Denitrifying Bacteria Isolated from Terrestrial Subsurface Sediments Exposed to Mixed-waste Contamination

In terrestrial subsurface environments where nitrate is a critical groundwater contaminant, few cultivated representatives are available 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 complete denitrifiers, whereas the Gram-positive isolates reduced nitrate to nitrous oxide. rRNA gene analyses coupled with physiological and genomic analyses suggest that bacteria from the genus Rhodanobacter are a diverse population of denitrifiers that are circumneutral to moderately acidophilic, with a high relative abundance in areas of the acidic source zone at the OR-IFRC site. Based on genome analysis, Rhodanobacter species contain two nitrite reductase genes and have not been detected in functional-gene surveys of denitrifying bacteria at the OR-IFRC site. Nitrite and nitrous oxide reductase gene sequences 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 and genomic and metagenomic data is essential to the in situ characterization of denitrifiers and that current PCR-based approaches are not suitable for deep coverage of denitrifiers. Our results indicate that the diversity of denitrifiers is significantly underestimated in the terrestrial subsurface.