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.

FIG. 1.

Bootstrapped (1,000 iterations) neighbor-joining tree of denitrifying isolate SSU rRNA gene sequences. Isolate sequences recovered in this study are in bold, with additional genetic and phenotypic properties provided in Table 2. Nodes supported by bootstrap values greater than 70% are indicated by numeric values, and nodes supported by Bayesian analysis, with posterior probability values greater than 95%, are indicated with gray circles. The scale bar represents 0.02 substitutions per nucleotide position.

FIG. 2.

Growth under denitrifying conditions of representatives of the five denitrifying organisms recovered in this study. (A) Growth curves, as measured by determining the optical density at 600 nm (OD₆₀₀), for each isolate. Lactate (10 mM) was used as the electron donor for Bacillus, Afipia, and Intrasporangium isolates. Acetate (10 mM) and ethanol (20 mM) were used for Rhodanobacter and Hyphomicrobium isolates, respectively. (B) Nitrate removal during growth of the five denitrifiers. Production of gaseous end products as a result of denitrification was demonstrated (Table 2).

FIG. 3.

Unrooted phylogeny of partial and complete nirK sequences. The trees were generated based on aligned amino acid (AA) sequences inferred from DNA sequences. The numerical values at the nodes represent bootstrap values (1,000 iterations), and nodes with gray circles are supported by Bayesian posterior probability values greater than 95%. The scale bar represents 5% distance after Poisson correction. Organism affiliations are in parentheses (Ac, Actinobacteria; Ba, Bacteroidetes; Cx, Chloroflexi; F, Firmicutes; Ge, Gemmatimonadetes; A, Alphaproteobacteria; B, Betaproteobacteria; D, Deltaproteobacteria; G, Gammaproteobacteria; Sp, Spirochaetes; V, Verrucomicrobia; Arc, Archaea; Fu, fungi). Accession numbers are in brackets, and the database is indicated (G, GenBank; J, JGI gene object ID). Adjacent to each organism is the DNA and inferred amino acid sequence for the class I nirK primer R3Cu (primer set 2 in Table 1), with the primer sequence shown for the sense strand. DNA and amino acid mismatches relative to the R3Cu primer sequence and inferred AA sequence are indicated. In addition, DNA and AA mismatches relative to the MG763-R3Cu primer sequence are shown (primer set 3 in Table 1). Differences from the primer DNA sequence and inferred AA sequence are indicated for each sequence by showing the different base or residue. No sequence data at the primer sites are included for sequences recovered in this study, as these primer sequences were used for amplification of the gene fragment.

FIG. 4.

Unrooted phylogeny of partial and complete nosZ sequences. The trees were generated based on aligned amino acid (AA) sequences inferred from DNA sequences. Numerical values at the nodes represent bootstrap values (1,000 iterations), and nodes with gray circles are supported by Bayesian posterior probability values greater than 95%. The scale bar represents 5% distance after Poisson correction. Organism affiliations are in parentheses (Ac, Actinobacteria; Aq, Aquificae; Ba, Bacteroidetes; Cx, Chloroflexi; Def, Deferribacteres; F, Firmicutes; Ge, Gemmatimonadetes; A, Alphaproteobacteria; B, Betaproteobacteria; G, Gammaproteobacteria; D, Deltaproteobacteria; E, Epsilonproteobacteria; S, Spirochaetes; V, Verrucomicrobia; Arc, Archaea; Fu, fungi). Accession numbers are in brackets, and the database is indicated (G, GenBank; J, JGI gene object ID). Adjacent to each organism is the DNA and inferred amino acid sequence for the primer nosZ2R (primer set 6 in Table 1), with the primer sequence shown for the sense strand. DNA and amino acid mismatches relative to the nosZ2R primer sequence and inferred AA sequence are indicated. Differences from the primer DNA sequence and inferred AA sequence are indicated for each sequence by showing the different base or residue. No sequence data at the primer sites are included for alphaproteobacterial sequences recovered in this study, as these primer sequences were used for amplification of the gene fragment. Amino acid positions indicated by an “X” represent multiple possible amino acid residues encoded by degeneracies in the DNA primer sequence. At the two “X” positions, the primer codon represents either an M or L.