Propionibacterium ruminifibrarum sp. nov., isolated from cow rumen fibrous content.

A novel propionate producing bacterium, strain JV5T, was isolated from the rumen fibrous content of a Holstein Friesian dairy cow. Cells of strain JV5T were Gram-stain-positive, non-motile and aerotolerant. Growth occurred between 35 and 45 °C, with an optimum at 39 °C. The pH range for growth was 6.5-8, with an optimum at pH 7. The 16S rRNA gene sequence of strain JV5T was 98.4 and 96.5 % identical to those of Propionibacterium australiense DSM 15818T and Propionibacterium acidifaciens DSM 21887T, respectively. Genome wide average nucleotide identity and digital DNA-DNA hybridization values were 88.3 and 35.5 %, respectively, against P. australiense DSM 15818T. The G+C content of strain JV5T was 68.9  mol%. Strain JV5T did not produce urease and was able to metabolize glutamate, but not aspartate and glycine. Strain JV5T was able to ferment a range of substrates including certain simple and complex carbohydrates, sugar alcohols and amino acids. Chemotaxonomic analysis of strain JV5T revealed the presence of meso-diamino pimelic acid isomers similar those found in P. australiense, but different from P. acidifaciens. The observed major (>10 %) cellular fatty acids in strain JV5T (C18 : 1 ω9c, anteiso-C15 : 1, C16 : 0, C17 : 0 and C16 : 0 alcohol) were also different from those observed in P. australiense and P. acidifaciens. Based on these findings, a novel species is proposed within the genus Propionibacterium, Propionibacterium ruminifibrarum sp. nov. (type strain JV5T=DSM 106771T=TISTR 2629T).

Characterization of dairy cow rumen bacterial and archaeal communities associated with grass silage and maize silage based diets.

The objective of the present study was to characterize the rumen bacterial and archaeal communities in dairy cows fed different ratios of maize silage (MS) and grass silage (GS), and place the findings in the context of ruminal fermentation as well as previously reported methane (CH4) emissions. Rumen fluid from 12 rumen cannulated dairy cows was collected after 10 and 17 days of feeding one of four diets, all of which had the same roughage to concentrate ratio of 80:20 based on dry matter (DM). Roughage in the four diets (GS100, GS0, GS67, GS33) consisted of either 1000 g/kg DM GS (GS100), 1000 g/kg DM MS (GS0), or a mixture of both silages in different proportions [667 g/kg DM GS and 333 g/kg DM MS (GS67); 333 g/kg DM GS and 677 g/kg DM MS (GS33)]. Total volatile fatty acid (VFA) concentrations and the molar proportions of the ruminal VFA were not affected by diet. Only the molar proportion of isovalerate was affected by time, being lower on day 17 than on day 10. Bacterial and archaeal concentrations were not affected by diet but increased from day 10 to day 17. The bacterial community composition was affected by diet, time and diet × time, whereas the archaeal community composition was only affected by diet. Several bacterial and archaeal genus level groups were associated with diet, but not with time. Analysis indicated the increased use of hydrogen by succinate and lactate producing bacteria is likely to at least partially explain the previously reported lower CH4 emissions from MS fed dairy cows. Furthermore, time had a significant effect on both bacterial and archaeal concentrations, and also bacterial community composition. This indicates that the rumen microbiota had not stabilized after 10 days of feeding the experimental diets.

The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows.

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.

Diurnal Dynamics of Gaseous and Dissolved Metabolites and Microbiota Composition in the Bovine Rumen.

Diurnal patterns of ruminal fermentation metabolites and microbial communities are not commonly assessed when investigating variation in ruminal CH4 production. The aims of this study were to monitor diurnal patterns of: (i) gaseous and dissolved metabolite concentrations in the bovine rumen, (ii) H2 and CH4 emitted, and (iii) the rumen microbiota. Furthermore, the effect of dietary inclusion of linseed oil on these patterns was assessed. Four rumen cannulated multiparous cows were used in a cross-over design with two 17 days periods and two dietary treatments: a control diet and a linseed oil supplemented diet [40% maize silage, 30% grass silage, 30% concentrate on dry matter (DM) basis for both diets; fat contents of 33 vs. 56 g/kg of DM]. On day 11, rumen contents were sampled for 10 h after morning feeding to profile gaseous and dissolved metabolite concentrations and microbiota composition. H2 and CH4 emission (mass per unit of time) was measured in respiration chambers from day 13 to 17. A 100-fold increase in ruminal H2 partial pressure (contribution to the total pressure of rumen headspace gases) was observed at 0.5 h after feeding. This peak was followed by a decline to basal level. Qualitatively similar patterns after feeding were also observed for H2 and CH4 emission, ethanol and lactate concentrations, and propionate molar proportion, although the opposite pattern was seen for acetate molar proportion. Associated with these patterns, a temporal biphasic change in the microbial composition was observed as based on 16S ribosomal RNA with certain taxa specifically associated with each phase. Bacterial concentrations (log10 16S ribosomal RNA gene copies based) were affected by time, and were increased by linseed oil supplementation. Archaeal concentrations (log10 16S ribosomal RNA gene copies based) tended to be affected by time and were not affected by diet, despite linseed oil supplementation decreasing CH4 emission, tending to decrease the partial pressure of CH4, and tending to increase propionate molar proportion. Linseed oil supplementation affected microbiota composition, and was most associated with an uncultivated Bacteroidales taxon. In summary, our findings support the importance of diurnal dynamics for the understanding of VFA, H2, and CH4 production.