This is the first investigation to show that oligotrophic denitrifying bacteria are dominant denitrifiers in subsurface upland soil. We examined the vertical distribution of denitrifying bacterial populations in upland soil using two kinds of enumeration media. The number of denitrifying bacteria, enumerated in subsurface soil layers by a 100-fold diluted nutrient broth (DNB) medium with NO3 −, was two to three orders of magnitude greater than those enumerated by a conventional nutrient broth medium with NO3 −, suggesting the dominance of oligotrophic denitrifying bacteria. Seventy-four percent of the total denitrifying bacterial isolates were DNB organisms of the oligotrophic type, which did not show appreciable growth on a conventional nutrient broth medium. The isolates were heterogeneous and were categorized as alpha (35 strains) and beta (19 strains) subdivisions of proteobacteria and high G+C gram-positive bacteria (7 strains) by 16S rRNA gene sequence analysis. The 35-alpha subdivision of proteobacterial isolates was of oligotrophic type and widely distributed from the surface to subsurface soil layers. Phylogenetic analysis indicated that some isolates belonged to groups with few or no cultivated representatives, and that one isolate may be a member of a new genus. This isolation procedure, using diluted media, is valuable in detecting diverse and novel denitrifying bacteria in the subsurface soil.
Keywords: Denitrification - Oligotrophic denitrifying bacteria - Soil biodiversity - Subsurface soil