Identification and isolation of active N 2 O reducers in rice paddy soil

Dissolved N 2 O is occasionally detected in surface and ground water in rice paddy fields, whereas little or no N 2 O is emitted to the atmosphere above these fields. This indicates the occurrence of N 2 O reduction in rice paddy fields; however, identity of the N 2 O reducers is largely unknown. In this study, we employed both culture-dependent and culture-independent approaches to identify N 2 O reducers in rice paddy soil. In a soil microcosm, N 2 O and succinate were added as the electron acceptor and donor, respectively, for N 2 O reduction. For the stable isotope probing (SIP) experiment, 13 C-labeled succinate was used to identify succinate-assimilating microbes under N 2 O-reducing conditions. DNA was extracted 24 h after incubation, and heavy and light DNA fractions were separated by density gradient ultracentrifugation. Denaturing gradient gel electrophoresis and clone library analysis targeting the 16S rRNA and the N 2 O reductase gene were performed. For culture-dependent analysis, the microbes that elongated under N 2 O-reducing conditions in the presence of cell-division inhibitors were individually captured by a micromanipulator and transferred to a low-nutrient medium. The N 2 O-reducing ability of these strains was examined by gas chromatography/mass spectrometry. Results of the SIP analysis suggested that Burkholderiales and Rhodospirillales bacteria dominated the population under N 2 O-reducing conditions, in contrast to the control sample (soil incubated with only 13 C-succinate). Results of the single-cell isolation technique also indicated that the majority of the N 2 O-reducing strains belonged to the genera Herbaspirillum (Burkholderiales) and Azospirillum (Rhodospirillales). In addition, Herbaspirillum strains reduced N 2 O faster than Azospirillum strains. These results suggest that Herbaspirillum spp. may have an important role in N 2 O reduction in rice paddy soils.