Dent corn Andisol at the Hokkaido University Shizunai Livestock Experimental Farm actively emits nitrous oxide (N
2O). In order to screen for culturable and active N
2O emitters with high N
2O emission potential, soft gel medium containing excess KNO
3 was inoculated with soil suspensions from farm soil samples collected at different land managements. Dominant bacterial colonies were searched for among 20 of the actively N
2O-emitting cultures from post-harvest soil and 19 from pre-tilled soil, and all isolates were subjected to the culture-based N
2O emission assay. Ten active N
2O-emitting bacteria, four from post-harvest soil and six from pre-tilled soil, out of 156 isolates were identified as genus
Pseudomonas by 16S rRNA gene sequencing. These N
2O emitters showed clear responses to NO
3− within a neutral pH range (5.5–6.7), and accelerated N
2O production with 1.5–15 mM sucrose supplementation, suggesting the production of N
2O during the denitrification process. However, the negative responses of 6 active N
2O emitters, 3 from post-harvest soil and 3 from pre-tilled soil, out of the 10 isolates in the acetylene-blocking assay suggest that these 6 N
2O emitters are incomplete denitrifiers that have lost their N
2O reductase (N
2OR) activity. Although the PCR assay for the denitrification-associated genes,
narG and
nirK/S, was positive in all 10
Pseudomonas isolates, those negative in the acetylene-blocking assay were
nosZ-negative. Therefore, these results imply that the high N
2O emission potential of dent corn Andisol is partly attributed to saprophytic,
nosZ gene-missing pseudomonad denitrifiers.
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