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.

FIG. 1

HPLC chromatogram of derivatized CoM (retention time, 2.9 min) from the hydrocarbon-contaminated aquifer. The buffer system used for both HPLC and LC-MS was ammonium acetate-acetonitrile (70:30). (Inset) LC-MS profile of the isoindole derivative of standard CoM (left panel) and the corresponding profile of the environmental sample (right panel). Both spectra show that the ionic molecular weight was 300, which is consistent with the structure of the fluorescent CoM derivative shown.

FIG. 2

Absorbance (●), CoM content per milliliter of culture (⧫), and CoM content normalized for culture protein content (■) in a Methanobacterium thermoautotrophicum Marburg culture growing on hydrogen.

FIG. 3

(A) Cultures of methanogens tested before and after freezing at −60°C. There was no decrease in the amount of CoM detected in cells due to freezing. (B) Environmental samples tested before and after freezing at −60°C. There were 46 to 83% decreases in the amount of detectable CoM depending on the matrix. Bars indicate standard errors. Abbreviations: Ms., Methanosarcina; Mb. therm, Methanobacterium thermoautotrophicum; Mc. therm., Methanococcus thermolithotrophicus; Mc., Methanococcus.