Long-term submergence of non-methanogenic oxic upland field soils helps to develop the methanogenic archaeal community as revealed by pot and field experiments

Takeshi WATANABE^a, Susumu ASAKAWA^a,b Koichi HAYANO^b

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464–8601 (Japan).

Abstract

The community structure of methanogenic archaea is relatively stable, i.e., it is sustained at a high abundance with minimal changes in composition, in paddy field soils irrespective of submergence and drainage. In contrast, the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils. This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments. In the pot experiment, a soil sample obtained from an upland field was incubated under submerged conditions for 275 d. Soil samples periodically collected were subjected to culture-dependent most probable number (MPN) enumeration, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of archaeal 16S rRNA gene, and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene (mcrA) of methanogenic archaea. The abundance of methanogenic archaea increased from 10² to 10³ cells g^-1 dry soil and 10⁴ to 10⁷ copies of mcrA gene g^-1 dry soil after submergence. Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis, members from Methanocellales, Methanosarcinaceae, and Methanosaetaceae proliferated in the soils, and the community structure was relatively stable once established. In the field experiment, the number of viable methanogenic archaea in a rice paddy field converted from meadow (reclaimed paddy field) was monitored by MPN enumeration over five annual cycles of field operations. Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer's paddy field was dressed onto the meadow (dressed paddy field) and an upland crop field converted from the meadow (reclaimed upland field). The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field. Then, the number gradually increased over five years and finally reached 10³–10⁴ cells g^-1 dry soil, which was comparable to that in the dressed paddy field. These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated, and the community structure was developed following repeated and long-term submergence. These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.

Keywords: denaturing gradient gel electrophoresis, mcrA gene, methanogenic archaea, microbial community structure, microbial habit, most probable number method, paddy field soil, qPCR.