Methanolinea tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic digester sludge

Abstract

Methanolinea tarda gen. nov., sp. nov., a methaneproducing archaeon isolated from a methanogenic digester sludge A novel methane-producing archaeon, strain NOBI-1 T was isolated from an anaerobic, propionate-degradation enrichment culture, which was originally obtained from a mesophilic methanogenic sludge digesting municipal sewage sludge. Cells were non-motile, rod-shaped, 0.7-1.0 mm by 2.0 mm, and formed multicellular filaments longer than 8 mm. Growth was observed between 35 and 55 6C (optimum 50 6C) and pH 6.7 and 8.0 (optimum pH 7.0). The G+C content of the genomic DNA was 56.3 mol%. The strain utilized H 2 and formate for growth and methane production. Based on comparative sequence analyses of the 16S rRNA gene and mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane-production pathway), strain NOBI-1 T was affiliated with the order Methanomicrobiales, but it was significantly distant from any other known species within the order. The most closely related species based on 16S rRNA and mcrA gene sequence similarity were respectively 'Candidatus Methanoregula boonei' (93.7 % 16S rRNA gene sequence similarity) and Methanocorpusculum parvum (74.2 % deduced McrA amino acid sequence similarity to the type strain). These phenotypic and genetic properties justified the creation of a novel species of a new genus for the strain, for which we propose the name Methanolinea tarda gen. nov., sp. nov. The type strain of Methanolinea tarda is strain NOBI-1 T (5DSM 16494 T 5JCM 12467 T 5NBRC 102358 T ).Methane-producing archaea (methanogens) are one of the key populations in methanogenic waste and wastewater treatment processes because they are responsible for the final step of the degradation of organic substances (Anderson et al., 2003;Garcia et al., 2000). So far, a number of methanogens, placed in a wide range of taxonomic groups within the phylum Euryarchaeota, have been isolated from various methanogenic treatment processes and characterized as novel species belonging to genera such as Methanobacterium, Methanoculleus and Methanosaeta (Garcia et al., 2000;Garrity & Holt, 2001). In addition, recent molecular surveys targeting the 16S rRNA gene and mcrA gene, encoding the alpha subunit of methyl coenzyme M reductase, have revealed that numerous unidentified methanogens may also exist in such ecosystems (Chouari et al., 2005;Sekiguchi & Kamagata, 2004;Sekiguchi, 2006; Shigematsu et al., 2004). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NOBI-1 T is AB162774 (other 16S rRNA gene clonal sequences have been deposited as accession numbers AB233309, AB233010 and AB233299-AB233302). The GenBank/EMBL/DDBJ accession numbers for the mcrA gene sequences of strains NOBI-1 T and SANAE are respectively AB300466 and AB300467.Details of 16S rRNA gene phylotypes obtained from the sludge, information on relative abundance of various groups of organisms and a graph of methane production by strain NOBI-1 T are available as supplementary material with the onl...

Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium

“…Using the 16S rRNA gene and McrA gene as biomarkers, temperature-dependent variations were also observed with acetoclastic methanogen populations found in the order Methanosarcinales (Supplementary Figure 4). The hydrogenotrophic methanogens identified here are closely related to methanogens found in mesophilic and thermophilic TA-degrading reactors (Wu et al, 2001;Chen et al, 2004), and together with Methanolinea tarda NOBI-1 recently isolated from anaerobic digestion processes (Imachi et al, 2008), form a novel cluster separate from other known hydrogenotrophic methanogens. The comparison of McrA genes further suggests that the methanogens found in the new cluster are likely different from M. tarda NOBI-1 (Supplementary Figure 4B). )…”

“…Several studies (Chan, 2000;Qiu et al, 2006;Imachi et al, 2008) have observed the existence of multiple bacterial populations in highly enriched methanogenic cultures degrading carbon substrates like formate, acetate, propionate and phthalate isomers. These observations were shown through a defined mixed culture (Dolfing et al, 2008), suggesting that syntrophic interactions in methanogenic enrichments are more complex than simple pairwise syntroph-methanogen relationships.…”

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method

“…In mesophilic reactors 1, 2, 4 and 6, the presence of Methanomicrobiales-related methanogens (MG1200m) was observed (Table 4), but their detailed phylogenetic affiliations within this family could not be properly identified using subfamily-, genus-and speciesspecific probes (Table 3), because major members of the order Methanomicrobiales in reactors 1 and 2 were related to uncultured environmental clones and a novel methanogen (Methanoline tarda) isolated and characterized very recently (Imachi et al, 2008) (Figure 3). Thus, additional scissor probes are needed to account for the presence of these yet-to-beidentified taxa.…”

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field

“…We, therefore, changed our strategy to isolation based on the co-culture technique in which an H 2 -producing bacterium and its substrate were co-inoculated together with the original sample in the primary enrichment and in further passages to fresh medium, so that H 2 was supplied to methanogens constantly at very low concentrations (,100 Pa). The underlying idea has been described in our previous reports (Sakai et al, 2007;Imachi et al, 2008). The method has been proven to be successful to eliminate non-target fast-growing methanogenic populations.…”