Physiological and Genotypic Characteristics of Nitrous Oxide (N2O)-Emitting Pseudomonas Species Isolated from Dent Corn Andisol Farmland in Hokkaido, Japan

Dent corn Andisol at the Hokkaido University Shizunai Livestock Experimental Farm actively emits nitrous oxide (N(2)O). In order to screen for culturable and active N(2)O emitters with high N(2)O 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(2)O-emitting cultures from post-harvest soil and 19 from pre-tilled soil, and all isolates were subjected to the culture-based N(2)O emission assay. Ten active N(2)O-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(2)O emitters showed clear responses to NO(3)(−) within a neutral pH range (5.5–6.7), and accelerated N(2)O production with 1.5–15 mM sucrose supplementation, suggesting the production of N(2)O during the denitrification process. However, the negative responses of 6 active N(2)O 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(2)O emitters are incomplete denitrifiers that have lost their N(2)O reductase (N(2)OR) 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(2)O emission potential of dent corn Andisol is partly attributed to saprophytic, nosZ gene-missing pseudomonad denitrifiers.