QUANTITATIVE-DETERMINATION OF METHANOGENIC CELLS BASED ON ANALYSIS OF ETHER-LINKED GLYCEROLIPIDS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A method for the quantitative determination of methanogenic cells in natural environments is described. The method is based on the determination of ether-linked glycerolipids that is specific to methanogens and other archaebacteria. Core lipids (archaeol and caldarchaeol), prepared from total lipids by a combination of acetolysis and acid methanolysis, were quantitated as the corresponding dinitrobenzoyl derivatives by high-performance liquid chromatography. The total amount of core lipids was proportional to the amount of methanogenic cells. This method was high sensitivity, and the lowest limit of detection of methanogenic cells was 2.5-mu-g (dry weight) in a 500-mu-l sample of culture or sludge. An identical standard curve was obtained for Methanobacterium thermoautotrophicum and Methanospirillum hungatei. With this method, it was possible to estimate methanogenic cells in a sample of sludge from an anaerobic digestor. In spite of contamination by large amounts of multifarious adulterants in the sludge, 83% of M. thermoautotrophicum cells added to the sludge was recovered and quantitated by this method.