Quantitative Determination of 2-Mercaptoethane Sulfonate as a Biomarker for Methanogens in Soil Using HPLC

All methanogens, irrespective of their preferred carbon sources, form coenzyme M (Co-M: 2-mercaptoethane sulfonate) as an intermediate to produce methane (CH₄). Generally, a real-time PCR technique is used for indirect quantification of methanogen abundance in soil. But the assay of mcrA genes, coding methyl Co-M reductase enzyme, by real-time PCR is sometimes limited by the necessity of sophisticated laboratory set-up and experienced operators. In this experiment, the method for quantifying Co-M was standardized by HPLC using UV detector, and the values of Co-M were compared with CH₄ emission flux and methanogen activity of soil. Our objectives were to standardize the Co-M quantification technique and to evaluate its feasibility as a biomarker for methanogens in soil. The Co-M was extracted from soil by rupturing the cell membrane of methanogens using lysis buffer. Trichloroacetic acid (0.5 M) and acetonitrile mixture (70:30, v/v) were used as a mobile phase for measuring Co-M by HPLC at 270 nm. The precision of the method was more than 97%, and 90.3% ± 8.1% of the added Co-M standard was recovered from soil by this method. The Co-M concentrations at different rice cultivation stages had shown significant correlations with CH₄ emission and methanogen activity in soil. The increases in methanogen activity and Co-M concentration following organic substrate incorporation were proportional to the changes in CH₄ flux from rice paddy soils. The conversion factor (155.03 ± 14.20 μg CH₄ produced mmol⁻¹ Co-M day⁻¹) could be used to calculate methanogen activity from Co-M concentrations in soil. Based on these results, it is proposed that Co-M can be used as a biomarker for methanogen activity, and the above-mentioned method can be used for an easy but precise quantification of Co-M in soil.