Estimation of methanogen biomass by quantitation of coenzyme M

Determination of the role of methanogenic bacteria in an anaerobic ecosyste m often requires quantitation of the organisms. Because of the extreme oxyg en sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We stand ardized a simple method for estimating methanogen biomass in a variety of e nvironmental matrices. In this procedure we used the thiol biomarker coenzy me M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in porewater (A. Vairavamurthy and M, Mopper, A nal. Chim. Acta 78:363-370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples, The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample, CoM was not adsorbed to sed iments, The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 +/- 0.07 fmol of CoM/cell, Environmental samples contai ned an average of 0.41 +/- 0.17 fmol/cell based on most-probable-number est imates, CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol , More than 90% of the CoM was recovered from pure cultures and environment al samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detecta ble CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively s imple way to estimate methanogenic biomass.