Phillip M. Jardine

Changes in bacterial and archaeal community structure and functional diversity along a geochemically variable soil profile.

ABSTRACT Spatial heterogeneity in physical, chemical, and biological properties of soils allows for the proliferation of diverse microbial communities. Factors influencing the structuring of microbial communities, including availability of nutrients and water, pH, and soil texture, can vary considerably with soil depth and within soil aggregates. Here we investigated changes in the microbial and functional communities within soil aggregates obtained along a soil profile spanning the surface, vadose zone, and saturated soil environments. The composition and diversity of microbial communities and specific functional groups involved in key pathways in the geochemical cycling of nitrogen, Fe, and sulfur were characterized using a coupled approach involving cultivation-independent analysis of both 16S rRNA (bacterial and archaeal) and functional genes (amoA and dsrAB) as well as cultivation-based analysis of Fe(III)-reducing organisms. Here we found that the microbial communities and putative ammonia-oxidizing and Fe(III)-reducing communities varied greatly along the soil profile, likely reflecting differences in carbon availability, water content, and pH. In particular, the Crenarchaeota 16S rRNA sequences are largely unique to each horizon, sharing a distribution and diversity similar to those of the putative (amoA-based) ammonia-oxidizing archaeal community. Anaerobic microenvironments within soil aggregates also appear to allow for both anaerobic- and aerobic-based metabolisms, further highlighting the complexity and spatial heterogeneity impacting microbial community structure and metabolic potential within soils.

Hydrogeochemical processes controlling the transport of dissolved organic carbon through a forested hillslope

Abstract The subsurface transport of dissolved organic carbon (DOC) through a proposed waste burial site during rain events was investigated in order to assess the role of colloid-mediated contaminant mobility. A sub-watershed (0.45 ha) located on a forested hillslope in eastern Tennessee, U.S.A., was instrumented with an isolated soil pedon for one-dimensional transport studies, and a subsurface weir monitoring system for three-dimensional transport studies. The source of DOC in the soils resulted from dissolution of organic matter in the surface horizon during, and between, rain events, as well as from a highly reactive B horizon which stored significant quantities of DOC in small pores. During large storms, the concentration of DOC was similar on ascending and descending limbs of the subface hydrograph, with a maximum concentration occuring at peak flow. During small storms however, chemical interactions with soil solution SO 4 2- caused DOC concentrations to be greater on the ascending limb of the hydrograph, with maximum DOC concenrations occuring before peak flow. Because subsurface lateral flow through preferential paths predominated in the Bt2 and the Bt3 horizons of the soil during storm events, the total cumullative flux of DOC downslope was generally much greater through the lower soil horizons. A significant component of mobile DOC consisted of hydrophobic organic solutes, even though this material was selectively adsorbed with soil depth relative to hydrophilic organic solutes. The implications of these findings on subsurface contaminant transport are discussed.

Field-scale transport from a buried line source in variably saturated soil

Lateral subsurface flow through the upper soil layers (stormflow zone) during storm events has been shown to be a dominant mechanism of contaminant transport in forested watersheds. Data bases for multi-region flow and transport modeling for hydrogeologic conditions where stormflow predominates are lacking. Direct measurement of the tracer flux under field-scale conditions are non-existent. The objective of this paper was to evaluate the significance of three hydrologic pore regions to stormflow. Two tracer releases were made from a buried line source during storm events and the spatial and temporal variability in solute concentration and the tracer fluxes were measured. During one of the injections, macropore flow was extremely rapid with solute transport to a downslope trench 65 m from the line source taking just 3.2h. Mesopore flow appeared to be significant for short distances in that tracer movement to solution samplers just 3 m downslope of the line source occurred within 3 h of the release. Soil sampling 6 months after the second release revealed that the tracer plume was refracted in the direction of the fractured bedding plane, and therefore did not coincide with the array of samplers for distances greater than 13 m downslope of the source. Soil sampling data suggested that micropores served as a sink/source for Br− with 47% of the non-reactive tracer remaining immobilized by micropores at the termination of the study. Interaction between the upper 2 m of the stormflow zone and the groundwater system was believed minimal; however, lateral flow below 2 m was concluded to be significant.

Reactive transport of EDTA-complexed cobalt in the presence of ferrihydrite

Abstract Many low-level radioactive wastes, historically disposed in shallow land trenches, are illdefined mixtures of radionuclides and organic chelating agents. The observed migration of nuclides, such as 60 Co, away from burial sites has been attributed, in part, to the formation of aqueous complexes with ethylenediaminetetraacetic acid (EDTA). The stability of Co-EDTA complexes, and thus the fate and transport: of 60 Co in the subsurface, is strongly dependent on the oxidation state of cobalt (log K co(II)EDTA = 18.3; log K Co(III)EDTA = 43.9). The factors that control the oxidation of Co(II) to Co(III) in subsurface environments are not well understood. We conducted a series of column flow experiments to provide an improved understanding of the geochemical processes that control the reactive transport of cobalt in the subsurface. A solution of 0.2 mM Co(II)EDTA 2− in 5 mM CaCl 2 was passed through saturated columns that were packed with ferrihydrite (Fe(OH) 3 )-coated Si0 2 . During transport through the column, a portion of the Co (II) EDTA 2− was oxidized to Co (III) EDTA − ; the amount of oxidation reached a steady-state under oxic conditions. Transport of the oxidized species, Co(III)EDTA − , was substantially more rapid than the transport of Co(II) EDTA 2− . The retardation of both Co-EDTA species and the extent of cobalt oxidation increased as the pH decreased. These results are consistent with the hypothesis that the association of Co(H)EDTA 2− with the ferrihydrite surface is essential for the charge-transfer involved in the oxidation reaction. Co(III)EDTA- exhibited less retardation because this monovalent anion had a lower affinity for the surface than the divalent Co(II)EDTA 2− . At faster flow rate, the retardation of Co(II)EDTA 2− decreased whereas Co (III) EDTA — breakthrough occurred later; the amount of Co(III)EDTA − formed decreased with increasing flow rate. Under anoxic conditions, the oxidation of Co(II)EDTA 2− was decreased, but was not eliminated, suggesting that ferric iron may serve as an oxidant in the system. The loss of oxidative sites under continuous exposure to Co(II)EDTP 2− and the blocking of oxidative sites by ions residing on the ferrihydrite surface resulted in a slow decline in the amount of oxidation under anoxic conditions. The oxidation of Co(II)EDTA 2− effectively competed with other geochemical reactions such as the Fe(III)-induced dissociation of Co(II)EDTA 2− complexes under oxic and anoxic conditions. These results indicate that an iron mineral can be more important for the formation of Co(III)EDTA 2− in the subsurface than the mineral is important for the dissociation of Co(II)EDTA − and the concomitant formation of Fe(III)EDTA − . The results suggest that conditions of pH and flow rate that inhibit the formation of the very stable Co(III)EDTA − also promote the undesirable rapid transport of Co(II)EDTA 2− posing a challenge to the selection of future waste sites and the development of remedial strategies for existing sites impacted by EDTA-complexed 60 Co.

Transport of Multiple Tracers in Variably Saturated Humid Region Structured Soils and Semi-arid Region Laminated Sediments

The processes governing physical nonequilibrium (PNE)—coupled preferential flow and matrix diffusion—are diverse between humid and semi-arid regions, and are directly related to climate and rock/sediment type, and indirectly related to subsequent soil profile development. The fate and transport of contaminants in these variably saturated undisturbed media is largely a function of the influence of PNE processes. Large cores of laminated silts and sands were collected from the US Department of Energy Pacific Northwest National Laboratory (PNNL) in semi-arid south central Washington. Additional cores of weathered, fractured interbedded limestone and shale saprolite were collected from the Oak Ridge National Laboratory (ORNL) in humid eastern Tennessee. PNNL cores were collected parallel (FBP) and perpendicular (FXB) to bedding, and the ORNL core was 30° to bedding. Saturated and unsaturated transport experiments were performed using multiple nonreactive tracers that had different diffusion coefficients (Br−, PFBA, and PIPES), in order to identify the influence of PNE on the fate and transport of solutes. In the ORNL structured saprolite, solute transport was governed by coupled preferential flow and matrix diffusion, as evidenced by tracer separation and highly asymmetric breakthrough curves (BTC). BTCs became more symmetric as preferential flowpaths became inactive during drainage. Tracer separation persisted during unsaturated flow suggesting the continued importance of nonequilibrium mass transfer between flowpaths and the immobile water that was held in the soil matrix. No evidence of PNE was observed under near-saturated conditions in the semi-arid region (PNNL) laminated silts and sands. Unsaturated flow in cores with discontinuous layering resulted in preferential flow and the development of perched, immobile water as evidenced by early breakthrough and separation of tracers. Conversely, transport parallel to laterally continuous beds did not result in preferential flow, the development of perched water, or tracer separation regardless of water content. These observations suggested that desaturation had two effects: (1) grain size variations between individual beds resulted in different antecedent water contents, and (2) the exchange of water and solutes between individual sedimentary beds was subsequently inhibited. Under unsaturated conditions, these effects may promote either stable lateral flow, or unstable vertical finger flow coupled with the development of perched, immobile water.

Diversity and Distribution of Anaeromyxobacter Strains in a Uranium-Contaminated Subsurface Environment with a Nonuniform Groundwater Flow

ABSTRACT 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 <105 cells/g sediment outside the ethanol biostimulation treatment zone to 108 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.

Uranium (VI) Reduction by Denitrifying Biomass

ABSTRACT Groundwater near the S3 ponds at the US Department of Energys Y-12 site in Oak Ridge, Tennessee, is contaminated by high levels of nitrate (up to 160 mM) and U(VI) (∼0.3 mM). To minimize nitrate inhibition, the authors proposed extraction of contaminated groundwater, nitrate removal in a denitrifying fluidized bed bioreactor (FBR), and return of nitrate-free effluent to the aquifer to stimulate in situ microbial reduction of U(VI). In the presence of carbonate, U(VI) sorption to biomass was negligible, but in its absence, sorption was significant. Biomass reduced U(VI) to U(IV), exhibiting slow first-order removal with respect to U(VI). Addition of electron donor increased rates. Addition of an inhibitor of sulfate reduction (molybdate) slowed the rate and inhibited sulfate reduction. Denitrifying β-Proteobacteria dominated clone libraries of SSU rRNA and dsrA gene sequences. Approximately 10% were low-G+C microorganisms that had 90% to 92% sequence identity with Sporomusa, Acetonema, and Propionispora. The dsrA sequences were dominated by a single clone with ∼80% nucleotide identity to dsrA of Desulfovibrio vulgaris sub sp. oxamicus. The authors conclude that some members of this denitrifyng community reduce uranium, and that sulfate-reducing bacteria likely contribute to this capability.

Elucidating Biogeochemical Reduction of Chromate via Carbon Amendments and Soil Sterilization

Sterilized and non-sterilized soil columns were amended with three different carbon sources to elucidate the potential for geochemical and biological Cr6+ reduction. Cr6+ was reduced to Cr3+ in the non-sterilized lactate, ethanol, and acetate-amended soils; however, soils amended with lactate reduced significantly more chromium. Soils sterilized by γ-irradiation reduced almost no Cr6+, indicating that Cr6+ reduction was at least indirectly biological in nature. Analyses of small subunit (ssu) rRNA genes amplified from the column sediments showed significantly different bacterial populations within the amended soils that may be due to carbon source or to aerobic micropockets within the sediment columns.

Micro‐Scale Heterogeneity in Biogeochemical Uranium Cycling

One method for the in situ remediation of uranium contaminated subsurface environments is the removal of highly soluble U(VI) from groundwater by microbial reduction to the sparingly soluble U(IV) mineral uraninite. Success of this remediation strategy will, in part, be determined by the extent and products of microbial reduction. In heterogeneous subsurface environments, microbial processes will likely yield a combination of U(IV) and U(VI) phases distributed throughout the soil matrix. Here, we use a combination of bulk X‐ray absorption spectroscopy (XAS) and micro‐focused XAS and X‐ray diffraction to determine uranium speciation and distribution with sediment from a pilot‐scale uranium remediation project located in Oak Ridge, TN.