Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N ₂ O) concentrations. N ₂ O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N ₂ O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N ₂ O to N ₂ reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N ₂ O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos ), and that previous nosZ -targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N ₂ O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N ₂ O consumption will advance understanding of the ecological controls on N ₂ O emissions and lead to refined greenhouse gas flux models.

Five strains were isolated which form a physiologically and phylogenetically coherent group of chlororespiring microorganisms and represent the first taxon in the Myxobacteria capable of anaerobic growth. The strains were enriched and isolated from various soils and sediments based on their ability to grow using acetate as an electron donor and 2-chlorophenol (2-CPh) as an electron acceptor. They are slender gram-negative rods with a bright red pigmentation that exhibit gliding motility and form spore-like structures. These unique chlororespiring myxobacteria also grow with 2,6-dichlorophenol, 2,5-dichlorophenol, 2-bromophenol, nitrate, fumarate, and oxygen as terminal electron acceptors, with optimal growth occurring at low concentrations (<1 mM) of electron acceptor. 2-CPh is reduced by all strains as an electron acceptor in preference to nitrate, which is reduced to ammonium. Acetate, H ₂ , succinate, pyruvate, formate, and lactate were used as electron donors. None of the strains grew by fermentation. The 16S ribosomal DNA (rDNA) sequences of the five strains form a coherent cluster deeply branching within the family Myxococcaceae within the class Myxobacteria and are mostly closely associated with the Myxococcus subgroup. With the exception of anaerobic growth and lack of a characteristic fruiting body, these strains closely resemble previously characterized myxobacteria and therefore should be considered part of the Myxococcus subgroup. The anaerobic growth and 9.0% difference in 16S rDNA sequence from those of other myxobacterial genera are sufficient to place these strains in a new genus and species designated Anaeromyxobacter dehalogenans . The type strain is 2CP-1 (ATCC BAA-258).

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to- cis -1,2-dichloroethene ( cis -DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [ cis -DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min ⁻¹ mg of protein ⁻¹ , respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H ₂ was consumed to threshold concentrations of 0.08 ± 0.03 nM, 0.16 ± 0.07 nM, and 0.5 ± 0.06 nM, respectively, and acetate was consumed to 3.0 ± 2.1 nM, 1.2 ± 0.5 nM, and 3.6 ± 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter ) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi . In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at −300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis -dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA -containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.