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

Abstracts of Poster Presentations
Tuesday, June 20, 2006

In Author Order

Continuous-Flow Column Studies of Reductive Dehalogenation of CAHs with Evanite Enriched Culture: Kinetics, Inhibition, and Monitoring of Microbial Activity

Mohammad Azizian, Department of Civil, Construction and Environmental Engineering, Oregon State University, Sebastian Behrens, Department of Civil and Environmental Engineering, Stanford University, Andy Sabalowsky, Department of Civil, Construction and Environmental Engineering, Oregon State University, Mark Dolan, Department of Civil, Construction and Environmental Engineering, Oregon State University, Alfred Spormann, Department of Chemistry, Oregon State University, Lewis Semprini, Department of Civil, Construction and Environmental Engineering, Oregon State University, Mark Dolan, Department of Civil, Construction and Environmental Engineering, Oregon State University

Anaerobic Continuous-flow column experiments were conducted with bioaugmented Evanite (EV) anaerobic culture and nonsterile Hanford aquifer material to evaluate the sequential reductive dechlorination of tetrachloroethylene (PCE). PCE concentrations of 0.09 mM in Hanford synthetic groundwater were tested. Retardation factors of 8.4 were calculated from bromide and PCE tracer breakthrough curves by using CXTFIT two-site-nonequilibrium model, which indicating strong sorption of PCE to Hanford aquifer material. PCE dechlorination to trichloroethylene (TCE), and cis-dichloroethylene (c-DCE) were observed when lactate concentrations increased from 0.35 mM to 0.67 mM. Rapid increases in cis-DCE concentration in the effluent compared to influent PCE concentrations indicated enhanced PCE desorption followed by reduction to TCE and cis-DCE. When lactate concentration was increased to 1.34 mM, propionate was observed from lactate fermentation and cis-DCE reduction to vinyl chloride (VC) and ethylene (ETH) occurred. After 170 days of operation flow was stopped and samples were obtained of the aquifer material within an anaerobic glove box. Microcosm studies were conducted to determine kinetic parameters under batch conditions to evaluate spatial rates of PCE transformation to TCE, cis-DCE, and VC at the end of column test. Real-time quantitative PCR was used to measure the distribution and abundance of subpopulations of the genus Dehalococcoides attached to the aquifer in the column. Dehalococcoides species 16S rRNA gene copy numbers dropped from ~ 73.5% of total Eubacterial 16S rRNA genes in the original inoculum, to about 0.5% to 4% through out the column. The results are consistent with the estimates of electron donor utilization, with 4.4% going towards dehalogenation reactions by the end of the column test. Our results provide support relationships between specific microbial dechlorinating activity and the removal of chlorinated ethenes from different locations of the flow column. These results suggest that the EV culture is able to transformation PCE to ETH under controlled reducing conditions in a continuous-flow system.

Microbes as Minerals: Thermodynamic Modeling of the Effects of Electron Donor Addition in Contaminated Aquifers

Angela Bice, Department of Civil, Construction and Environmental Engineering, Oregon State University

At many DOE legacy waste sites U and Tc subsurface contamination persists and presents a serious concern given the mobility of U(VI) and Tc(VII), as well as their very long half lives. It has been shown that some iron reducing bacteria fortuitously reduce U and Tc to their immobile forms, U(IV) and Tc(IV). Given that most natural systems are oligotrophic, in this case e- donor limited, the addition of a donor can act to stimulate the precipitation of the contaminants effectively removing them from solution. However, determining which donor to add and how much can be costly and time consuming. Additionally, the long term effects of continued donor addition and ultimate cessation on the overall system are difficult to predict, suggesting the need for the development of a predictive tool. Our model will use the Rittman and McCarty method to generate an overall cell reaction from donor, cell, and acceptor half reactions. The approach combines stoichiometric mass balancing with energetics. During heterotrophic reactions some substrate harvested electrons are used to generate energy and some are used for synthesis. In order to account for this dual requirement the energetic model partitions electrons into feo and fso, those used for energy and those used for synthesis. The partitioning numbers are determined from energetic considerations and then applied to the overall reaction equation, which is normalized to one mole of cells. This equation is bridged into REACT, which models geochemical processes thermodynamically, and is thereafter treated like a mineral and is subjected to mineral saturation rules. The basis can be modified to reflect system changes, (pH, the addition of an e- donor, etc.), and the biogeochemical effects observed. The project will be conducted in three stages: 1) A literature review will be conducted to constrain ?, the e- transfer efficiency, (a variable that can greatly impact the overall reaction equation), for iron, sulfate, and manganese reducers, 2) An iron reducing model will be developed within REACT and compared with an existing kinetic model, and 3) Laboratory tests will be conducted on simple and complex systems to determine if model predictions reflect reality.

Design and Characterization of Spectrometers for Determination of Oxygen and Reduced Species in Anaerobic Systems

Defne Çakin and James Ingle Jr., Department of Chemistry, Oregon State University

Unique portable spectrometers were constructed to monitor low level of oxidants and reductants (e.g. O2, Fe (II), S(-II) etc.) in anaerobic systems. Oxygen contamination of samples and reagents were critically evaluated and minimized with double containment. Dissolved oxygen (DO) at low levels was chosen as target analyte due to its significance in groundwater monitoring. Oxygen determination was based on measuring the absorbance increase after injecting a water sample with a manual syringe or pump into a solution of the reduced redox indicator indigo carmine. Two distinct configurations of spectrometers were built: 1) a flow-based circulating system with a reactor and 24-cm liquid core waveguide capillary cell (LWCC) and 2) a vial serving as 1-cm reactor-cell. These systems are compared and calibration data are presented. Due to its longer path length, the LWCC circulating system provided 20 times higher absorbance when compared to the 1-cm reactor-cell, allowed smaller sample volumes (e.g., microliters), but exhibited more sensitivity to low levels of oxygen contamination. A detection limit of 0.04 ppm (k = 3, n = 7) was achieved for the determination of DO with the 1-cm reactor-cell. Contamination of samples and reagents with O2 during sample transfer was minimized but still detectable. Contamination of the reduced indicator was greatly reduced by continuous reduction of excess oxygen with H2 and a Pt catalyst. Coupled with automatic sample introduction, these portable spectrometers can provide robust and accurate analysis for low level redox-active species in anaerobic cultures and groundwater.

Metabolism of Alkanes and Degradation of Chlorinated Ethenes by the Bacterium Pseudomonas butanovora

D. Davidson, K. Bateman, D. M. Doughty, L. A. Sayavedra-Soto, D. J. Arp, and P. J. Bottomley, Oregon State University

Previous studies have shown that there are great physiological and biochemical differences between metabolism of even and odd length alkanes in the bacterium Pseudomonas butanovora which, in turn, affects the rate at which the bacterium is able to degrade trichloroethylene (TCE). For bioremediation purposes, the goal is to find a suitable alkane to act as a substrate for the bacterium Pseudomonas butanovora and still permit a steady rate of degradation of the environmental contaminant TCE. The affinity of the BMO active site for the alkane and the chlorinated contaminant must be such that the alkane is capable of supporting bacterial growth and the BMO enzyme will still degrade chlorinated ethenes, or chloroethenes at the same time by cometabolism. In other words, to manage a balance such that competetion between the alkane and chloroethene for the enzymatic active site neither inhibits growth of the culture to any great extent, nor reduces the enzyme's ability to degrade chloroethenes.

Inactivation of Butane Monooxygenase by Propionate

D. M. Doughty, C. Vievelle, K. H. Halsey, L. A. Sayavedra-Soto, D. J. Arp, and P. J. Bottomley, Oregon State University

Incubation of the alkane oxidizing bacterium, Pseudomonas butanovora, with butyrate or propionate lead to irreversible, time-, and oxygen-dependent loss of butane monooxygenase (BMO) activity. In contrast, BMO activity was insensitive to the presence of lactate and acetate. BMO activity could be protected from propionate by the addition of the enzymes natural substrate, butane. Treatment of butane-grown cells with acetylene (14C2H2), a know suicide substrate of the BMO enzyme, resulted in the specific labeling of a 58 kDa peptide as analyzed by SDS page. Because the 58 kDa peptide corresponded to the migration distance of the alpha peptide of purified BMO hydroxylase we concluded that BMO was specifically inactivated by acetylene. Further propionate [2-14C] and 14C2H2 labeling studies indicated that 14C2H2 but not propionate [2-14C] remained associated with the BMO enzyme in SDS-page protein gels. While treatment with propionate did not completely inactivate all BMO activity in butane-grown cells the residual BMO activity remained C2H2 sensitive. These data could not distinguish between BMO inactivation vs. a reduced rate of BMO turnover. Therefore we treated butane grown cells with propionate, washed the cells in fresh phosphate buffer, and then exposed cells to 14C2H2 for two hours. Propionate treatment reduced 14C2H2 labeling of the 58 kDa peptide indicating that BMO was inactive during exposure to the 14C2H2. The BMO enzyme of P. butanovora mutant strain G113N, in which an individual amino acid substitution had been made to the ?-subunit of the hydroxylase, was determined to be insensitive to propionate.

Investigating the Basis of Substrate Specificity of Butane Monooxygenase and Chlorinated Ethene Toxicity in Pseudomonas Butanovora

Kimberly H. Halsey, Molecular and Cellular Biology Program, David M. Doughty, Department of Microbiology, Luis A. Sayavedra-Soto, Department of Botany and Plant Pathology, Peter J. Bottomley, Department of Microbiology, and Daniel J. Arp, Department of Botany and Plant Pathology, Oregon State University

Butane monooxygenase (BMO) is a member of the soluble methane monooxygenase (sMMO) subfamily of soluble diiron monooxygenases. Because BMO and sMMO comprise a group of powerful oxidative systems capable of activating highly stable hydrocarbons, they garner serious attention for their potential in bioremediation and bioindustrial catalysis. Although BMO and MMO share extended substrate ranges including alkanes, alkenes, aromatics, and chlorinated xenobiotics, BMO is the only member of the sMMO subfamily that does not appreciably oxidize methane. To investigate the fundamental differences in substrate specificity between the two homologous monooxygenase systems, BMO and MMO, single amino acid substitutions were made to the hydroxylase -subunit of butane monooxygenase (BMOH- ) in Pseudomanas butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH- were altered to the corresponding residues from the -subunit of methane monooxygenase (MMOH- ). Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were 7-fold lower than Rev WT. Strains G113N and L279F also showed 278 and 5.5-fold increases in the ratio of rates of 2-butanol to 1-butanol production as compared to the Rev WT strain. Propane oxidation by strain G113N was also predominantly sub-terminal, resulting in acetone accumulation which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although 23 M methanol completely inhibited methane oxidation in the Rev WT strain. In contrast, 83 M methanol was required to inhibit methane oxidation in mutant strain G113N. A three-dimensional model of BMOH- , revealed a region of flexibility including residue G113 in Helix B affecting the rotation of F185 in the leucine gate and the geometry of the active site. We are now studying the degradation of chlorinated ethenes by these mutant P. butanovora strains to further probe the catalytic mechanism of BMO. In addition, measurement of whole cell responses to DCE and TCE degradation will enable identification of the basis of chlorinated ethene toxicity on P. butanovora.

Dispersion through Highly Heterogeneous Porous Media

Stephanie Harrington, Environmental Engineering PhD Student, Oregon State University

This project focuses on how solute transport, specifically the dispersion process, is affected by heterogeneities in porous media. In order for media to be termed 'highly heterogeneous,' there needs to be a high variance in the log-conductivities of the different media within the system. Solute transport behavior through highly heterogeneous media is easy to distinguish due to its non-Fickian behavior. This is observed experimentally as tailing in the spatial concentration field or the solute concentration history (i.e. breakthrough curve). For non-Fickian behavior, mathematically the conventional dispersive flux term in the conservation of mass equation does not capture all of the solute transport behavior. It is hoped through this research to develop a mathematical model for a binary spatial conductivity field. The binary field will be represented as a high-conductivity matrix material (the -phase) with low-conductivity inclusions (the -phase). A 100 liter flow cell will be filled with course silica sand, with a particle diameter of ~0.2 cm, with sintered spherical inclusions of a much finer silica sand, with particle diameter of ~80 m, placed 'randomly' within the flow cell in order to create this highly heterogeneous binary system. This system will be used to provide data correlating the experimental and the mathematical models developed through the upscaling and volume averaging techniques. 3-D computer models will also be developed to simulate the processes occurring within the experimental system and ensure correlations between experimental and mathematical results.

Redox Sensors for the Subsurface Environment

Peter Ruiz-Haas and James Ingle Department of Chemistry, Oregon State University

New methods, sensors, and devices based on redox indicators were developed to monitor redox conditions in anoxic and anaerobic laboratory systems and the sub-surface environment. The primary application was monitoring redox conditions during the dechlorination of tetrachloroethene (PCE) to ethene (ETH) in packed columns and microcosm bottles containing an enriched chlororespiring culture (Evanite culture). Redox levels were monitored by measuring the absorbance of the redox indicators thionine (THI) or cresyl violet (CV) immobilized on a cellulose acetate film that was placed in a unique spectrometric flow cell. This flow cell was incorporated into a flow loop with a pump to create a versatile, flow-based redox sensor for samples in bottles and columns. All components and materials in the flow sensor loop were optimized to reduce O2 contamination of pumped solutions to very low levels (30 nmol/h). The flow sensor was a critical tool for evaluating the integrity and O2 contamination in many laboratory systems and types of sample containers and for monitoring of redox levels of highly O2-sensitive dechlorinating cultures. The application of a novel fiber-optic redox probe, based on redox indicator film, is also reported. During dechlorination experiments in both columns and bottles, complete reduction of THI indicates redox conditions are appropriate for active dechlorination, and partial reduction of CV is indicative of dechlorination of cis-dichloroethene and vinyl chloride. A new technique was developed and applied to determine a "redox capacity" rather than "redox level". An environmental sample is mixed with a known amount of redox indicator to determine the amount of indicator reduced and an effective concentration of reductant, termed reductive capacity (RC). RC measured with THI ranged from 100 - 400 µM during active dechlorination of PCE in microcosms and packed columns and increased by a factor of two or greater as dechlorination progressed from PCE to ETH. RC likely probes reductants that are located on cell membranes or within cells because the RC drops significantly when solutions obtained from a microcosm bottle or columns are filtered. A large fraction of the reductants measured may be attached to the particles that were retained by the filter.

Quantitative Determination of 1,4-Dioxane and Tetrahydrofuran in Water by Activated Carbon Solid Phase Extraction GC-MS/MS

Carl Isaacson, Department of Chemistry, and Jennifer A. Field, Department of Chemistry and Department of Environmental and Molecular Toxicology, Oregon State University

The presence of oxygenated solvent stabilizers in groundwater is a topic of increasing interest. 1,4-dioxane (dioxane), a probable human carcinogen, and tetrahydrofuran (THF) are examples of such solvent stabilizers. The environmental fate and transport of these compounds is poorly understood, but a limited number of observations to date indicate that these compounds are the most readily transported contaminants in chlorinated solvent and mixed waste plumes and these compounds are not readily biodegraded. Quantitative determination of these compounds in water is complicated by their physical chemical properties (eg high water solubility, low Kow and Henry's Constant). A quantitative analytical method was developed for the determination of dioxane and THF based on activated carbon solid phase extraction. The quantitation limit of the method is 0.31 ?g/L for dioxane and 3.1 ?g/L for THF, based on signal to noise of 10/1. Furthermore, spike and recovery experiments (n=5) yielded 98% and 95% recoveries for dioxane and THF, respectively with precision, as determined by relative standard deviation, of 1.8% and 5.5% for THF, all while using only 1.2ml of solvent. The method was applied to samples from mixed solvent and chlorinated solvent contaminated sites.

Methodological Developments in Meso-scale Visualization of Colloid Transport Dynamics in the Subsurface

E.L. Kraft, N. Ochiai, N. Tufillaro, and J.S. Selker, Oregon State University

Measurements that are representative of colloid transport processes in the subsurface are difficult to acquire due both to the small size of the particles and to the inaccessible nature of the soil environment. Most colloid transport studies to date have been conducted in closed columns with destructive sampling where the responses of the system to various stimuli are modeled as the proverbial black box. For mass balance analyses this approach appears to be entirely sufficient, however when questions arise that address the underlying process at play in the system, visualization of the particle movement becomes requisite. In the past decade, developments of non-invasive methods of monitoring have been transformative to the breadth of questions that can be addressed regarding colloid transport phenomena. These approaches include but are not limited to the use of transparent micromodels, epi-fluorescence, magnetic resonance imaging, X-ray computed tomography, and the light-transmission and fluorescence technique. The field of method development in this area of subsurface hydrology is both diverse and dynamic and justifies synthesis as a body of work in its own right. It is this breadth of methodology that is propelling colloid transport experimentation forward into a new era that will reveal new and potentially valuable information about contamination and its remediation in the subsurface. Here we present a review of these methods, including the novel capabilities, advantages, and limitations of each. Particular attention is directed towards the sensitivity of each method and its concomitant signal-to-noise ratio, which is a vital parameter to minimize in a successful visualization system. Advances made in our lab to reduce the signal-to-noise ratio of the light-transmission and fluorescence technique will also be presented. These specific advancements are framed in the context of the capacity of the light-transmission technique to quantify and visualize colloid transport phenomena in variably saturated porous media at the meso-scale under heterogeneous conditions.

Pore-scale Visualization of Colloid Transport

N. Ochiai, Department of Crop and Soil Science, E. Kraft, Department of Bioresource Engineering, M. Dragila, Department of Crop and Soil Science and J.L. Parke, Department of Crop and Soil Science, Oregon State University

Colloids, generally defined as particles less than 2 m which do not readily settle in water, comprise various materials including mineral fragments, organic macromolecules, and microorganisms (protozoa, fungi, bacteria, viruses). Transport of such particles through porous media is of interest to a wide range of researchers in diverse fields such as contaminant transport, waste water treatment, and bioremediation. Investigation of colloid transport has traditionally relied on column studies which allow observation of changes in colloid concentrations exiting a column relative to influent concentrations, but yield no direct information regarding mechanisms which influence these changes. After completion of an experiment, distribution of colloids within the column can be examined by column dissection, however, this process does not preserve information about specific locations where colloids are retained. Visualization provides direct information about particle movement within pore spaces as well as interaction of particles with water-solid, air-water, and air-water-solid interfaces over time, and thus enables investigation of pore-scale mechanisms governing colloid behavior. Here we review developments in pore-scale visualization methods over the past decade, focusing on design of artificial pore networks (micro-models) and media-packed flow-cells, including description of a flow-cell system under development by our group to observe transport and movement of Phytophthora zoospores in porous media, and present some representative results of visualization studies.

Nitrification Inhibition of Nitrosomonas europaea by Toluene: Kinetics and Proteomics

Tyler Radniecki, Mark Dolan and Lew Semprini, Department of Civil, Construction and Environmental Engineering, Oregon State University

Nitrosomonas europaea completes the first step of the removal of toxic ammonia from wastewater treatment plant (WWTP) by oxidizing ammonia to nitrite. However, N. europaea is sensitive to many nitrification inhibitors, including toluene. Designing a robust nitrification inhibition biosensor based on the detection of "sentinel genes" could be a valuable tool for WWTP operators. Sentinel genes are genes expressed only in the presence of a specific inhibitor or class of inhibitors. In this work, N. europaea was exposed to various concentrations of ammonia and toluene for one hour. Toluene degradation and the production of benzyl alcohol and benzaldehyde were observed. Plotting the calculated apparent Kmax vs. the apparent Ks revealed that an uncompetitive inhibition model best described the mechanism of nitrification inhibition by toluene. The EC50 of toluene, the concentration of toluene which results in a 50% reduction in the nitrite production rate, was measured to be 20 M in the presence of 173 M NH3. To explore for the production of sentinel genes, N. europaea was exposed to 20 M toluene in the presence of 173 M NH3 for a three-hour period. At the end of three hours, cells were extracted for 2-demensional gel electrophoresis analysis. Proteins showing up-regulation in the 2-D gels have been excised and will be sequenced via mass-spectrometry. Confirmation of the up-regulation of these proteins will be performed via quantitative PCR (qPCR) using Total RNA extracted at selected time points throughout the three-hour experiment. Using molecular techniques, such as 2-D gels and qPCR, will permit the discovery of several potential sentinel genes for toluene derived nitrification inhibition.

Modeling cDCE Toxicity to a Dehalococcoides-like Anaerobic Culture and Comparison of Dechlorination Performance in Suspended vs. Attached Growth

Andrew Sabalowsky and Lewis Semprini, Department of Civil, Construction and Environmental Engineering, Oregon State University

The chlorinated ethenes trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) are ubiquitous groundwater contaminants known for both toxicity and carcinogenicity. Anaerobic biological reductive dechlorination of these compounds is a popular strategy for in situ remediation. However, the more chlorinated compounds are known to inhibit dechlorination of the lesser chlorinated compounds, and high concentrations have been observed to be inhibitory towards dechlorination. This can make complete dechlorination to ethene difficult. A flowthrough chemostat and batch experiments were operated with TCE-fed, reductively dechlorinating, Dehalococcoides-containing mixed anaerobic cultures in suspension. Both systems demonstrated an apparent inhibition or toxicity on TCE dechlorination by extreme cDCE concentrations (>2000 uM). Modeling both systems suggests the cessation of dechlorination most likely results from toxicity and not reversible inhibition. Present work is devoted to testing the nature of this toxicity. We are also evaluating whether attached growth is more resistant to toxicity or inhibition than suspended growth. Batch systems have been constructed comparing small non-disturbed soil packets in mixed solution, verses thoroughly mixed soils in suspension. Both of these systems were seeded with Dehalococcoides-containing anaerobic soils from a previous column study. Non-disturbed soils produced faster cDCE and VC dechlorination compared to the continually mixed soil systems. It is presently hypothesized that diffusion limitations, either across the distance of the soil suspension apparatus, or across undisturbed biofilm thickness, buffer inhibition of more chlorinated ethenes on lesser chlorinated ethene dechlorination. Present modeling and experimentation are devoted to determining the reason for apparent differences in dechlorination behavior of attached vs. suspended growth. Future work will determine if diffusion through a biofilm dominate the differences in attached vs. planktonic dechlorination.

Metabolic Uncoupling of Shewanella oneidensis MR-1 Under the Presence of Excess Substrate and 3, 3', 4', 5 Tetrachlorosalicylanilide (TCS)

Gaurav Saini, Department of Civil, Construction and Environmental Engineering, Oregon State University

Dissociation between catabolism and anabolism, generically termed "metabolic uncoupling", has been studied for aerobic cultures of Shewanella oneidensis MR-1. Under conditions promoting metabolic uncoupling, the cell yield diminishes while the substrate utilization rate remains largely unchanged. The effects of excess substrate conditions and TCS addition on the metabolism of pyruvate by S. oneidensis MR-1 were examined and modeled. In the first phase, the observed cell yield (Yobs) was successfully modeled as a function of relative substrate concentration (S0/X0), under excess substrate conditions. Cellular yield was reduced by about 82% as the substrate concentration increased from 5 mM to 100 mM pyruvate and more than 75% of the substrate was utilized for non-growth activities. Preliminary experiments illustrated the effectiveness of TCS in reducing the cell concentration. In the later phase, an expression is proposed to model the uncoupling effect of excess substrate as well as TCS addition, simultaneously. This model was verified by using the experimental data obtained by introduction of three different TCS doses to cells grown under excess substrate conditions. The uncoupling coefficient (Eu) was used to distinguish between the uncoupling effects of excess substrate conditions and TCS addition. Excess substrate conditions were observed to be more effective in uncoupling the cellular metabolism, as compared to TCS addition. In our experiments, it was noticed that acetate accumulated as a metabolic intermediate. Average protein content of the cells was also found to increase with an increase in concentrations of either the substrate or the uncoupler. These could be the possible pathways for consumption of substrate for non-growth associated activities. Determining an optimum combination of substrate and uncoupler concentrations could be very useful in controlling the biomass growth in engineered microbial practices like wastewater treatment and bioremediation.

Effect of Chlorobenzene on Nitrosomonas europea Physiology and Protein Expression

Sean Sandborgh, Oregon State University

Nitrification by autotrophic ammonia-oxidizing bacteria, such as Nitrosomonas europea, is an important biological process in subsurface ecosystems and is a key step in the global nitrogen cycle. The enzyme responsible for the initial step of nitrification, ammonia monooxygenase (AMO), which oxidizes ammonia to hydroxylamine, is a reasonably non-specific enzyme which acts on a variety of different substrates. This non-specificity can cause inhibition of ammonia oxidation by the presence of a wide variety of compounds, including chlorobenzene. In this study, chlorobenzene was shown to inhibit AMO, while not significantly inhibiting downstream processes. At 2 ?M chlorobenzene concentrations, N. europea was inhibited in nitrite production by approximately 50%. Toxicity was implied by increased loss of activity under longer duration exposures and with higher chlorobenzene concentrations. Two-dimensional protein gels are presented with differentially expressed proteins marked. Further proteomic research, as well as quantitative PCR and microarray experiments are planned under similar experimental conditions to better understand the genetic and proteomic responses to chlorobenzene stress.

Pathway, Inhibition And Regulation of MTBE Oxidation by a Filamentous Fungus, /Graphium sp.

K. M. Skinner, M. R. Hyman, L. M. Ciuffetti, Oregon State University

/Graphium/ sp. is the only fungus currently known to cometabolically degrade the fuel oxygenate, methyl /tertiary/ butyl ether (MTBE). It is also one of the few organisms known to generate both /tertiary/ butyl formate (TBF) and /tertiary/ butyl alcohol (TBA) from MTBE. However, little is known about the final metabolic fate of TBF and TBA in this organism. It is also unclear whether these intermediates have regulatory effects on MTBE oxidation similar to those claimed for some MTBE-oxidizing bacteria. In this study we refined the pathway of MTBE oxidation in propane-grown mycelia and examined the independent effects of TBA, TBF, gaseous inhibitors and environmental variables on the MTBE-oxidizing activity of this fungus. Our hypothesis was that the pathway of MTBE oxidation would be similar to bacterial systems that generate TBF as an intermediate in MTBE oxidation. *Methods.* Mycelia of /Graphium/ sp. were grown on propane on fiberglass filter paper and were exposed to reactants in sealed glass serum vials. Rates of propane, MTBE, TBA and TBF consumption were determined by GC analysis of the reaction headspace. *Results.*/ /Propane-grown mycelia rapidly oxidized MTBE to TBF aerobically but not under anaerobic conditions. This activity was fully inhibited by acetylene and propane but was unaffected by high concentrations of TBF or TBA. In contrast, TBF was rapidly hydrolyzed to TBA under both aerobic and anaerobic conditions and this process was unaffected by acetylene, propane and TBA. TBA was not further oxidized under any conditions. TBF consumption resulted in substoichiometric (<30%) TBA accumulation when exposed to low TBF concentrations but was close to stoichiometric (>90%) at high TBF concentrations. *Conclusions.* Our results suggest MTBE oxidation by propane-grown /Graphium/ /sp/. is a novel variant of other MTBE-oxidation pathways as it involves TBF production but no further detectable oxidation of TBA. Our results also suggest this organism has a limited capacity for further TBA detoxification but unlike some bacterial systems, there is no evidence for a competitive or alternative regulatory effect of TBA or TBF on MTBE oxidation.

Geochemical Status of a Remedial Cap on Creosote-Contaminated Sediments in Portland Harbor and Its Potential Link to Natural Attenuation

Kiara Smith, Portland State University, Angela Bice, Oregon State University, Lisa Brutcher, Oregon State University, Stephanie Harrington, Oregon State University, Mandy Michalson, Oregon State University, Hollie Oakes-Miller, Portland State University, Rebecca Poulson, Oregon State University, Hap Pritchard, Portland State University, Jack Istok, Oregon State University

Creosote is a dark oily mixture comprised of hundreds of chemicals that make it an excellent wood preservative and, when not handled properly, a problematic toxic environmental contaminant. It is currently only authorized for commercial usage within the United States and serves primarily as a preservative for railroad ties and utility poles (US EPA, web site). The predominant constituents of creosotes are polycyclic aromatic hydrocarbons (PAHs), making up approximately 85% of the mixture, as well as phenolics and various heterocyclics (Mueller, 1989). The fact that PAHs are the major constituents in creosote and, along with their resulting metabolites, are considered possible carcinogens and mutagens, often makes them the chemicals of interest when characterizing and monitoring creosote contaminated sites. Of the PAHs commonly found within creosote naphthalene, phenanthrene, and anthracene typically make up the largest weight percents. PAHs consist of fused ring structures whose solubility and vapor pressure decrease as the number of rings increase making them fairly insoluble and persistent at contaminated sites, especially as the site ages and lighter PAHs have mobilized (WHO, 2004). Removing this residual contamination is extremely difficult especially in aqueous environments, such as aquifers, where excavation is not always an option. This has generated more interest in natural attenuation processes, particularly biodegradation, as a method for removing PAH contamination in situ. The McCormick and Baxter Creosoting Company was founded in 1944, and operated at the site until 1991. The company treated wood with a variety of preservative solutions, the remnants of which present substantial environmental hazards today. These preservatives included organic substances such as creosote and pentachlorophenol (PCP), as well as inorganic metal-based solutions high in arsenic, chromium, copper, and zinc. Wastewater from the plant was originally discharged directly into the Willamette River, and other wood preservative sludge products were dumped into an on site disposal trench. As a result of these practices, soil and groundwater at the site, as well as nearby sediments along the river, have been significantly contaminated. The site was listed on the National Priorities List by the United States Environmental Protection Agency (EPA) in June of 1994, and the Oregon Department of Environmental Quality (DEQ) was designated as the lead agency for implementing all remediation projects. DEQ has engaged in a variety of remedial actions, including creosote removal from groundwater aquifers, removal of contaminated sludge and soil from the site, and full demolition of the original plant. In 2004, a sediment cap was emplaced over 23 acres of contaminated sediment at the McCormick and Baxter site by the Oregon DEQ (MB_CSM, 2005). The cap is comprised of a sand layer (2 to 5 feet thick), a layer of hydrophobic organoclay with an affinity for non-soluble organic materials, and a layer of armoring material. The DEQ has determined from the results of sampling and coring that the dense non-aqueous phase liquid (DNAPL) contaminants are non-mobile and are not a concern for most of the site. It was also determined that the light non-aqueous phase liquids (LNAPLs) and dissolved contaminants are mobile with a flow rate of approximately 0.06gal/day. At this rate, it will take nearly 133 years for all of the mobile phase to be absorbed by the organoclay layer of the cap, which could continue to absorb NAPL for an additional 382 years afterward. The purpose of this project was to geochemically characterize the McCormick and Baxter Creosoting Company Superfund site, a site known to have extensive creosote and metal co-contamination, to determine if anaerobic biodegradational activity can or is occurring beneath the previously installed cap.

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.