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 H-2/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 C-13-labeled rice callus cells (78 at.%) as a substrate. Soils with C-13-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 C-13-labeled and unlabeled treatments. The shift of many bands to the fractions of heavier buoyant density (BD) was observed in the C-13-labeled treatment. DNA in the heaviest fractions of the C-13-labeled treatment with a BD from 1.754 to 1.759 g ml(-1) was subjected to population analysis of methanogens (C-13-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 (C-12) 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 C-13-labeled acetoclastic Methanosaeta and Methaosarcina and hydrogenotrophic Methanobacterium and Methanocella indicates the growth of methanogens on acetate and H-2/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. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.