A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether‐linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9‐anthroyl derivatives by high‐performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α‐hydroxyarchaeol and β‐hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H2/CO2‐consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.

The kinetics of acetate utilization by concentrated suspensions of cells was examined in five strains of Methanothrix soehngenii. The rate of acetate utilization by all strains was dependent on the initial acetate concentration and followed simple Michaelis-Menten kinetics. The ability to utilize acetate differed among the various strains of M. soehngenii and was highest in the strain designated MTAS.

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Mesophilic Methanothrix sp. strains MTAS and MTKO were isolated as pure cultures from the sludge of anaerobic digestors. We examined the relationships between these two isolates and three other Methanothrix strains (strains Opfikon' [T = type strain], GP(il', and FE) by using an immunological method, by performing DNA homology experiments, and by analyzing polar lipids. Our results indicate that all five strains belong to the same species.Acetate is the precursor of at least 60% of the methane produced during anaerobic digestion of organic compounds (4, 20). Kaspar and Wiihrmann (13) reported that acetate splitting is the rate-limiting reaction in this system. On the other hand, the members of three genera, Methanothrix, Methanosaeta, and Methanosarcina, can convert acetate to methane. The members of the genus Methanothrix, in particular, have a high affinity for acetate (&, 0.7 mM), so these bacteria seem to play an important role in methane fermentation (8, 12).The genus Methanothrix includes two mesophilic species, Methanothrix soehngenii (type strain, Opfikon) (12) and Methanothrix concilii (type strain, GP6) (17). Touzel et al. indicated that strains OpfikonT (T =type strain) and GPGT belong to the same species, as judged by the results of DNA-DNA hybridization experiments, and proposed that the name Methanothrix concilii should be rejected as a later synonym of Methanothrix soehngenii (22). Recently, Patel i$nd Sprott proposed that the genus name Methanothrix should be rejected and described a new genus, Methanosaeta, with Methanosaeta concilii as its type species, because no axenic culture of Methanothrix soehngenii seems to exist ((18). In this paper we describe the isolation of two new Methanothrix strains as pure cultures (strains MTAS and MTKO), and we compare these strains with other Methanothrix strains. MATERIALS AND METHODS Sources of strains.Methanothrix concilii GP6T (= OCM69T) was obtained from the Oregon Collection of Methanogens, Beaverton. Methanothrix soehngenii Opfikon (= DSM 2139T) was a gift from A. J. B. Zehnder (Agricultural University, Wageningen, The Netherlands). Methanobacterium thermoautotrophicum AHT (= DSM 1053T), Methanosarcina barkeri MST (= DSM 800T), and Methanothrix soehngenii FE (= DSM 3013) were obtained from the Deutsche Sammulung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Federal Republic of Germany. Methanothrix sp. strain MTAS was isolated from the anaerobic digestor (in which the temperature is not controlled) that is used for treating domestic wastewater in the town of Ashiya (Fukuoka, Japan). Strain MTKO was isolated from the anaerobic fixed-bed digestor (in which the temperature is not controlled) that is used for treating wastewater from a flush toilet in our laboratory. * Corresponding author.Media and cultivation. We used Deutsche Sammlung von Mikroorganismen und Zellkulturen medium no. 334 (7) for isolating and cultivating all Methanothrix strains except strain GPtiT. This medium was dispensed into serum vials under 100% nitrogen atmosphere and ...

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