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

Abstract

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.

Metabolic interdependencies between phylogenetically novel fermenters and respiratory organisms in an unconfined aquifer

“…Nitrate is the most prevalent groundwater contaminant and impacts drinking water resources on a global scale (Rivett et al, 2008). Despite this, the diversity and activity of nitrate-reducing bacteria remains surprisingly understudied in the subsurface relative to other ecosystems, such as soils or wastewater treatment systems (Green et al, 2010). In our samples, only members of the Proteobacteria encoded the capacity for dissimilatory nitrate reduction.…”

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

“…However, the primers used for the amplification of nirK from the peat soils do not target nirK of Rhodanobacter sp. (Green et al, 2010). Thus, it is still unclear whether Rhodanobacter-like denitrifiers occur in acidic peat soils.…”

“…However, the molecular data are largely dependent on the choice of primers. Although the primer systems used in this study are well evaluated and widely applied for estimating denitrifier diversity, and four denitrification-associated genes were analyzed in parallel to maximize the detectability of denitrifiers, it is known that not all denitrifers are detectable by the primer systems used (Throbäck et al, 2004;Enwall et al, 2010;Green et al, 2010;Palmer et al, 2010). Considering the rather high threshold distances used in this study for calling OTUs, the diversity analyses of cryoturbated and unturbated peat soil denitrifiers might be regarded as a minimal estimate of the 'real' denitrifier diversity.…”

Insights into Denitrification in Methylotenera mobilis from Denitrification Pathway and Methanol Metabolism Mutants

“…However, in none of the later studies employing this organism was denitrification further linked to methylotrophy (6). Likewise, studies of denitrification in other methylotroph species, including classic denitrifiers, such as Hyphomicrobium spp., were inconclusive in terms of whether denitrification is directly linked to methylotrophy (8,9). Recently, methanol-dependent denitrification by Methyloversatilis has been investigated using proteomics (10).…”