A novel methane-producing archaeon, strain SMSP T , was isolated from an anaerobic, propionatedegrading enrichment culture that was originally obtained from granular sludge in a mesophilic upflow anaerobic sludge blanket (UASB) reactor used to treat a beer brewery effluent. Cells were non-motile, blunt-ended, straight rods, 1.0-2.6 mm long by 0.5 mm wide; cells were sometimes up to 7 mm long. Asymmetrical cell division was observed in rod-shaped cells. Coccoid cells (0.5-1.0 mm in diameter) were also observed in mid-to late-exponential phase cultures. Growth was observed between 10 and 40 6C (optimum, 30-33 6C) and pH 7.0 and 7.6 (optimum, pH 7.4). The G+C content of the genomic DNA was 56.2 mol%. The strain utilized formate and hydrogen for growth and methane production. Based on comparative sequence analyses of the 16S rRNA and mcrA (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane-producing pathway) genes, strain SMSP T was affiliated with group E1/E2 within the order Methanomicrobiales. The closest relative based on both 16S rRNA and mcrA gene sequences was Methanoregula boonei 6A8 T (96.3 % 16S rRNA gene sequence similarity, 85.4 % deduced McrA amino acid sequence similarity). The percentage of 16S rRNA gene sequence similarity indicates that strain SMSP T and Methanoregula boonei 6A8 T represent different species within the same genus. This is supported by our findings of shared phenotypic properties, including cell morphology and growth temperature range, and phenotypic differences in substrate usage and pH range. Based on these genetic and phenotypic properties, we propose that strain SMSP T represents a novel species of the genus Methanoregula, for which we propose the name Methanoregula formicica sp. nov., with the type strain SMSP T (5NBRC 105244The group E1/E2, originally called the R10 group or fen cluster, is a family-level clade within the order Methanomicrobiales, which comprises H 2 /CO 2 -using methanogenic archaea (Hales et al., 1996; Bräuer et al., 2006b;Galand et al., 2002;Cadillo-Quiroz et al., 2006). This clade has long been recognized as an uncultured group; however, to date, three methanogenic strains have been isolated and characterized: Methanoregula boonei 6A8 T (Bräuer et al., 2006a(Bräuer et al., , 2011, Methanosphaerula palustris E1-9cT (Cadillo-Quiroz et al., 2008) and Methanolinea tarda NOBI-1 T (Imachi et al., 2008). Methanoregula boonei and Methanosphaerula palustris were isolated from peatlands, while Methanolinea tarda was isolated from a methanogenic digester. In addition to the these isolates, many 16S rRNA gene clones belonging to group E1/E2 have been retrieved from a wide variety of anoxic environments, such as bogs and fens (Bräuer et al., 2006a, b;Cadillo-Quiroz et al., 2006Chan et al., 2002;Galand et al., 2002), methanogenic sludges (Imachi et al., 2008; Chen et al., 2004Chen et al., , 2009Narihiro et al., 2009) Supplementary Fig. S1, available in IJSEM Online), indicating the widespread distribution of E1/E2 methanogens.Recen...

Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with 13 C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect 13 C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in 13 C-labeled and unlabeled rRNA fractions. The bacterial populations in the 13 C-labeled rRNA fractions were identified by cloning and sequencing of reversetranscribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.Lipid is a one of the major organic fractions of wastes/ wastewaters, and lipid-rich wastes/wastewaters are widely found in certain food processing industries, such as dairy, edible oil, and slaughterhouses (20). Because lipids have a high theoretical methane yield in comparison with other organic substances, methanogenic treatment has been applied to lipidrich wastes/wastewaters but resulted in low organic loading rates (see, for example, references 16 and 50) compared to that seen for other types of wastes/wastewaters. This is at least partly due to the acute toxicity of long-chain fatty acids (LCFA), which are the main constituent and hydrolysate of lipids in the anaerobic consortium. LCFA can cause substrate toxicity in anaerobic microorganisms (see, for example, references 18 and 44) and tend to adsorb onto the biomass and flow out of the reactor.Under methanogenic conditions, LCFA degradation requires a syntrophic association of LCFA-degrading anaerobes and hydrogenotrophic methanogens, because the oxidation of LCFA is thermodynamically unfavorable in such environments unless the consumption of reducing equivalents (hydrogen and/or formate) is coupled with oxidation (37). Due to the syntrophic metabolism and toxicity of LCFA, isolation of LCFA-degrading syntrophs is difficult. Thus, information on LCFA-degrading bacteria in pure culture is based on Syntrophomonas species (10, 21, 36, 47, 54) and on Thermosyntropha lipolytica...

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the 13 C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.Butyrate is one of the important intermediates in the degradation of organic matter under methanogenic conditions (14,16). Under these conditions, butyrate degradation is carried out by a syntrophic association of butyrate-oxidizing bacteria and hydrogenotrophic methanogens, because of thermodynamic constraints (17). Due to the fastidious nature of this syntrophic metabolism, isolation of butyrate-degrading syntrophs has been difficult and thus information on butyratedegrading bacteria is based on some isolates belonging to the family Syntrophomonadaceae. Due to this lack of knowledge and the lack of appropriate molecular markers, culture-independent studies have focused only on species of the family Syntrophomonadaceae (5, 15, 25). Consequently, the natural diversity of syntrophic butyrate-degrading bacteria has not been studied in any detail.The recent development of stable isotope probing (SIP) enables metabolic function and taxonomic identity to be examined concurrently (3). Only one study on methanogenic butyrate degradation has been reported with this technique (1), but not within waste/wastewater-treated methanogenic sludges. SIP provides a potentially fruitful tool for identifying potential butyrate-degraders in a methanogenic environment. In this study, therefore we used RNA-based SIP (RNA-SIP) with [ 13 C 4 ]butyrate as a substrate to explore the microorganisms involved in butyrate degradation in four methanogenic sludges.Four methanogenic sludges were used in this study. Mesophilic granular sludge MP and thermophilic granular sludge TP were taken from two lab-scale multistage upflow anaerobic sludge blanket reactors treating palm oil mill effluent. Mesophilic anaerobic digester sludge treating palm oil mill effluent (sludge MBF) and thermophilic digester sludge treating municipal solid waste (sludge JET) were taken from commercial plants. Detailed properties of these sludges were described in our previous report (7). Incubation was carried out anaerobically at 37°C (for mesophilic sludges) or 55°C (for thermophilic sludges). The granular sludges TP and MP were preincubated with 5 mM butyrate because of prolonged storage at 4°C for over 2 years. Degradation of butyrate was monitored by measuring methane production using gas chromatography as described previously (6). Preincubation was conducted for 14 days, added butyrate was completely converted to methane, and then the sludge was sampled as an unlabeled control microbial consortium. Digester sludges MBF and JET were used immediately after sampling, and RNA extracted from unincubated sludges was used as an unlabeled control RNA. Incubation of stable isotope-label...