Growth of methanogens in an oxic soil microcosm: Elucidation by a DNA-SIP experiment using 13C-labeled dried rice callus

Methanogens are not expect to grow in oxic soil because they are obligate anaerobes that require high concentrations of H2/acetate and low oxidation–reduction potentials. However, we verified their growth in an oxic soil microcosm by a DNA-SIP (stable isotope probing) experiment using 13C-labeled rice callus cells (78 at.%) as a substrate. Soils with 13C-labeled and unlabeled dried rice callus were sampled six times during oxic incubation for 56 days. After isopycnic centrifugation of the extracted DNA and PCR amplification of fractionated DNA with methanogen-specific primers, we compared DGGE band patterns of respective fractions between 13C-labeled and unlabeled treatments. The shift of many bands to the fractions of heavier buoyant density (BD) was observed in the 13C-labeled treatment. DNA in the heaviest fractions of the 13C-labeled treatment with a BD from 1.754 to 1.759 g ml−1 was subjected to population analysis of methanogens (13C-assimilating populations). Partial 16S rRNA genes from methanogens were successfully PCR-amplified from day 3 onwards, and 35 DGGE bands sequenced were closely related to Methanosaeta, Methaosarcina, Methanobacterium and Methanocella. Because the estimated BD of non-labeled (12C) DNA of the genera Methanosarcina, Methanosaeta and Methanobacterium was less than 1.72 g ml−1, these results verified the growth of methanogens in the oxic soil microcosm. Detection of 13C-labeled acetoclastic Methanosaeta and Methaosarcina and hydrogenotrophic Methanobacterium and Methanocella indicates the growth of methanogens on acetate and H2/CO2 in the soil microcosm. Based on these results, the growth of acetoclastic and hydrogenotrophic methanogens was discussed in dried callus under oxic soil conditions.

► This study verified the growth of methanogen in an oxic soil microcosm.
► Soil incubation with 0.5% 12C- and 13C-callus under oxic condition.
► We used DNA-SIP and PCR–DGGE analysis.
► The 13C-labeld acetoclastic and hydrogenotrophic methanogen were detected.