Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils.

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.

Unique ecophysiology among U(VI)-reducing bacteria as revealed by evaluation of oxygen metabolism in Anaeromyxobacter dehalogenans strain 2CP-C.

Anaeromyxobacter spp. respire soluble hexavalent uranium, U(VI), leading to the formation of insoluble U(IV), and are present at the uranium-contaminated Oak Ridge Integrated Field Research Challenge (IFC) site. Pilot-scale in situ bioreduction of U(VI) has been accomplished in area 3 of the Oak Ridge IFC site following biostimulation, but the susceptibility of the reduced material to oxidants (i.e., oxygen) compromises long-term U immobilization. Following oxygen intrusion, attached Anaeromyxobacter dehalogenans cells increased approximately 5-fold from 2.2x10(7)+/-8.6x10(6) to 1.0x10(8)+/-2.2x10(7) cells per g of sediment collected from well FW101-2. In the same samples, the numbers of cells of Geobacter lovleyi, a population native to area 3 and also capable of U(VI) reduction, decreased or did not change. A. dehalogenans cells captured via groundwater sampling (i.e., not attached to sediment) were present in much lower numbers (<1.3x10(4)+/-1.1x10(4) cells per liter) than sediment-associated cells, suggesting that A. dehalogenans cells occur predominantly in association with soil particles. Laboratory studies confirmed aerobic growth of A. dehalogenans strain 2CP-C at initial oxygen partial pressures (pO2) at and below 0.18 atm. A negative linear correlation [micro=(-0.09xpO2)+0.051; R2=0.923] was observed between the instantaneous specific growth rate micro and pO2, indicating that this organism should be classified as a microaerophile. Quantification of cells during aerobic growth revealed that the fraction of electrons released in electron donor oxidation and used for biomass production (fs) decreased from 0.52 at a pO2 of 0.02 atm to 0.19 at a pO2 of 0.18 atm. Hence, the apparent fraction of electrons utilized for energy generation (i.e., oxygen reduction) (fe) increased from 0.48 to 0.81 with increasing pO2, suggesting that oxygen is consumed in a nonrespiratory process at a high pO2. The ability to tolerate high oxygen concentrations, perform microaerophilic oxygen respiration, and preferentially associate with soil particles represents an ecophysiology that distinguishes A. dehalogenans from other known U(VI)-reducing bacteria in area 3, and these features may play roles for stabilizing immobilized radionuclides in situ.

Electron donor-dependent radionuclide reduction and nanoparticle formation by Anaeromyxobacter dehalogenans strain 2CP-C.

Anaeromyxobacter dehalogenans strain 2CP-C reduces U(VI) and Tc(VII) to U(IV)O(2(s)) (uraninite) and Tc(IV)O(2(S)) respectively. Kinetic studies with resting cells revealed that U(VI) or Tc(VII) reduction rates using H(2) as electron donor exceeded those observed in acetate-amended incubations. The reduction of U(VI) by A. dehalogenans 2CP-C resulted in extracellular accumulation of approximately 5 nm uraninite nanoparticles in association with a lectin-binding extracellular polymeric substance (EPS). The electron donor did not affect UO(2(S)) nanoparticle size or association with EPS, but the utilization of acetate as the source of reducing equivalents resulted in distinct UO(2(S)) nanoparticle aggregates that were approximately 50 nm in diameter. In contrast, reduction of Tc(VII) by A. dehalogenans 2CP-C cell suspensions produced dense clusters of TcO(2) particles, which were localized within the cell periplasm and on the outside of the outer membrane. In addition to direct reduction, A. dehalogenans 2CP-C cell suspensions reduced Tc(VII) indirectly via an Fe(II)-mediated mechanism. Fe(II) produced by strain 2CP-C from either ferrihydrite or Hanford Site sediment rapidly removed (99)Tc(VII)O(4)(-) from solution. These findings expand our knowledge of the radionuclide reduction processes catalysed by Anaeromyxobacter spp. that may influence the fate and transport of radionuclide contaminants in the subsurface.

Diversity and distribution of anaeromyxobacter strains in a uranium-contaminated subsurface environment with a nonuniform groundwater flow.

Versaphilic Anaeromyxobacter dehalogenans strains implicated in hexavalent uranium reduction and immobilization are present in the fractured saprolite subsurface environment at the U.S. Department of Energy Integrated Field-Scale Subsurface Research Challenge (IFC) site near Oak Ridge, TN. To provide insight into the in situ distribution of Anaeromyxobacter strains in this system with a nonuniform groundwater flow, 16S rRNA gene-targeted primers and linear hybridization (TaqMan) probes were designed for Oak Ridge IFC Anaeromyxobacter isolates FRC-D1 and FRC-W, along with an Anaeromyxobacter genus-targeted probe and primer set. Multiplex quantitative real-time PCR (mqPCR) was applied to samples collected from Oak Ridge IFC site areas 1 and 3, which are not connected by the primary groundwater flow paths; however, transport between them through cross-plane fractures is hypothesized. Strain FRC-W accounted for more than 10% of the total quantifiable Anaeromyxobacter community in area 1 soils, while strain FRC-D1 was not detected. In FeOOH-amended enrichment cultures derived from area 1 site materials, strain FRC-D1 accounted for 30 to 90% of the total Anaeromyxobacter community, demonstrating that this strain was present in situ in area 1. The area 3 total Anaeromyxobacter abundance exceeded that of area 1 by 3 to 5 orders of magnitude, but neither strain FRC-W- nor FRC-D1-like sequences were quantifiable in any of the 33 area 3 groundwater or sediment samples tested. The Anaeromyxobacter community in area 3 increased from <10(5) cells/g sediment outside the ethanol biostimulation treatment zone to 10(8) cells/g sediment near the injection well, and 16S rRNA gene clone library analysis revealed that representatives of a novel phylogenetic cluster dominated the area 3 Anaeromyxobacter community inside the treatment loop. The combined applications of genus- and strain-level mqPCR approaches along with clone libraries provided novel information on patterns of microbial variability within a bacterial group relevant to uranium bioremediation.

U(VI) reduction to mononuclear U(IV) by Desulfitobacterium species.

The bioreduction of U(VI) to U(IV) affects uranium mobility and fate in contaminated subsurface environments and is best understood in Gram-negative model organisms such as Geobacter and Shewanella spp. This study demonstrates that U(VI) reduction is a common trait of Gram-positive Desulfitobacterium spp. Five different Desulfitobacterium isolates reduced 100 microM U(VI) to U(IV) in <10 days, whereas U(VI) remained soluble in abiotic and heat-killed controls. U(VI) reduction in live cultures was confirmed using X-ray absorption near-edge structure (XANES) analysis. Interestingly, although bioreduction of U(VI) is almost always reported to yield the uraninite mineral (UO(2)), extended X-ray absorption fine structure (EXAFS) analysis demonstrated that the U(IV) produced in the Desulfitobacterium cultures was not UO(2). The EXAFS data indicated that the U(IV) product was a phase or mineral composed of mononuclear U(IV) atoms closely surrounded by light element shells. This atomic arrangement likely results from inner-sphere bonds between U(IV) and C/N/O- or P/S-containing ligands, such as carbonate or phosphate. The formation of a distinct U(IV) phase warrants further study because the characteristics of the reduced material affect uranium stability and fate in the contaminated subsurface.

Microbial colonization of an in situ sediment cap and correlation to stratified redox zones.

In situ capping is a management technique for contaminated sediments involving the placement of clean material at the sediment-water interface. This work combined porewater geochemical profiling with quantitative microbial data to investigate the intrinsic microbial colonization of a sand cap. Geochemical characterization using voltammetric microelectrodes indicated vertical stratification of biogeochemical processes within a capped sediment column. Following dissection of the column, quantitative real-time PCR (qPCR) enumerated microbial populations within each discrete redoxzone and was accompanied by terminal-restriction fragment length polymorphism (T-RFLP) to elucidate general community shifts. Bacteria and Archaea were present within the cap according to qPCR, with higher concentrations generally observed in the underlying sediment. Iron-reducing populations were detected and quantified using newly designed qPCR primer pairs for Anaeromyxobacter spp. and Shewanella spp. and published primer sets for delta-Proteobacteria and Geobacteracea. Results confirmed geochemical measurements indicating that microbial Fe(III) reduction was a major process in the overlying cap. Genes encoding microbial sulfate reduction (dsrA) and methanogenesis (mcrA) were also present within the cap but were more prevalent in the sediment. Canonical correspondence analysis of terminal-restriction fragment length polymorphism (T-RFLP) patterns verified that spatial changes in bacterial community composition were significantly correlated to depth and Fe2+ and Mn2+ concentration gradients. Cumulatively, results demonstrate that microorganisms indigenous to aquatic sediments colonized the overlying cap to form complex communities mirroring redox stratification. Implications of capping for biogeochemical cycling, contaminant fate and transport, and remedial design are discussed.

The mosaic genome of Anaeromyxobacter dehalogenans strain 2CP-C suggests an aerobic common ancestor to the delta-proteobacteria.

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta-Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb(3) oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.

Hexavalent uranium supports growth of Anaeromyxobacter dehalogenans and Geobacter spp. with lower than predicted biomass yields.

The stimulation of bacteria capable of reducing soluble U(VI) to sparingly soluble U(IV) is a promising approach for containing U(VI) plumes. Anaeromyxobacter dehalogenans is capable of mediating this activity; however, its ability to couple U(VI) reduction to growth has not been established. Monitoring the increase in 16S rRNA gene copy numbers using quantitative real-time PCR (qPCR) in cultures provided with U(VI) as an electron acceptor demonstrated growth, and 7.7-8.6 x 10(6) cells were produced per mumole of U(VI) reduced. This biomass yield was lower than predicted based on the theoretical free energy changes associated with U(VI)-to-U(IV) reduction. Lower than predicted growth yields with U(VI) as electron acceptor were also determined in cultures of Geobacter lovleyi and Geobacter sulfurreducens suggesting that U(VI) reduction is inefficient or imposes an additional cost to growing cells. These findings have implications for U(VI) bioremediation because Anaeromyxobacter spp. and Geobacter spp. contribute to radionuclide immobilization in contaminated subsurface environments.