Subsurface Biosphere Initiative Workshop/ IGERT Retreat
June 18-21, 2006

Abstracts of Talks
June 18-21, 2006

In Agenda Order

Center of Excellence for Subsurface Biosphere Education and Research

Lewis Semprini, Daniel Arp, Peter Bottomley, Martin Fisk, and David Myrold, Oregon State University

The Center of Excellence for Subsurface Biosphere Education and Research, supports several themes of OSU's Strategic Plan, including the I) Understanding of the dynamics, and sustainability of the Earth and its resources; II) Realizing fundamental contributions in the life sciences and optimizing health and well-being of the public; and III) Managing natural resources that contribute to Oregon's quality of life and growing and sustaining natural resource based industries. The specific goals are to 1) Promote the development of a Center of Excellence for Subsurface Biosphere Education and Research; 2) Continue to develop Top Tier multidisciplinary graduate education relevant to Subsurface Biosphere; 3) Foster the development of externally funded Research Centers and Programs and large interdisciplinary research grants; and 4) Support undergraduate and graduate research experiences that promote student diversity. The Center of Excellence for Subsurface Biosphere Education and Research is one focal point of OSU Strategic Plan themes that directly relates to the mission of OSU as a Land Grant University. The Subsurface Biosphere Initiative interconnects the topical areas, including existing educational and research activities. The three focus areas are: Global Biogeochemical Cycles, Sustainable Natural Resources, and Engineered Environmental Processes. The Initiative involves twenty-eight faculty members in five OSU Colleges (Agricultural Sciences, Engineering, Forestry, Oceanic and Atmospheric Science, and Science). The Center of Excellence for Subsurface Biosphere Education and Research will serve to tie the biological and earth scientists and environmental engineers closer together to make fundamental contributions to the better understanding of biogeochemical cycles; foster new technologies in interdisciplinary efforts to study soil processes in a more holistic fashion; emphasize the interaction between microorganisms, water, and the physico-chemical properties of the subsurface; and promote the engineering of environmental processes for clean-up of contaminants, as well as fostering the development of nanotechnology.

Session 1: Global Geochemical Cycles and the Subsurface Biosphere

Cycles and the Subsurface Biosphere Biogeochemical Transformations of Metals: New Mechanistic Insights from Coupled Microbial-Geochemical Research

Jim Fredrickson and John Zachara, Pacific Northwest National Laboratory

The microbial oxidation and reduction of metals are recognized as globally significant processes that control the biogeochemical cycling of a variety of elements. These processes have also been highly touted for their potential to alter the solubility of metals and radionuclides, and hence to alter the transport of these contaminants in the environment. Over the past few decades, numerous microorganisms capable of catalyzing these reactions have been isolated and characterized with regards to their physiology and phylogeny. Many of these organisms have also been used extensively for laboratory-based biogeochemical investigations including our own research involving several species of dissimilatory metal-reducing Shewanella. Early investigations provided new insights into the geochemical factors controlling these reactions but largely lacked insight into the biological controls due to the absence of detailed knowledge on biochemical mechanisms catalyzing these reactions. Nonetheless, these early experiments identified important mechanistic questions regarding the molecular nature of biogeochemical interactions such as electron transfer that are now becoming accessible through vastly more sensitive microscopies and spectroscopies, molecular modeling, and genomic information. The availability of whole genome sequence for microbes involved in biogeochemical cycling of metals, including Shewanella oneidensis MR-1 and other strains of Shewanella is providing numerous new biological insights into the function of these model dissimilatory metal-reducing bacteria, and how they accomplish varied biogeochemical activities. Many of these findings, including the identification of a high number of c-type cytochromes in both organisms, have resulted from comparative genomic analyses, and several have been experimentally confirmed. These genome sequences have also aided the identification of genes important for the reduction of metal ions and other electron acceptors utilized during anaerobic growth, by facilitating the identification of genes disrupted by random insertions. Technologies for assaying global expression patterns for genes and proteins have also been employed, but their application has been limited mainly to the analysis of the role of global regulatory genes and to identifying genes expressed or repressed in response to specific electron acceptors. It is anticipated that details of the mechanisms of metal ion respiration, and metabolism in general, will eventually be revealed by comprehensive, systems-level analyses enabled by functional genomics data in combination with increasingly detailed geochemical measurements and carefully conceived in vitro and in vivo experiments.

Microbes, the First Miners

Radu Popa, Portland State University

The interaction of life with solid phases includes surface recognition, attachment but also phase transformations. Such processes are common in the repertoire of Bacteria. In fact, the subsurface biosphere is mostly the result of microbes acting at solid-liquid interfaces. Prokaryotes may either form minerals (induced and controlled biomineralization) or participate to the bioweathering of various solid phases (minerals and amorphous solids). Due primarily to the small scales involved the mechanisms used by microbes to interact with solid phases are poorly understood. Sometimes, even the benefits of these interactions for the microbes are unclear. The connections between the microbes and solid interfaces are discussed, particularly energetic benefits derived from these interactions. The outcome of microbe mineral interactions also helps identify biosignatures at the micron scale; examples include weathering patterns and intracellular crystallites.

Life in the Slow Lane: Methanogenesis in Permanently Cold Marine Sediments

Melissa M. Kendall, Department of Microbiology, University of Texas Southwestern Medical Center

Methanogenesis in cold marine sediments is a globally-important process, leading to methane hydrate deposits, cold seeps, physical instability of the sediment, and atmospheric methane emissions. To investigate methanogenesis in such an environment, we employed a multidisciplinary approach that combined culture-dependent analyses with geochemical assays in the cold, anoxic sediments of Skan Bay, Alaska (53 N, 167 W). Enrichment cultures were prepared in 5 cm increments from the sediment-water interface to 69 cm depth with formate, acetate, or trimethylamine as catabolic substrate. Most probable number enumeration indicated that methanogens were present in numbers of 102 to 103 per gram of sediment. These cultures produced methane after year-long incubations, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant, marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO2-reducing methanogens: AK-3, AK7, and AK-8. Additionally, two psychrotolerant, syntrophic strains were enriched from these sediments. One strain, AK-B, oxidized butyrate syntrophically and was isolated in defined co-culture with a H2-using methanogen or in a dixenic co-culture that also contained an acetate-scavenging methanogen. The other enrichment culture syntrophically oxidized propionate. Growth of these syntrophic cultures was very slow, approximately 1 year for co-cultures of strain AK-B to form colonies and >1 year for the propionate-oxidizing enrichment to form colonies. The cultivation techniques used to grow the methanogens and syntrophs as well as their phylogenetic positions and physiological characteristics will be discussed. The distribution and abundance of methanogens and syntrophic bacteria in the sediments of Skan Bay provides evidence for the importance of CO2-reducing and methylotrophic methanogens as well as syntrophic bacteria in cold marine sediments.

Experimentally Determined Rates of Methanogenesis in Methane Hydrate-Bearing Sediments

F.S. Colwell, Idaho National Laboratory, T. Lorenson, U.S. Geological Survey, S. Boyd, University of Idaho, M. Delwiche, Idaho National Laboratory, D. Reed, Idaho National Laboratory, D. Newby

Hydrate modelers require data on in situ rates of methanogenesis and abiotic controls on these rates. For this work we estimated the in situ methane production rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. A real time polymerase chain reaction assay that targeted the methanogen-specific mcr gene indicated that 75% of the HR samples analyzed were below the detection limits for the assay (<100 methanogens/g of sediment). Samples with higher numbers of methanogens were either from sediments <10 meters below seafloor or from a few deeper locations that appeared to be associated with notable geological features such as the boundary of gas hydrate and free gas (known as the bottom-simulating reflector or BSR), and an ash-bearing zone with high fluid movement. Acetate concentrations in sample porewaters ranged from 3.17 to 2515??M with the highest concentrations measured just above the BSR at a control site relative to HR. Locally high concentrations of acetate were evident at all BSRs, and often corresponded with gas hydrate presence. High acetate concentrations sometimes corresponded to low methanogen biomass. Acetate concentrations were typically lowest near the seafloor. Porewater hydrogen concentrations ranged from 16.45 to 1036 parts per million by volume (ppmv). Sometimes hydrogen and acetate concentrations were elevated concurrently. By combining methanogenesis rates for starved cells and the numbers of methanogens at selected depths we derived a maximum estimate of 1.7 x 10-6 nmol methane produced/g sediment/day (where methanogens could not be detected), a rate that is lower than previous methanogenic rate estimates in hydrate-bearing sediments. Where methanogen numbers are higher the rates may exceed 7.4 x 10-2 nmol methane produced/g sediment/day. These data will improve models intended to predict the location of hydrates in marine sediments, the amount of biogenic gas that can accumulate in the sediments, and the potential influence that this microbially-mediated source of methane has on the global carbon cycle.

Metal Precipitation and Mobility in Systems with Fluid Flow and Mixing: Illustrating Coupling and Scaling Issues

G.D. Redden, Idaho National Laboratory, Y. Fang, Pacific Northwest National Laboratory, T.D. Scheibe, Pacific Northwest National Laboratory, A.M. Tartakovsky, Pacific Northwest National Laboratory, D.T. Fox, Idaho National Laboratory , T.A.White, Idaho National Laboratory

Two- and three-dimensional experiments in packed-sand media were conducted where solutions containing calcium and carbonate ions came into contact along a parallel flow boundary and mixed by dispersion and diffusion. The result is the propagation of calcium carbonate precipitates along the solution-solution boundary in the direction of flow. As carbonate precipitates fill the pore space transport of solutes between the two solutions is restricted and therefore precipitation, flow, and transport are coupled. The distribution of carbonate phases is a complex interaction involving precipitation and dissolution kinetics, which are functions of pore-scale saturation indices and solute ratios, heterogeneous vs. homogeneous nucleation and growth mechanisms and changes in porosity and flow. Experimental and modeling results illustrate challenges in understanding the macroscopic and microscopic phenomena that depend on solute mixing, the relevance of molecular and pore-scale processes to the macroscopic behavior, and potential impact on metal mobility in porous media. Ultimately, the principles involved in the deposition of mass in fluid-fluid mixing zones will have applications to experiments on the distribution and function of biological activity in porous media.

Session 2: Plants as Drivers

Woody Plant Invasion of Grassland: Impacts on Soil Carbon and Nitrogen Storage and Dynamics

Thomas W. Boutton, Department of Rangeland Ecology and Management, Texas A&M University

Woody plant invasion of grasslands is prevalent worldwide, but the biogeochemical consequences of this vegetation change remain largely unquantified. In the Rio Grande Plains, TX, grasslands and savannas dominated by C4 grasses have undergone succession over the past century to subtropical thorn woodlands dominated by C3 trees/shrubs. To elucidate mechanisms of soil organic carbon (SOC) and soil total N (STN) storage and dynamics in this ecosystem, the mass and isotopic composition (?13C, ?15N) of C and N in whole-soil and soil size/density fractions were measured in chronosequences consisting of remnant grasslands (Time 0) and woody plant stands ranging in age from 10-130 years. Rates of SOC and STN storage averaged 10-30 g C m-2 yr-1 and 1-3 g N m-2 yr-1, respectively, in the upper 15 cm of soil. Soil microbial biomass (SMB-C) also increased after woody invasion. Decreasing Cmic/Corg and higher qCO2 in woodlands relative to grasslands suggests that woody litter is of poorer quality than grassland litter. Greater SOC and STN following woody invasion was also due to stabilization of organic matter by soil physical structure. Soil aggregation increased following woody encroachment; however, considerable C and N accumulated in free particulate organic matter (POM) fractions not protected within aggregates. Mean residence times (MRTs) of soil fractions were determined from changes in their 13C with time after woody encroachment. Free POM had the shortest average MRTs (30 yrs) and silt+clay the longest (360 yrs). Intra-aggregate POM had MRTs of about 60 yrs, reflecting protection by soil physical structure. 15N values of soil fractions were positively correlated with their MRTs, suggesting that higher 15N values reflect an advanced state of decay. Increases in SOC and STN are probably driven by greater inputs, protection of organic matter by association with silt and clay and inclusion in aggregate structure, and biochemical recalcitrance of woody inputs. Grassland-to-woodland conversion during the past century has been geographically extensive in grassland ecosystems worldwide, suggesting that changes in soil C and N dynamics and storage documented here could have significance for global C and N cycles.

Mycorrhizal Fungi and Their Influence on Soil Dynamics

Kathleen K. Treseder, Dept. of Ecology and Evolutionary Biology, University of California, Irvine, and Katie M. Turner, Dept. of Biological Sciences, Stanford University

Global change can alter the capacity of soils to store organic carbon and to resist erosion. Mycorrhizal fungi can also mediate soil responses to global change, and in doing so they may offset or accentuate carbon sequestration and erosion. For instance, arbuscular mycorrhizal fungi are the only organisms known to produce glomalin, a glycoprotein that can reside in the soil for years to decades. Because it has a long residence time, glomalin accumulates until it represents as much as 5% of soil carbon. As such, glomalin may sequester a large amount of carbon globally. In addition, glomalin appears to increase soil aggregation, which deters erosion. Nevertheless, examinations of environmental controls over glomalin are still in their early stages. Arbuscular mycorrhizal fungi rely almost completely on their host plants as a source of carbon. Therefore, environmental conditions that influence the carbon status of plants can also influence arbuscular mycorrhizal fungi. One example is atmospheric CO2 enrichment, which increases photosynthetic rates in plants and accentuates nitrogen or phosphorus limitation of plant growth. In response, plants appear to allocate a greater proportion of photosynthate to mycorrhizal fungi to improve nutrient acquisition. Elevated CO2 often is accompanied by an increase in abundance of arbuscular mycorrhizal fungi. Concentrations of soil glomalin are likewise enhanced. In contrast, anthropogenic nitrogen deposition has the opposite effect on arbuscular mycorrhizal fungi and glomalin, most likely because plants allocate fewer resources to their fungal symbionts when soil nutrients are less limiting. Globally, soil glomalin stocks increase linearly with precipitation across ecosystems; water relations of the fungi or the plants may be another important consideration. By examining the ecology of mycorrhizal fungi and their relations with host plants, we may better predict and manipulate soil conditions under various elements of global change.

Links Between the Subsurface Biosphere and the Atmosphere: Examples from DIRT and Airshed

Elizabeth Sulzman, Crop and Soil Science, Oregon State University, Barbara Bond, Forest Science, Oregon State University, Kate Lajtha, Botany and Plant Pathology, Oregon State University, Bruce Caldwell, Forest Science, Oregon State University, Zac Kayler, Forest Science, Oregon State University, Mark Hauck, Forest Science, Oregon State University, Tom Pypker, College of Oceanic and Atmospheric Sciences, Oregon State University, Mike Unsworth, College of Oceanic and Atmospheric Sciences, Oregon State University, and Alan Mix, College of Oceanic and Atmospheric Sciences, Oregon State University

Carbon storage in, and release from soils is a major control of atmospheric concentrations of CO2, with possible ramifications for global climate. Two studies being conducted in the H.J. Andrews Experimental Forest in the central Cascade Range of Oregon are presented as examples of attempts to quantify the carbon budget and monitor landscape scale carbon transfers among vegetation- soil- and atmospheric pools. In the Detrital Inputs Removal and Transfer (DIRT) study we measured soil CO2 efflux biweekly to monthly for three years, modeled daily fluxed, and calculated annual budgets. On average, root and rhizospheric respiration contributed 22%; aboveground litter decomposition contributed 19%, and belowground litter decomposition contributed 58% to total soil CO2 efflux, respectively. Calculations of efflux due to the exponential decay of litter indicate a positive priming effect that has strong implications for soil C storage, as it is clearly incorrect to assume that increases in aboveground productivity will translate directly into additional belowground storage. In the Airshed study we are taking advantage of cold air drainage that regenerates daily to trap and mix ecosystem respired CO2; we are testing the hypothesis that we can infer ecosystem-level responses to climatic drivers from measurements of 13CO2. Data indicate that isotopic values of ecosystem respiration reflect soil moisture more than vapor pressure deficit, contrary to results published for other systems. Surprisingly, soil moisture appears to be a weaker control on 13CO2 respired by soil organisms. Our measurements show pronounced diel variation in carbon isotopes in soil-respired CO2 that may indicate a very rapid response (on the scale of hours) of belowground respiration to carbon substrates provided by plants. Also intriguing is the finding that the isotopic composition of soil-respired CO2 is consistently enriched (by at least 1 per mil) on the south-facing, as compared to the north-facing slope. Current efforts are focused on teasing apart landscape-scale differences in soil properties, microclimate, and species composition as controls on the isotopic composition of ecosystem respiration. Our long-term goal is to "invert" an understanding of isotopic variation in ecosystem respiration to monitor intra- and inter-annual variations in ecosystem metabolism on a basin scale.

Oaks Below Ground: Mycorrhizas, Truffles and Small Mammals

Darlene Southworth, Jonathan Frank, and Seth Barry, Department of Biology, Southern Oregon University

Among the microscopic subsurface life forms and biogeochemical processes, larger organisms also interact. Although they are more recognizable, their subsurface activities are also unknown and overlooked. Oaks depend subsurface organisms and processes for survival and reproduction. These organisms include mycorrhizal fungi, with hyphae, mycorrhizal roots, and fruiting bodies, and small mammals that eat the fruiting bodies and run tunnels through the woodland. Oregon white oak (Quercus garryana) forms ectomycorrhizas with over 40 species of fungi, many of which are hypogeous forming fruiting bodies or truffles in the upper layer of mineral soil. Truffles do not release spores directly into the air, but remain closed below ground. We collected 21 species of truffles near Oregon white oak, three of which were undescribed species. We hypothesized that in oak woodlands small mammals eat hypogeous fungi and defecate the fungal spores. We trapped small mammals near Oregon white oak and examined fecal pellets for hypogeous fungal spores. Three species of rodents, California vole (Microtus californicus), deer mouse (Peromyscus maniculatus), and harvest mouse (Reithrodontomys megalotis), had spores from twelve species of fungi in their fecal pellets. The most common truffle spores found in fecal pellets were Tuber candidum/T. quercicola, Hydnotryopsis setchellii, and Cazia flexiascus, all Ascomycota. All oak seedlings require mycorrhizal inoculum. Seedlings growing in the root zone of mature oaks have access to the mycorrhizal network of parent trees, but seedlings outside the root zone lack mycorrhizal sources. If the mycorrhizal community on saplings located away from mature oaks includes hypogeous fungi, then small mammals may be dispersing fungal spores into shrublands where saplings are located. We examined roots of oak saplings at distances up to 72 meters from mature oaks and found mycorrhizas of hypogeous species, suggesting that small mammals eat fungal fruiting bodies and disperse the spores for mycorrhizal inoculum. Thus we demonstrate small mammal mycophagy of hypogeous fungi in Oregon white oak woodlands and provide evidence that mycorrhizal inoculum is distributed in fecal pellets. Regeneration of oak woodlands may depend on subsurface events-production of hypogeous fruiting bodies and dispersal of mycorrhizal fungal spores by small mammals.

Session 3: Bioremediation

Bioremediation - The Solution from Within

Frank E. Löffler, Environmental Engineering, Georgia Institute of Technology

Human activities release large amounts of toxic organic and inorganic chemicals into the environment. Toxic waste streams threaten dwindling drinking water supplies and impact terrestrial, estuarine, and marine ecosystems; hence, economically feasible remedies are needed. Naturally occurring bacteria that grow at the expense of anthropogenic pollutants have been enriched and isolated from contaminated soils and sediments. Some of these organisms are highly specialized and require the contaminant as an energy source. Interestingly, bacteria with such highly specialized metabolisms are members of natural microbial communities in pristine environments that never experienced exposure to anthropogenic contaminants. There is mounting evidence that most pollutants also have natural origins, and probably have been around long before human activities altered their environmental distribution and concentrations. Hence, it is not surprising to find bacteria that readily profit from anthropogenic pollutants. Bioremediation takes advantage of such naturally occurring bacteria that catalyze transformation reactions that lead to detoxification. To fully exploit the power of these organisms for environmental cleanup, the physiology and ecology of the key players must be understood. Research in microbial ecology currently emphasizes "omics" approaches; however, meaningful genomic analysis hinges on the detailed understanding of the target organism's physiology, and isolates are desirable. Recent efforts identified unique microbes that transform and detoxify specific environmental pollutants under anaerobic conditions. The Dehalococcoides and Anaeromyobacter groups have been chosen as examples to demonstrate how traditional cultivation and isolation efforts combined with "omics" approaches provide a blueprint for innovative hypotheses and solutions. These clues generate a critical scientific understanding of the existing reservoir of catalytic diversity that can be exploited in engineering approaches, and further demonstrates the ecological relevance of these organisms.

Bioremediation of Atomic Bomb Wastes

Craig Criddle, Stanford University

Mixed wastes and extreme conditions pose a significant challenge for remediation of uranium-contaminated soils at many contaminated sites. We evaluated the potential for field-scale in situ biological reduction of uranium (VI) in Area 3 of the DOE NABIR Field Research Center in Oak Ridge, TN. At this site, high levels of uranium are present in the groundwater (~60 mg/L) and soil (up to 700-1,000 mg/kg). Many other contaminants are also present. Notably, the groundwater contains high levels of nitrate (8-10 g/l), calcium (0.9 g/l), and aluminum (0.5 g/L) at a pH of 3.4. To evaluate potential remediation processes, we used a three-step approach: (1) flushing with a low pH water to remove aluminum, calcium, and bulk nitrate; (2) adjustment of pH to near 6, a range favorable for microbial activity; and (3) ethanol addition to stimulate U (VI) reduction. After flushing of the subsurface and pH adjustment to near 6, in situ bioremediation was initiated by weekly ethanol additions. Nitrate levels dropped to 0.3 mM or lower. After three months, ethanol addition was accompanied by a reduction in aqueous U (VI). Sulfate consumption and sulfide formation occurred concomitantly. A tracer test with bromide and ethanol confirmed that the removal of ethanol was due to biological activity. Most Probable Number analyses and clone libraries confirmed the presence of denitrifying bacteria, sulfate reducing bacteria, and iron reducing bacteria. X-ray absorption near edge structure spectroscopy of the sediments confirmed that U (VI) was reduced to U (IV) at the injection, monitoring and extraction wells. After more than one year of operation, more than 60% of the uranium in injection well sediment samples was present as U (IV). U (IV) in extraction well sediments samples increased over time, indicating expansion of the zone of reduction. At the monitoring wells, uranium concentrations dropped near or below the US EPA maximum contaminant limit for drinking water (30 µg/l).

Thermodynamics and Growth: Is it Possible to Predict Changes in Microbial Community Composition Using Equilibrium-Based Reaction Paths?

Jack Istok, Oregon State University

Rationale: In situ field experiments at the NABIR Field Research Center have shown that cooperative metabolism of denitrifiers and Fe(III)/sulfate reducers is essential for creating subsurface conditions favorable for U(VI) and Tc(VII) bioreduction (Istok et al., 2004). Although much has been learned about the physiology and metabolic potential of specific microorganisms with these capabilities that have been isolated from the FRC and other sites using pure cultures of microorganisms, major gaps exist in our understanding of the functioning of these organisms when they are present in intact microbial communities. For example, although ongoing NABIR studies have demonstrated the large genetic diversity of subsurface microorganisms at the FRC, many of these have never been isolated in pure culture. Yet it is the collective metabolic capability of these largely uncharacterized microorganisms that must be relied on for effective U(VI) and Tc(VII) bioimmobilization. Research Goal and Hypotheses: The overall goal of this project is to develop and test a thermodynamic network model for predicting the effects of substrate additions and environmental perturbations on the composition and functional stability of subsurface microbial communities. The overall scientific hypothesis is that a thermodynamic analysis of the energy-yielding reactions performed by broadly defined groups of microorganisms can be used to make quantitative and testable predictions of the change in microbial community composition that will occur when a substrate is added to the subsurface or when environmental conditions change. Approach: The proposed research will be conducted at the FRC using four intermediate-scale (~ 2 m) bioreactor models currently deployed in Areas 1 and 2. The network model will be used to predict the effects of substrate additions on the microbial community composition in the bioreactors by predicting the growth of major metabolic groups of organisms (aerobes, fermenters, denitrifiers, Fe(III)/sulfate/metal reducers). Model predictions will be tested by quantifying changes in the abundance of each of these groups using a combination of functional genes and lipid analysis. The network model will also be used to examine the stability of the U(VI) and Tc(VII) reducing microbial communities to changing environmental conditions. These predictions will be tested by challenging the microbial community in the bioreactors with a series of perturbations representative of those likely to occur (due to heterogeneity in groundwater geochemistry) in a full-scale bioreactor at the FRC. These will include variations in pH, nitrate, and sulfate concentrations in the bioreactor influent. Project Significance: As will be shown below, the ability to predict the effects of donor addition on change in microbial community composition is essential for creating conditions that favor the long-term stability of bioreduced U and Tc. Moreover, the ability of a microbial community to maintain functional stability (i.e. maintain high rates of U(VI) and Tc(VII) reduction) when subjected to various environmental perturbations (e.g., fluctuating pH and concentrations of electron acceptors) is of critical importance for the ultimate use of bioimmobilization at DOE legacy waste sites. The principal immediate significance of this project will be in establishing and testing a theoretical framework for designing and interpreting complex field experiments and to aid in "bridging-the-gap" between basic NABIR laboratory and field research.

Microbially Facilitated Calcite Precipitation for Immobilization of Strontium-90 in the Subsurface

Yoshiko Fujita, Idaho National Laboratory

Contamination of groundwater by radionuclides and metals from past weapons processing activities is a significant problem for the United States Department of Energy. Removal of these pollutants from the subsurface is prohibitively expensive, and therefore in situ containment and stabilization of the contaminants is an attractive remediation alternative. One potential approach for the immobilization of certain radionuclides and metals (e.g., 90Sr, 60Co, Pb, Cd) is to induce geochemical conditions that promote co-precipitation in the mineral calcite. Many aquifers in the arid western US are calcite-saturated, and calcite precipitated under an engineered remediation scheme in such aquifers is expected to remain stable even after return to pre-manipulation conditions. We have proposed that an effective way to promote calcite precipitation is to take advantage of native groundwater microorganisms capable of hydrolyzing the compound urea with the enzyme urease. Urea hydrolysis results in carbonate and ammonium production, and an increase in pH. The increased carbonate alkalinity favors calcite precipitation, and the ammonium serves the additional role of promoting the desorption of sorbed metal ions from the aquifer matrix by ion exchange. The desorbed metals are then accessible to co-precipitation in calcite, which can be a longer-term immobilization mechanism than sorption. The ability to hydrolyze urea is common among environmental microorganisms, and we have shown in the laboratory that microbial urea hydrolysis can be linked to calcite precipitation and co-precipitation of the trace metal strontium. In the field, we have performed experiments to demonstrate that urea hydrolysis in groundwater can be stimulated by the addition of low levels of molasses and urea, and we have obtained evidence for calcite precipitation. This presentation will provide a summary of our work in this area.

Evaluation of Fluoroethene as a Surrogate Indicator of Vinyl Chloride Degradation

Anne E. Taylor, Mark Dolan, Peter J. Bottomley, Lewis Semprini, Oregon State University

Aerobic utilization of vinyl chloride (VC) as a source of energy and carbon results in the end products CO2 and Cl-. Background Cl- concentrations in most aquifers makes it infeasible to measure the in situ rates of VC degradation by monitoring the release of Cl-. We evaluated fluoroethene (FE), the fluorinated analog of VC as a surrogate indicator of VC degradation. Aerobic degradation of FE releases F- that can easily be measured in most aquifers where there generally is a low background level of this ion. To show that FE degradation could function as surrogate, three strains of ethylene (Eth) degrading bacteria were utilized. EE13A was isolated from contaminated groundwater from Ft. Lewis, Washington and can utilize Eth as a growth substrate and cometabolically degrade VC and FE. Mycobacterium strain JS60 utilizes Eth and VC as growth substrates and will degrade FE. Nocardioides strain JS614 will directly utilize Eth, VC and FE as sole carbon and energy sources. For each isolate initial rates of degradation of VC and FE were similar whether there was direct or indirect metabolism of the substrate. Additionally, the Ks for VC and FE were comparable for each isolate. Halide release measured during short-term experiments was substantial for all three phenotypes. Halogen release was not stoicheometric to the amounts of substrate degraded by EE13a and JS60 when these strains were grown on Eth, but Eth-grown JS614 had stoicheometric release of Cl- and F- ions. VC-grown JS60 and JS614, and FE-grown JS614 did not have stoicheometric release of halide. Less halide released than substrate consumed during the degradation of VC and FE is an indication that some fraction of the halide remains associated with unknown metabolites. This is even true of strains that will grow on VC, FE or both, as a sole carbon and energy source. Similar rates of utilization and Ks values of FE and VC by each strain, and measurable F- ion release, regardless of growth phenotype, indicate that FE will prove to be a valid surrogate for VC degradation in the subsurface.