Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Abstract

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme 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 pore water (A. Vairavamurthy and M. Mopper, Anal. 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 sediments. 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 contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental 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 detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.

Methanogenesis in subglacial sediments

“…The quantity of CoM determined in RG east sediments corresponds to~3 ¥ 10 3 methanogen cells g -1 dry weight sediment, when converted using the CoM cell -1 values previously determined for Methanosarcina barkeri str. Fusaro when grown on acetate (Elias et al, 1999). It is important to note that it has been suggested that CoM may be involved in the AOM (Hallam et al, 2004); however, this hypothesis remains untested.…”

“…Coenzyme M is present in all methanogens and is involved in the final steps of methane formation, accepting methyl groups from methylcobalamin to form methylcoenzyme M, which is subsequently reductively demethylated, yielding methane (Taylor and Wolfe, 1974). The concentration of CoM in pure cultures of taxonomically and metabolically diverse methanogens is proportional to the growth status of the cells, the concentration of viable cells, and to cellular protein (Elias et al ., 1999). Coenzyme M is labile and not likely to be preserved in environmental samples, as indicated by an approximate 46–83% decrease in detectable CoM in a variety of environmental samples following a single freeze‐thaw cycle when compared with unfrozen samples (Elias et al ., 1999).…”

“…The concentration of CoM in pure cultures of taxonomically and metabolically diverse methanogens is proportional to the growth status of the cells, the concentration of viable cells, and to cellular protein (Elias et al ., 1999). Coenzyme M is labile and not likely to be preserved in environmental samples, as indicated by an approximate 46–83% decrease in detectable CoM in a variety of environmental samples following a single freeze‐thaw cycle when compared with unfrozen samples (Elias et al ., 1999). Thus, CoM recovered from environmental samples is likely to be associated with not only viable, but active populations of methanogens.…”

Effective Suppression of Methane Emission by 2-Bromoethanesulfonate during Rice Cultivation

“…Coenzyme M and mcrA gene copy numbers are the biomarkers of methaogens [ 23 ] and Co-M only found in methanogens. BES is a structural analogue of Co-M and application of it makes competitive inhibition for methyl group during methanogenesis and thereby inhibits methanogenesis.…”

Effects of microbial community, lipids and pretreatment on the performance of the two-phase anaerobic digestion

“…However, the content of CoM varies with the growth phase of methanogens and it is not accurate in samples after freezing (Elias et al 1999).…”