Identification and isolation of active N2O reducers in rice paddy soil

Abstract

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.

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

“…Previous studies utilized conventional primer sets to analyze nirK and nirS of denitrifiers that belonged primarily to alpha-, beta-, and gamma-proteobacteria from various environmental samples (Braker et al, 2000;Heylen et al, 2006;Smith et al, 2007;Ishii et al, 2011;Palmer et al, 2012). However, our phylogenetic analysis of the available nirK and nirS showed that a greater diversity of microorganisms possess nirK or nirS, including Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Spirochetes, Planctomycetes, NC10, and even the archaeal phylum Euryarchaeota (Figure 1).…”

Bacterial community structure in maize stubble-amended soils with different moisture levels estimated by bar-coded pyrosequencing

“…In addition, the amended NoFer soil at low moisture level had an abundant Burkholderiales at days 20 and 200, as well as Actinomycetales at day 80 of the incubation period. Most Burkholderiales are identified as the reducers of soil dissolved N 2 O (Ishii et al, 2011), so in the presence of stubble these bacteria maybe have strong N 2 O-reducing ability in soils with low fertility and moisture levels. Sometimes the growth of Actinomycetales can be well maintained at low fertility and moisture levels, because Actinomycetales can explore soil space with the mycelium for nutrient acquisition at the soil-straw interface (Frey et al, 2003).…”

Nitrogen Cycling in Rice Paddy Environments: Past Achievements and Future Challenges

“…In order to elucidate the microbes responsible for N2O reduction in rice paddy soils, Ishii et al (44) employed both culture-independent (SIP analysis) and culture-dependent (FSC isolation) methods. Similar to the results obtained in rice paddy soils under denitrification conditions, Herbaspirillum sp.…”