Advantages of a diluted nutrient broth medium for isolating N 2 -producing denitrifying bacteria of α-Proteobacteria in surface and subsurface upland soils

This research aimed to determine whether a diluted nutrient broth (DNB) medium was different from a conventional nutrient broth (NB) medium when counting and isolating denitrifying bacteria in surface and subsurface upland soils. To this end, we investigated populations of denitrifying bacteria isolated from the surface to a depth of 4 m of subsurface upland soil that had received slurry. The DNB medium gave higher viable counts of denitrifying bacteria than the NB medium and a higher isolation ratio of denitrifying bacterial isolates. In total, 74 isolates from the DNB medium (D-isolates) and 26 isolates from the NB medium (N-isolates) were collected. We characterized their denitrifying activity and analyzed the diversity of 16S rDNA and denitrifying-related genes. Seventy-three percent of the D-isolates were oligotrophic denitrifying bacteria. The N2-producing, oligotrophic denitrifying bacteria, largely of α-Proteobacteria, increased in the D-isolates. The D-isolates and the N-isolates had some taxonomic overlapping on a phylogenetic tree based on 16S rDNA. It was not possible to identify the denitrification phenotype (N2-producing or N2O-producing) on the phylogenetic tree. Phylogenetic groups of isolates corresponded to nirK groups, except in some isolates in which horizontal gene transfer might have occurred. The terminal gas emission of the isolates was consistent with the existence of the nosZ gene. The DNB medium may be very useful in isolating N2-producing denitrifying α-Proteobacteria. Its use highlights the ecological significance of oligotrophic isolates and the different viable counts resulting from the selectivity of conventional and diluted media.