Abstract
In terrestrial subsurface environments where nitrate is a critical groundwater
contaminant, few cultivated representatives are available with which to verify the metabolism of
organisms that catalyze denitrification. In this study, five species of denitrifying bacteria from
three phyla were isolated from subsurface sediments exposed to metal radionuclide and nitrate
contamination as part of the U.S. Department of Energy’s Oak Ridge Integrated Field Research
Challenge (OR-IFRC). Isolates belonged to the genera Afipia and Hyphomicrobium
(Alphaproteobacteria), Rhodanobacter (Gammaproteobacteria), Intrasporangium
(Actinobacteria) and Bacillus (Firmicutes). Isolates from the phylum Proteobacteria were
confirmed as complete denitrifiers, whereas the Gram-positive isolates reduced nitrate to nitrous
oxide. Ribosomal RNA gene analyses reveal that bacteria from the genus Rhodanobacter
comprise a diverse population of circumneutral to moderately acidophilic denitrifiers at the ORIFRC
site, with a high relative abundance in areas of the acidic source zone. Rhodanobacter
species do not contain a periplasmic nitrite reductase and have not been previously detected in
functional gene surveys of denitrifying bacteria at the OR-IFRC site. Sequences of nitrite and
nitrous oxide reductase genes were recovered from the isolates and from the terrestrial
subsurface by designing primer sets mined from genomic and metagenomic data and from draft
genomes of two of the isolates. We demonstrate that a combination of cultivation, genomic and
metagenomic data are essential to the in situ characterization of denitrifiers and that current
PCR-based approaches are not suitable for deep coverage of denitrifying microorganisms. Our
results indicate that the diversity of denitrifiers is significantly underestimated in the terrestrial
subsurface.