Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

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Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences ( approximately 1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson's gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella-Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.Morphologically unique, large bacteria of the genus Oscillospira, which have not been grown in pure culture yet, can be routinely detected microscopically in the rumen contents of cattle and sheep (11,18). A number of Oscillospira spp. are responsive to the advent of green pastures and fluctuate seasonally (13, 17) (see also Table 1). The only species of Oscillospira described in Bergey's Manual of Systematic Bacteriology (8) is Oscillospira guilliermondii, and this entry refers to the original morphological description by Chatton and Perard in 1913. Warner (34) proposed that at least two strains or species be included in the Oscillospira group based on cell diameter and the tendency to form spores as the main characteristic differences. This implies that there are different morphological forms or species related to different diets or in different gut ecosystems.Detection and identification of microbial populations are the most basic prerequisites for microbial ecology studies. Over the last decade, several molecular techniques have been developed that when applied to the rumen microbiota have revealed enormous microbial diversity not recognized previously because of limitations and biases inherent in the cultivation approach (24,(30)(31)(32)37). The cultivation-independent approach provides technology for detection and monitoring of microorganisms such as Oscillospira, for which growth requirements are unknown and undetermined. The only requirement for the development of molecular detection techniques is the availability of a marker molecule. Fortunately, several 16S rRNA gene sequences have recently been retrieved from this large bacterium isolated by flow cytometric sorting (39), and this enabled the design of various molecular probes for detection and monitoring. We designed and validate here PCR and PCR-denaturing gradient gel electroph...