A sensitive method for quantification of aceticlastic methanogens and estimation of total methanogenic cells in natural environments based on an analysis of ether-linked glycerolipids

Abstract

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.

Diether and Tetraether Phospholipids and Glycolipids as Molecular Markers for Archaebacteria (Archaea)

“…^These genera also contain hydroxyarchaeol lipid cores. (14,15). This method can also be applied to environmental samples containing methanogens (15).…”

Biomass Measurement of Methanogens in the Sediments of Tokyo Bay Using Archaeol Lipids

“…Archaeol in the sediment samples was prepared by the following method modified from that of Ohtsubo et al (1993). Crude lipids were extracted from each sample by acid Bligh and Dyer methods (Nishihara and Koga, 1987).…”

“…Their membrane lipids are composed of a unique diphytanyl glycerol ether, called archaeol, which is different from eubacteria and eukaryotes (Tornabene and Langworthy, 1978). Ohtsubo et al (1993) developed a method to estimate total methanogenic biomass in anaerobic sludge by measuring ether-linked glycerolipids, which is based on findings that archaeol content per cell carbon is relatively constant.…”

“…For the determination of archaeol content, several methods have been developed, such as high-performance liquid chromatography (HPLC; Martz et al, 1983;Demizu et al, 1992;Ohtsubo et al, 1993), supercritical liquid chromatography (Holzer et al, 1988;Hedrick et al, 1991), and gas chromatography (Smith and Gloodgate, 1992). The main purposes of those studies were to obtain better sensitivity and to eliminate interference by other compounds in their analyses.…”

“…The main purposes of those studies were to obtain better sensitivity and to eliminate interference by other compounds in their analyses. In particular, Ohtsubo et al (1993) achieved high sensitivity of the analysis with HPLC by using fluorescent derivatives.…”