Evaluation of alternative substrates for determining methane-oxidizing activities and methanotrophic populations in soils

Abstract

The magnitude of methane emission is a net result of methane production and the oxidation rate. The possibility of measuring oxidized products of alternative substrates of methane monooxygenase was examined to determine methane-oxidizing ability of soils, and to count methanotrophic populations in soils. Wetland rice soils were incubated under methane containing air to enirch the methanotrophs. Methane loss and oxygen uptake were inhibited by acetylene, dimethylether, and nitrapyrin (N-Serve). Acetylene was used routinely, because it inhibited methane oxidation even at a low concentration of 0.03 to 0.06 μl ml^-1 in the incubation headspace. Propylene at 10 kPa was used as an alternative substrate of methane monooxygenase, and the formation of propylene oxide was measured. When soils were incubated under methane, their methane-oxidizing activity increased. Propylene oxide formation increased simultaneously. Acetylene also blocked propylene oxidation. The results of several experiments and propylene oxide formation (r=0.87 after long-transformation). These results indicate that propylene oxide formation can be used as a semiquantitative measure of the methane-oxidizing activity of soils. The colonies of soluble methane monooxygenase-forming methanotrophs were counted on Cu-deficient methanotroph agar medium by the formation of naphthol from haphthalene. The counts increased from 10⁴ (0 days) to 10⁷ (21 days) g^-1 soil during oxic incubation under methane.