Two dense and highly enriched (up to 10(9) cells ml(-1), <10% of bacterial satellites) acido-tolerant (pH 4.0-6.5) methanogenic consortia, '26' and 'K', were isolated from the peat beneath a Sphagnum-Eriophorum-Carex community in West Siberia. Both consortia produced methane from CO2:H2 on chemically defined, diluted N-free media containing Ti(III)citrate as reducing agent. The phylogenetic analysis of 16S ribosomal DNA revealed three archaeal and nine bacterial sequence types. Consortium '26' contained single archaea Methanobacterium sp., represented by rods of 1.5-10x0.5-1.0 microm. In consortium 'K', there were two archaeal phylotypes, the respective methanogens were further differentiated morphologically with the fluorescence in situ hybridization technique: one less abundant (<2%) population of the long-curved rods (50-100x0.3-0.4 microm) fell into the order of Methanomicrobiales, while the dominant organism ( approximately 98%), represented by straight rods with abrupt rectangular ends (3-9x0.5 microm), was affiliated with earlier uncultured 'Rice cluster I'. The main bacterial satellite used citrate as a single carbon and energy source; it was similar in both consortia, and after isolation in pure culture, it was identified as a new member of the alpha-subclass of Proteobacteria. The other bacterial satellites were distributed among four taxonomic groups: the delta-subclass of Proteobacteria, the Flavobacterium-Bacteroides-Cytophaga line of descent, the Acidobacterium-Fibrobacter line of descent and the Green non-sulfur bacteria line of descent. At least 11 out of 12 components of acido-tolerant consortia are new to science at the species, genus and order levels; their existence until now was evident only from environmental gene retrievals. The Sphagnum wetlands, attracting attention only recently because of their global environmental role, are shown to be an especially valuable source of novel prokaryotic organisms.

A significant number of microorganisms from the human oral cavity remain uncultivated. This is a major impediment to the study of human health since some of the uncultivated species may be involved in a variety of systemic diseases. We used a range of innovations previously developed to cultivate microorganisms from the human oral cavity, focusing on anaerobic species. These innovations include (i) in vivo cultivation to specifically enrich for species actively growing in the oral cavity (the "minitrap" method), (ii) single-cell long-term cultivation to minimize the effect of fast-growing microorganisms, and (iii) modifications of conventional enrichment techniques, using media that did not contain sugar, including glucose. To enable cultivation of obligate anaerobes, we maintained strict anaerobic conditions in most of our cultivation experiments. We report that, on a per cell basis, the most successful recovery was achieved using minitrap enrichment (11%), followed by single-cell cultivation (3%) and conventional plating (1%). Taxonomically, the richest collection was obtained using the single-cell cultivation method, followed by minitrap and conventional enrichment, comprising representatives of 13, 9, and 4 genera, respectively. Interestingly, no single species was isolated by all three methods, indicating method complementarity. An important result is the isolation and maintenance in pure culture of 10 strains previously only known by their molecular signatures, as well as representatives of what are likely to be three new microbial genera. We conclude that the ensemble of new methods we introduced will likely help close the gap between cultivated and uncultivated species from the human oral cavity.

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Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.