolved hydrogen was positively correlated (r = 0.93; P < 0.001) with Gibbs free energy changes of reactions producing greater acetate and hydrogen, but not correlated with those reactions producing more propionate without hydrogen. CONCLUSIONS: Changes in fermentation pathways from acetate toward propionate production and in microbiota from fibrolytic toward amylolytic species were closely associated with ruminal dissolved hydrogen in lactating dairy cows. An unresolved paradox was that greater dissolved hydrogen was associated with greater numbers of methanogens but with lower gaseous methane emissions.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW. © 2016 Society of Chemical Industry.

Rumen microbial abundance and fermentation profile during severe subacute ruminal acidosis and its modulation by plant derived alkaloids in vitro.

Rumen microbiota have important metabolic functions for the host animal. This study aimed at characterizing changes in rumen microbial abundances and fermentation profiles using a severe subacute ruminal acidosis (SARA) in vitro model, and to evaluate a potential modulatory role of plant derived alkaloids (PDA), containing quaternary benzophenanthridine and protopine alkaloids, of which sanguinarine and chelerythrine were the major bioactive compounds. Induction of severe SARA strongly affected the rumen microbial composition and fermentation variables without suppressing the abundance of total bacteria. Protozoa and fungi were more sensitive to the low ruminal pH condition than bacteria. Induction of severe SARA clearly depressed degradation of fiber (P < 0.001), which came along with a decreased relative abundance of fibrolytic Ruminococcus albus and Fibrobacter succinogenes (P < 0.001). Under severe SARA conditions, the genus Prevotella, Lactobacillus group, Megasphaera elsdenii, and Entodinium spp. (P < 0.001) were more abundant, whereas Ruminobacter amylophilus was less abundant. SARA largely suppressed methane formation (-70%, P < 0.001), although total methanogenic 16S rRNA gene abundance was not affected. According to principal component analysis, Methanobrevibacter spp. correlated to methane concentration. Addition of PDA modulated ruminal fermentation under normal conditions such as enhanced (P < 0.05) concentration of total SCFA, propionate and valerate, and increased (P < 0.05) degradation of crude protein compared with the unsupplemented control diet. Our results indicate strong shifts in the microbial community during severe SARA compared to normal conditions. Supplementation of PDA positively modulates ruminal fermentation under normal ruminal pH conditions.

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.

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by ß-glucanases and other cellulases.

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.

Microbial populations and fermentation profiles in rumen liquid and solids of Holstein cows respond differently to dietary barley processing.

AIMS: To evaluate the effects of treating barley grain with lactic acid (LA) and heat on postprandial dynamics of 19 microbial taxa and fermentation in the rumen of dairy cows. METHODS AND RESULTS: This study was designed as a double 3 × 3 Latin square with six rumen-cannulated cows and three diets either containing untreated control barley or barley treated with 1% LA and 1% LA and heat (LAH, 55°C). Microbial populations, pH and volatile fatty acids were assessed in rumen liquid and solids during the postprandial period. Propionate increased and butyrate decreased in rumen solids of cows fed LA and LAH treated barley compared to the control barley. The LA but not LAH treatment depressed Fibrobacter succinogenes in rumen liquid and solids, whereas the opposite effect was observed for Ruminococcus albus in both fractions and Ruminococcus flavefaciens in rumen solids. LA promoted Ruminobacter amylophilus with the effect being more pronounced with LAH. The Lactobacillus group and Megasphaera elsdenii increased in both fractions with LA but not with LAH. CONCLUSIONS: LA and LAH treatment of barley differently altered ruminal abundance of certain bacterial taxa and fungi and increased propionate fermentation in rumen solids, whereby LA and LAH effects were consistent and mostly independent of the rumen fraction and time after barley feeding. SIGNIFICANCE AND IMPACT OF THE STUDY: Results provided evidence that LA and LAH treatment of barley can enhance rumen propionate fermentation without adversely affecting rumen pH. As propionate is the major contributor to gluconeogenesis in ruminants, the present barley treatment may have practical application to enhance energy supply in dairy cows.

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.

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.

Integrated 'omics analysis for studying the microbial community response to a pH perturbation of a cellulose-degrading bioreactor culture.

Integrated 'omics have been used on pure cultures and co-cultures, yet they have not been applied to complex microbial communities to examine questions of perturbation response. In this study, we used integrated 'omics to measure the perturbation response of a cellulose-degrading bioreactor community fed with microcrystalline cellulose (Avicel). We predicted that a pH decrease by addition of a pulse of acid would reduce microbial community diversity and temporarily reduce reactor function in terms of cellulose degradation. However, 16S rDNA gene pyrosequencing results revealed increased alpha diversity in the microbial community after the perturbation, and a persistence of the dominant community members over the duration of the experiment. Proteomics results showed a decrease in activity of proteins associated with Fibrobacter succinogenes 2 days after the perturbation followed by increased protein abundances 6 days after the perturbation. The decrease in cellulolytic activity suggested by the proteomics was confirmed by the accumulation of Avicel in the reactor. Metabolomics showed a pattern similar to that of the proteome, with amino acid production decreasing 2 days after the perturbation and increasing after 6 days. This study demonstrated that community 'omics data provide valuable information about the interactions and function of anaerobic cellulolytic community members after a perturbation.

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.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.