Oak Ridge Integrated Field Research Research Articles

Stress-related ecophysiology of members of the genus Rhodanobacter isolated from a mixed waste contaminated subsurface

This work examines the physiologic basis of stress tolerance in bacterial strains of the genus Rhodanobacter that dominate in the acidic and highly metal contaminated near-source subsurface zone of the Oak Ridge Integrated Field Research Challenge (ORIFRC) site. Tolerance of R. denitrificans to levels of different stresses were studied in synthetic groundwater medium and R2A broth. Two strains of R. denitrificans, strains 2APBS1T and 116–2, tolerate low to circumneutral pH (4-8), high Uranium (1 mmol/L), elevated levels of nitrate (400 mmol/L) and high NaCl (2.5%). A combination of physiologic traits, such as growth at low pH, increased growth in the presence of high organics concentration, and tolerance of high concentrations of nitrate, NaCl and heavy metals is likely responsible for dominance of Rhodanobacter at the ORIFRC site. Furthermore, extended incubation times and use of low carbon media, better approximating site groundwater conditions, are critical for accurate determination of stress responses. This study expands knowledge of the ecophysiology of bacteria from the genus Rhodanobacter and identifies methodological approaches necessary for acquiring accurate tolerance data.

Comparative metagenomics reveals impact of contaminants on groundwater microbiomes.

To understand patterns of geochemical cycling in pristine versus contaminated groundwater ecosystems, pristine shallow groundwater (FW301) and contaminated groundwater (FW106) samples from the Oak Ridge Integrated Field Research Center (OR-IFRC) were sequenced and compared to each other to determine phylogenetic and metabolic difference between the communities. Proteobacteria (e.g., Burkholderia, Pseudomonas) are the most abundant lineages in the pristine community, though a significant proportion ( >55%) of the community is composed of poorly characterized low abundance (individually <1%) lineages. The phylogenetic diversity of the pristine community contributed to a broader diversity of metabolic networks than the contaminated community. In addition, the pristine community encodes redundant and mostly complete geochemical cycles distributed over multiple lineages and appears capable of a wide range of metabolic activities. In contrast, many geochemical cycles in the contaminated community appear truncated or minimized due to decreased biodiversity and dominance by Rhodanobacter populations capable of surviving the combination of stresses at the site. These results indicate that the pristine site contains more robust and encodes more functional redundancy than the stressed community, which contributes to more efficient nutrient cycling and adaptability than the stressed community.

Distribution of uranium and thorium in dolomitic gravel fill and shale saprolite

The objectives of this study were to examine (1) the distribution of U and Th in dolomitic gravel fill and shale saprolite, and (2) the removal of uranium from acidic groundwater by dolomitic gravel through precipitation with amorphous basaluminite at the U.S. DOE Oak Ridge Integrated Field Research Challenge (ORIFRC) field site west of the Oak Ridge Y-12 National Security Complex in East Tennessee. Media reactivity and sustainability are a technical concern with the deployment of any subsurface reactive media. Because the gravel was placed in the subsurface and exposed to contaminated groundwater for over 20 years, it provided a unique opportunity to study the solid and water phase geochemical conditions within the media after this length of exposure. This study illustrates that dolomite gravel can remove U from acidic contaminated groundwater with high levels of Al3+, Ca2+, NO3−, and SO42− over the long term. As the groundwater flows through high pH carbonate gravel, U containing amorphous basaluminite precipitates as the pH increases. This is due to an increase in groundwater pH from 3.2 to ∼6.5 as it comes in contact with the gravel. Therefore, carbonate gravel could be considered as a possible treatment medium for removal and sequestration of U and other pH sensitive metals from acidic contaminated groundwater. Thorium concentrations are also high in the carbonate gravel. Thorium generally shows an inverse relationship with U from the surface down into the deeper saprolite. Barite precipitated in the shallow saprolite directly below the dolomitic gravel from barium present in the acidic contaminated groundwater.

Geochemical and geophysical responses during the infiltration of fresh water into the contaminated saprolite of the Oak Ridge Integrated Field Research Challenge site, Tennessee

At the Oak Ridge Integrated Field Research Challenge (IFRC) site, Tennessee, the saprolitic aquifer was contaminated by leaks from the former S‐3 disposal ponds between 1951 and 1983. The chemistry of the contaminant plume is also episodically impacted by fresh meteoritic water infiltrating vertically from a shallow variably saturated perched zone and the ditch surrounding the former S‐3 ponds. We performed a column experiment using saprolite from the contaminated aquifer to understand the geochemical and complex electrical conductivity signatures associated with such events. The changes in the pH and pore water ionic strength are responsible for measurable changes in both the in‐phase and quadrature conductivities. The pore water conductivity can be related to the nitrate concentration (the main ionic species in the plume) while the release of uranium is controlled by the pH. We developed a simple model to determine the pore water conductivity and pH from the recorded complex conductivity. This model is applied to time‐lapse resistivity data at the IFRC site. Time‐lapse inversion of resistivity data, performed with an active time constrain approach, shows the occurrence of an infiltration event during the winter of 2008–2009 with a dilution of the pore water chemistry and an increase of the pH. A simple numerical simulation of the infiltration of fresh water into the unconfined contaminated aquifer is consistent with this scenario.

Genome Sequences for Three Denitrifying Bacterial Strains Isolated from a Uranium- and Nitrate-Contaminated Subsurface Environment

Genome sequences for three strains of denitrifying bacteria (Alphaproteobacteria—Afipia sp. strain 1NLS2 and Hyphomicrobium denitrificans strain 1NES1; Firmicutes—Bacillus sp. strain 1NLA3E) isolated from the nitrate- and uranium-contaminated subsurface of the Oak Ridge Integrated Field Research Challenge (ORIFRC) site, Oak Ridge Reservation, TN, are reported.

Hydrogeophysical investigations of the former S-3 ponds contaminant plumes, Oak Ridge Integrated Field Research Challenge site, Tennessee

At the Oak Ridge Integrated Field Research Challenge site, near Oak Ridge, Tennessee, contaminants from the former S-3 ponds have infiltrated the shallow saprolite for over 60 years. Two- and three-dimensional DC-resistivity tomography is used to characterize the number and location of the main contaminant plumes, which include high concentration of nitrate. These contaminant plumes have typically an electrical resistivity in the range 2–20 ohm-m while the background saprolite resistivity is in the range 60–120 ohm-m, so the difference of resistivity can be easily mapped using DC-resistivity tomography to locate the contaminant pathways. We develop a relationship to derive the in situ nitrate concentrations from the 3D resistivity tomograms accounting for the effect of surface conductivity. The footprint of the contamination upon the resistivity is found to be much stronger than the local variations associated with changes in the porosity and the clay content. With this method, we identified a total of five main plumes (termed CP1 to CP5). Plume CP2 corresponds to the main plume in terms of nitrate concentration (∼50,000 [Formula: see text]). We also used an active time constrained approach to perform time-lapse resistivity tomography over a section crossing the plumes CP1 and CP2. The sequence of tomograms is used to determine the changes in the nitrate concentrations associated with infiltration of fresh (meteoritic) water from a perched aquifer. This study highlights the importance of accounting for surface conductivity when characterizing plume distributions in clay-rich subsurface systems.

Petrophysical properties of saprolites from the Oak Ridge Integrated Field Research Challenge site, Tennessee

At the Oak Ridge Integrated Field Research Challenge site, near Oak Ridge, Tennessee, the shallow saprolitic aquifer is contaminated by nitric acid, uranium, and metals originating from the former S3 settling ponds. To interpret low-frequency geophysical methods used to image contaminant plumes, we have characterized the petrophysical properties of three representative saprolite core samples. Their hydraulic conductivity ranges from [Formula: see text] to [Formula: see text] in agreement with field data. Complex conductivity measurements, in the frequency range of 1 mHz to 45 kHz, were performed with NaCl solutions with electrical conductivities in the range [Formula: see text] to [Formula: see text], a range representative of field conditions. The electrical conductivity data were well reproduced with a simple linear conductivity model between the saprolite conductivity and the pore water conductivity. The conductivity plots were used to estimate the formation factor (the cementation exponent was about [Formula: see text]) and the surface conductivity ([Formula: see text]). The magnitude of the surface conductivity depended on the degree of weathering and therefore on the amount of smectite and mixed layer (illite-smectite) clays present in the saprolite. The chargeability of the core samples was in the range of [Formula: see text] and is strongly dependent on the salinity. We also performed streaming potential measurements with the same pore fluid composition as that used for the complex conductivity measurements. We found an excess of movable electrical charges on the order of 100 to [Formula: see text] in agreement with previous investigations connecting the movable excess charge density to permeability. The zeta potential was in the range of [Formula: see text] to [Formula: see text] independent on the salinity. The electrical measurements were consistent with an average cation exchange capacity in the range of 1.4 to [Formula: see text] and a specific surface area on the order of 4000 to about 30,000 [Formula: see text].

Long-term electrical resistivity monitoring of recharge-induced contaminant plume behavior

Geophysical measurements, and electrical resistivity tomography (ERT) data in particular, are sensitive to properties that are related (directly or indirectly) to hydrological processes. The challenge is in extracting information from geophysical data at a relevant scale that can be used to gain insight about subsurface behavior and to parameterize or validate flow and transport models. Here, we consider the use of ERT data for examining the impact of recharge on subsurface contamination at the S-3 ponds of the Oak Ridge Integrated Field Research Challenge (IFRC) site in Tennessee. A large dataset of time-lapse cross-well and surface ERT data, collected at the site over a period of 12months, is used to study time variations in resistivity due to changes in total dissolved solids (primarily nitrate). The electrical resistivity distributions recovered from cross-well and surface ERT data agrees well, and both of these datasets can be used to interpret spatiotemporal variations in subsurface nitrate concentrations due to rainfall, although the sensitivity of the electrical resistivity response to dilution varies with nitrate concentration. Using the time-lapse surface ERT data interpreted in terms of nitrate concentrations, we find that the subsurface nitrate concentration at this site varies as a function of spatial position, episodic heavy rainstorms (versus seasonal and annual fluctuations), and antecedent rainfall history. These results suggest that the surface ERT monitoring approach is potentially useful for examining subsurface plume responses to recharge over field-relevant scales.

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO− 3 most rapidly (Vmax = 15.8 μmol e−·min−1·mg protein−1) and had the fastest generation time (gt) on NO− 3 (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO− 2 tolerant and grew rapidly on NO− 2 (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO− 2 rapidly (Vmax = 10.6 μmol e−·min−1·mg protein−1) but was far less NO− 2 tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO− 3 more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO− 2 accumulation in high-NO− 3 groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.

Gene Expression Correlates with Process Rates Quantified for Sulfate- and Fe(III)-Reducing Bacteria in U(VI)-Contaminated Sediments

Though iron- and sulfate-reducing bacteria are well known for mediating uranium(VI) reduction in contaminated subsurface environments, quantifying the in situ activity of the microbial groups responsible remains a challenge. The objective of this study was to demonstrate the use of quantitative molecular tools that target mRNA transcripts of key genes related to Fe(III) and sulfate reduction pathways in order to monitor these processes during in situ U(VI) remediation in the subsurface. Expression of the Geobacteraceae-specific citrate synthase gene (gltA) and the dissimilatory (bi)sulfite reductase gene (dsrA), were correlated with the activity of iron- or sulfate-reducing microorganisms, respectively, under stimulated bioremediation conditions in microcosms of sediments sampled from the U.S. Department of Energy’s Oak Ridge Integrated Field Research Challenge (OR-IFRC) site at Oak Ridge, TN, USA. In addition, Geobacteraceae-specific gltA and dsrA transcript levels were determined in parallel with the predominant electron acceptors present in moderately and highly contaminated subsurface sediments from the OR-IFRC. Phylogenetic analysis of the cDNA generated from dsrA mRNA, sulfate-reducing bacteria-specific 16S rRNA, and gltA mRNA identified activity of specific microbial groups. Active sulfate reducers were members of the Desulfovibrio, Desulfobacterium, and Desulfotomaculum genera. Members of the subsurface Geobacter clade, closely related to uranium-reducing Geobacter uraniireducens and Geobacter daltonii, were the metabolically active iron-reducers in biostimulated microcosms and in situ core samples. Direct correlation of transcripts and process rates demonstrated evidence of competition between the functional guilds in subsurface sediments. We further showed that active populations of Fe(III)-reducing bacteria and sulfate-reducing bacteria are present in OR-IFRC sediments and are good potential targets for in situ bioremediation.

Denitrifying Bacteria from the Genus Rhodanobacter Dominate Bacterial Communities in the Highly Contaminated Subsurface of a Nuclear Legacy Waste Site

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface was investigated at the field scale at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN. The abundance, community composition, and distribution of groundwater microorganisms were examined across the site during two seasonal sampling events. At representative locations, subsurface sediment was also examined from two boreholes, one sampled from the most heavily contaminated area of the site and another from an area with low contamination. A suite of DNA- and RNA-based molecular tools were employed for community characterization, including quantitative PCR of rRNA and nitrite reductase genes, community composition fingerprinting analysis, and high-throughput pyrotag sequencing of rRNA genes. The results demonstrate that pH is a major driver of the subsurface microbial community structure and that denitrifying bacteria from the genus Rhodanobacter (class Gammaproteobacteria) dominate at low pH. The relative abundance of bacteria from this genus was positively correlated with lower-pH conditions, and these bacteria were abundant and active in the most highly contaminated areas. Other factors, such as the concentration of nitrogen species, oxygen level, and sampling season, did not appear to strongly influence the distribution of Rhodanobacter bacteria. The results indicate that these organisms are acid-tolerant denitrifiers, well suited to the acidic, nitrate-rich subsurface conditions, and pH is confirmed as a dominant driver of bacterial community structure in this contaminated subsurface environment.

Nitrate-Reducing Bacteria at the Nitrate and Radionuclide Contaminated Oak Ridge Integrated Field Research Challenge Site: A Review

Mining and enrichment of uranium (U) for nuclear weapons and energy has left this radionuclide an important groundwater contaminant in the United States and worldwide. The use of nitric acid in these processes has also resulted in low pH and high nitrate concentrations in U-contaminated subsurface sediments. This presents problems for bioremediation strategies to control the migration of U in groundwater, usually achieved through microbial reduction of soluble U(VI) to insoluble U(IV) upon electron donor addition to the subsurface. Nitrate, which serves as a competitive electron acceptor in the subsurface, can inhibit or retard U(VI) reduction efforts; as well, intermediates of nitrate respiration (or denitrification), such as nitrite, can lead to U(IV) oxidation and remobilization. The Integrated Field Research Challenge site in Oak Ridge, Tennessee provides an ideal location to address the challenges nitrate poses to uranium bioreduction as the site encompasses several different geochemical conditions: two acidic U- high-nitrate U-contaminated sites (both Areas 1 and 3), a low-nitrate U-contaminated site (Area 2), and a pristine (uncontaminated) background site. In this paper, we review 24 studies examining the microbial communities from these sites within the OR-IFRC as well as denitrifying fluidized bed reactors (FBRs) treating high-nitrate groundwater in an effort to describe the overall potential denitrifying community composition at these sites. Pseudomonas was the most widely detected genus among all sites, but was not detected in either of two studies describing metabolically active populations from community total RNA extracts. Collectively, 16S rRNA gene surveys indicate the following genera may be of potential importance in nitrate reduction and denitrification at the OR-IFRC: Ralstonia and Dechloromonas in the low nitrate neutral pH Area 2, Castellaniella and Burkholderia in Area 1, Thiobacillus and Ferribacterium in Area 3, and Acidovorax in FBRs. This work begins to help us understand how geochemical conditions can determine the composition of nitrate-reducing microbial communities at uranium contaminated sites as well as how population structure and physiologies of the microorganisms present affect in situ rates of denitrification and radionuclide immobilization.

Coupled modeling of hydrogeochemical and electrical resistivity data for exploring the impact of recharge on subsurface contamination

The application of geophysical methods, in particular, electrical resistivity measurements, may be useful for monitoring subsurface contamination. However, interpreting geophysical data without additional data and without considering the associated hydrogeochemical processes is challenging since the geophysical response is sensitive to not only heterogeneity in rock properties but also to the saturation and chemical composition of pore fluids. We present an inverse modeling framework that incorporates the simulation of hydrogeochemical processes and time‐lapse electrical resistivity data and apply it to various borehole and cross‐borehole data sets collected in 2008 near the S‐3 Ponds at the U.S. Department of Energy's Oak Ridge Integrated Field Research Challenge site, where efforts are underway to better understand freshwater recharge and associated contaminant dilution. Our goal is to show that the coupled hydrogeochemical‐geophysical modeling framework can be used to (1) develop a model that honors all the available data sets, (2) help understand the response of the geophysical data to subsurface properties and processes at the site, and (3) allow for the estimation of petrophysical parameters needed for interpreting the geophysical data. We present a series of cases involving different data sets and increasingly complex models and find that the approach provides useful information about soil properties, recharge‐related transport processes, and the geophysical response. Spatial heterogeneity of the petrophysical model can be described sufficiently with two layers, and its parameters can be estimated concurrently with the hydrogeochemical parameters. For successful application of the approach, the parameters of interest must be sensitive to the available data, and the experimental conditions must be carefully modeled.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

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.

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.