1999 RESEARCH PROJECTS Program Element 5 Assessment PROJECT: Field-Portable Immunoassay Instruments and Reagents to Measure Chelators and Mobile Forms of Uranium PRINCIPAL INVESTIGATOR: Diane A. Blake PROGRAM ELEMENT 5 Assessment The goal of this research is to develop to new techniques for measuring the rate and effectiveness of microbial bioremediation efforts. Efforts will focus upon the development of reagents (monoclonal antibodies) and instruments (hand-held immunosensors) for the speciation and quantification of ionic uranium and for the detection of EDTA and DTPA chelators present in radionuclide wastes. The Principal Scientists involved in this collaborative project are: D.A. Blake, PI, Dept. Ophthalmology and Biochemistry, Tulane University School of Medicine R.C. Blake II, Co-PI, College of Pharmacy, Xavier University of Louisiana Previous studies in our laboratories have demonstrated the feasibility of immunoassays for identification and quantification of specific metal ions. In the most complete study to date, a competitive immunoassay for Cd(II) was assembled and validated for ambient water samples. Further, prototype immunoassays for ionic uranium and lead were recently developed using monoclonal antibodies that are highly specific for the respective chelated metal ions. We seek to extend these studies (i) to investigate and compare different formats and field-portable instruments for the U(VI) immunoassay to maximize assay performance, and (ii) to prepare new monoclonal antibodies to the primary chelators (EDTA and DTPA) found in DOE wastes. Accordingly, the specific aims for the current grant period are as follows: To exploit existing anti-uranium antibodies to develop optimized immunoassays for U(VI) in both laboratory and field applications To assemble, test, and validate a new field-portable immunosensor based on the KinExATM instrument. This instrument will be developed in collaboration with private industry. Performance characteristics (sensitivity, precision, and ease-of-use) of the new hand-held instrument will be compared with those of a portable 96-well microtiter plate reader, a piezoelectric crystal micro-balance immunosensor (than can be readily converted to a portable unit), and a portable evanescent wave fiber optic fluorescent immunosensor; and To generate new monoclonal antibodies directed toward generic metal-EDTA and metal-DTPA complexes. Individual immunoassays will then be developed with each new antibody. The on-site field assays envisioned herein could greatly reduce analysis costs associated with site characterization before, during, and after bioremediation processes. This project has the potential to develop a marketable product, a stable of field tests for uranium and related toxic wastes that could be exploited both in governmental and commercial sectors. Research collaborations are either in progress or pending regarding the adaptation of our prototype immunoassays to biosensor technologies under development therein. Further, Sapidyne Instruments, Inc., has indicated its interest in collaborating on the development of a portable instrument to conduct competitive immunoassays in the field. We anticipate that further research and development will heighten this interest in technology transfer. PROJECT: Coupled Use of DNA Microarrays, Voltammetry, and X-ray Studies for Profiling Changes in Microbial Community Structure and Metal Speciation in Response to Metal Contamination PRINCIPAL INVESTIGATOR: Darrell P. Chandler PROGRAM ELEMENT 5 Assessment Detailed direct (non-culture based) information on changes in the types and activity of microorganisms, as a result of contamination and/or nutrient addition during engineered bioremediation of metals/radionuclides, are needed to improve our understanding of microbial community structure and desired bioremediation endpoints. Nucleic acid microarrays are a powerful new technology for assessing microbial processes because they provide phylogenetic and functional information for a virtually unlimited number of different target sequences (corresponding to particular genes or microbial groups) in a sample in a single hybridization reaction. The objectives of the research are to optimize and apply Genometrix DNA microarray technology (DNA chips) to rapidly profile microbial communities in process-level microcosms dosed with increasing metal (chromate, lead) over time, and to simultaneously monitor microbial populations and their effect on metal speciation/mobility. Metal speciation and mobility in sediment microcosms will be measured by voltammetry. When mobility increases in a particular microcosm over time, the potential role of microbially-produced ligands will be examined; when mobility decreases over time, the local molecular environment of immobile species will be determined by X-ray absorption fine structure (XAFS) measurements. By coupling the two approaches, general microbial populations responsible for specific changes in metal speciation that are associated with desired bioremediation endpoints (mobilization, immobilization). In a more general sense, the microarray technology will be useful for rapid and economical site assessment and monitoring, for building a data-rich information base that will improve the probability of bioremediation success at other locations, and for microbial ecology research. PROJECT: Stable Isotope and Fluorescent Labeling and Detection Methodologies for Tracking Injected Bacteria During in situ Bioremediation PRINCIPAL INVESTIGATOR: Mark E. Fuller PROGRAM ELEMENT 5 Assessment Understanding of the great diversity of microbial activities, both natural and engineered, continues to grow, especially with respect to organic compound degradation and transformation. The diversity of organisms which have been found to possess the ability to transform metals and radionuclides, is also expanding, creating the possibility that these compounds can be immobilized within a defined area, or mobilized for collection and disposal. With the realization that conventional (non-biological) remediation technologies are not always effective for all classes of pollutants or under all site-specific conditions, more research has been initiated to develop and expand the use of this wide range of microorganisms for in situ bioremediation. Bioremediation efforts usually focus on either assisting indigenous microorganisms to degrade, detoxify or immobilize the pollutant (biostimulation), or injecting microbial strains with well characterized degradative abilities directly into the polluted area (bioaugmentation). Both of these technologies have been used to remediate sites contaminated with organic pollutants, and there is a growing interest in applying them at heavy metal- and radionuclide-contaminated sites, many of which are owned by the U.S. DOE. One of the areas that is critical to effective and efficient bioaugmentation is a better understanding of the factors that control the transport of degradative organisms through subsurface environments. Research on bacterial transport is currently being conducted under the auspices of the Acceleration Element of the NABIR Program at the DOE South Oyster, Va. site. One of the primary needs to enable research such as this to yield quality data is a reliable, sensitive and specific method to quantitatively detect the injected bacteria. Selective plate counts, fluorescent cell stains, and whole-cell labeling with the stable isotope of carbon (13C), have all been used to monitor microbial transport in groundwater, however each of these techniques have potential limitations. The detection limit for selective plate counts may be higher than is needed to document the presence of the injected target organism in a downgradient sampling well, and may not be able to enumerate those target organisms which are still able to carry out the function for which they were injected but are no longer able to form colonies on solid media (Oliver, 1993). Fluorescent cell stains allow all the injected cells to be detected regardless of their culturability, but most of these compounds adversely affect the activity, viability or adhesive properties of the cells (Parolin et al., 1990). Injection of cells with high amounts of 13C incorporation, with subsequent detection of the stable isotope enrichment of the carbon downgradient is not dependent on cell culturability and is not expected to alter cell activity, viability or adhesive properties. However, these bulk determinations of 13C enrichment are not able to unequivocally document that the 13C represents live target cells, since the 13C in the injected organisms may have been transferred or incorporated into other microbes as target organisms died and lysed or became prey for protozoa. There is, therefore, a need to develop and evaluate new bacterial tracking methodologies, as well as refine traditional ones, in light of scientific and technological advances. The proposed research will examine modifications to the stable isotope and fluorescent stain cell labeling methods. A new methodology for analyzing stable isotopes, gas chromatography-chemical reaction interface mass spectrometry (GC-CRIMS) has recently become available. Cellular components (fatty acids, proteins and nucleic acids) of 13C - and 15N-labeled cells are extracted, purified and separated via gas chromatography and converted in the chemical reaction interface to oxidized products (CO2, NOx). These products then enter the mass spectrometer, where the amounts of 12CO2, 13CO2, 14NOx, and 15NOx are measured. By comparing the isotopic enrichment in specific cellular components of the target cells prior to injection with that of downgradient samples, the presence and quantity of the target cells in the post-injection samples can be determined. The major advantage of this method is that 13C - and 15N-labeled cellular components establish a "signature" for the target cells. Variations in this signature indicate changes in the target cells themselves, cell death, or incorporation of labeled cellular materials by other microbes, all of which would allow for adjustments to be made in the calculated number of target organisms without further analyses being required. One disadvantage is that the detection limit may be higher than for whole-cell 13C analysis, since only a subset of the cellular components are being analyzed. New fluorescent dyes have been and continue to be developed which may allow cells to be stained without loss of activity, viability or changes in adhesive properties. Many of these dyes have been developed specifically for eukaryotic cell staining, but the principles underlying their use make them applicable to prokaryote staining as well. Some of the newer dyes specifically stain cell membranes, while others cross the membrane and covalently bond to intracellular proteins. In either case, some of these dyes have been shown to be retained in cells for up to 3-4 weeks, without loss of cell viability or alterations in cell function or adhesion (Haugland, 1996). PROJECT: In Situ Determination of Microbial Metabolic Activity PRINCIPAL INVESTIGATOR: Jonathan D. Istok PROGRAM ELEMENT 5 Assessment Quantitative information on subsurface microbial metabolic activities is required to support site characterization and feasibility assessments for both intrinsic and enhanced bioremediation strategies. The single-well, push-pull test method, recently developed by the PIs at Oregon State University (OSU) is a fundamentally new and powerful in situ test method for obtaining microbiological information on groundwater aquifers. A push-pull test consists of the controlled injection of a prepared aqueous test solution into a selected depth interval using a drive-point screen or existing monitoring well, followed by the recovery of the test solution/groundwater mixture from the same location. The results of several field studies conducted at OSU have demonstrated that the push-pull test method is useful for quantifying rates of aerobic respiration and denitrification at sites contaminated with petroleum hydrocarbons, chlorinated solvents, and heavy metals. Moreover, the utility of the method for quantifying spatial variability in these processes at the site scale also has been demonstrated. The overall goal of this project is to further develop the capabilities of the push-pull test so that it can be used as a routine field assay for quantifying microbial biomass and metabolic activity in both pristine and contaminated subsurface environments. Estimates of metabolically-active microbial biomass will be obtained by monitoring the transformation rates of injected substituted nitrophenols selected to assay for constitutive enzymatic activities; estimates of Fe(III)- and sulfate-reducing activity will be estimated by measuring the transformation rates of injected combinations of electron donors and acceptors in the presence and absence of specific inhibitors. Objectives: To select and optimize the use of substituted nitrophenols as enzyme substrates for the in situ measurement of metabolically-active microbial biomass, To select and optimize concentrations and combinations of electron donors, electron acceptors, and inhibitors for measuring in situ Fe(III) - and sulfate-reducing activity, and To quantify the field-scale spatial and temporal variability in microbial biomass and activity as a function of mineralogy, stratigraphy, geochemistry, and contaminant levels at selected field sites. Approach: In collaboration with other NABIR investigators, laboratory studies will utilize groundwater and sediment samples obtained from the Oyster, Virginia Analog Field Site (South Oyster Site) and one or more Field Research Centers (FRC) or other DOE sites as they become available. Laboratory studies will be used to optimize concentrations and conditions for assays to be used to measure microbial biomass and activity in field tests; to develop rapid, low-cost methods for monitoring enzyme substrate and electron donor and acceptor disappearance and product formation; and to compare measurements of enzyme substrate reactivity with other indicators of microbial activity and redox indicators developed by other NABIR investigators. Field studies will be designed to parallel laboratory experiments and will be conducted at the same sites. A preliminary series of push-pull tests will be performed to optimize field protocols; an extensive series of push-pull tests then will be performed to quantify spatial and temporal variability in reactivity of injected substrates and electron donors and acceptors as a function of mineralogy, stratigraphy, geochemistry, and contaminant levels. This project is consistent with the goals of the NABIR program because (a) it will provide quantitative in situ information on microbial metabolic activities that control the mobility, bioavailability, and toxicity of contaminants, (b) it will provide a rapid, low- cost field method for studying interactions between microbial biomass, metabolic activity, geochemistry, and contamination in the subsurface, and (c) it will provide a standardized approach for screening microbial activity at candidate Field Research Center sites and other DOE sites, which will be useful in FRC site characterization or in the design of larger-scale field experiments at these sites. PROJECT: Core-Scale Interrogation of Permeability and Geochemical Heterogeneity for Assessment of Bioremediation Effectiveness PRINCIPAL INVESTIGATOR: Philip E. Long PROGRAM ELEMENT 5 Assessment Quantitative, field-scale understanding of reactions between microbes and natural porous media is critical to solving many contemporary subsurface environmental problems. Because these reactions occur at water-mineral-cell interfaces and are strongly controlled by local biogeochemical conditions, knowledge of small-scale variations (heterogeneity) in natural porous media properties and their net effect on field-scale transport is needed. However, small-scale heterogeneity of physical properties such as permeability and porosity combines with that of biogeochemical properties to give rise to complex behaviors that are difficult to quantify at relevant field scales. Detailed descriptions of small-scale heterogeneity, and observations of their relationship to bacterial attachment, are needed to form a defensible foundation for quantitative modeling and theoretical developments. Integration of a number of innovative, core-scale imaging technologies will significantly enhance detailed assessment of physical and biogeochemical heterogeneity at sub-core scales. The technologies proposed have been applied, in varying degrees, to geological characterization problems, but have not been integrated and applied to quantify joint physical and biogeochemical core- and outcrop-scale heterogeneity. Basic issues to be addressed by this research include the interpretability of mineral abundance in natural porous media from spectral response of sediments, relationships among observations of physical properties (especially permeability) at several scales, the significance of preferential flow paths in microbial transport and attachment, and determination of optimal moisture contents for estimation of permeability using air mini-permeameters and infrared imaging methods. Products of this research will include development and validation of the integrated core interrogation method, collection of a unique and much needed detailed dataset describing millimeter to centimeter scale joint physical and biogeochemical heterogeneity, and new insights into the controls on microbial attachment in heterogeneous porous media. This research is being conducted in close collaboration with NABIR-supported research in bacterial transport at the Oyster field site near Oyster, Virginia. These new core interrogation methods will be developed and tested using intact cores and small outcrop images from the Oyster site. Project personnel participated in a recent excavation characterization and core sampling field campaign conducted by Oyster site investigators. Initial data collection using ultrasensitive infrared (IR) methods has been completed on sediments from the Narrow Channel Site, Oyster, VA including approximately 250 IR images (256 X 256 pixels) of split cores and outcrop exposures. Preliminary analysis suggests that the capillary fringe dominates the IR images at the lower part of the outcrop exposure, a feature that will have to be digitally removed to establish a systematic relationship between permeability and IR intensity. Preliminary data have also been collected on split cores from the Narrow Channel flow cell using a flat bed scanner, providing distortion-free, 0.05 mm resolution images with 30 bit color depth. Because of the distinct reddish color of some Fe-oxide coatings, this method may provide a quantifiable estimate of Fe-oxide heterogeneity. The IR and scanner data are the first of several datasets that will be integrated to develop for the first time, a coherent, detailed, quantitative description of correlated physical and biogeochemical heterogeneity at the core scale. Data collected using this integrated imaging approach will fill a key gap in the knowledge required for assessment of in situ field-scale bioremediation. PROJECT: Spatial Heterogeneity of Microbial Iron Reduction Potential in Subsurface Sediments PRINCIPAL INVESTIGATOR: Christopher J. Murray PROGRAM ELEMENT 5 Assessment This research is expected to provide an improved understanding of the field-scale spatial distribution of microbial iron reducers in Atlantic coastal plain aquifers, and of the implications posed by that heterogeneity for bioremediation by microbial iron reduction. The geostatistical methodology developed for the study will be applicable to a wide range of DOE sites, and could be readily adapted to the study of other important microbial processes. The approach will be to analyze the spatial heterogeneity of microbial iron reduction potential (MIRP) in subsurface sediments at a field site, and determine if it can be correlated to geological factors that can be identified geophysically. Planned research activities include: Identify a method for measurement of microbial iron reduction potential in batch sediment samples from cores. This research will be performed in collaboration with Dr. Eric Roden of the University of Alabama. Because of the need to characterize the spatial heterogeneity of MIRP, the method must be inexpensive, allowing a large number of batch samples to be analyzed. As a related task, address the relationship between measurements from the batch method and the in situ potential by a series of static and dynamic intact core experiments. Perform the batch measurement of MIRP on closely-spaced samples from several boreholes. The study will be performed at a well-characterized DOE study site, probably the South Oyster location. Sample boreholes will be located along a line of previously measured geophysical data (ground penetrating radar (GPR)). Measure a limited number of additional geological and geochemical properties on each sample (e.g., facies type, porosity, conductivity, grain size, and extractable iron content). Identify facies variation in MIRP using multivariate statistical methods. Determine the spatial heterogeneity of the MIRP using geostatistical methods, principally variogram analysis. Also, determine the spatial cross-correlation between MIRP and the geological and geochemical properties. If MIRP can be predicted from knowledge of the sedimentary facies or other geological properties that can be mapped from the geophysical data, develop an estimate of MIRP between the sampling boreholes from geophysical data. Test the estimates by drilling infill boreholes at the estimate locations, sampling them, and comparing the results with the predictions. PROJECT: Assessment of Effective Reactive Surface Area of Chemically Heterogeneous Porous Media PRINCIPAL INVESTIGATOR: Robert W. Smith PROGRAM ELEMENT 5 Assessment The relationship between effective reactive surface area (i.e., surface area that reacts with locally advected solutes) of heterogeneous porous media and advecting groundwater will be evaluated using reactive tracer experiments on Oyster and Abbots Pit cores. Inverse reactive transport modeling techniques relating tracer breakthrough to effective reactive surface area will be developed. Research results will provide a validated physicochemical scaling approach to assess the role of variable reactive surface area for field-scale contaminant and bacterial transport. PROJECT: SIMS for Direct Interrogation of Microbe/Mineral Interfaces PRINCIPAL INVESTIGATOR: Jani C. Ingram PROGRAM ELEMENT 5 Assessment The goal of this work is to evaluate static secondary ion mass spectrometry (SIMS) as a tool for direct assessment of microbial populations at mineral surfaces. Because SIMS is a sensitive, surface analysis technique, it has the potential to directly interrogate interfacial interactions between microorganism and the mineral substrate. A number of controlled microbial samples have been characterized; this benchmark research is leading toward the microbial surface characterization of sediments found at the Uranium Mill Tailings Remedial Action (UMTRA) site near Shiprock, New Mexico. The basis for our approach is to use static SIMS to probe phospholipid fatty acids and other biomolecules associated with the cell membrane of intact microorganisms. We hypothesize that since static SIMS probes only the top layers of the sample surface, it could be used to collect unique mass spectral signatures of the cell membrane chemistry of a microorganism by analyzing intact cells (no sample preparation). In order to test this hypothesis, SIMS spectra of >50 microorganisms were collected, and the results were compared to a standard method for probing the cell membrane chemistry (Microbial Identification System, MIS). The mass spectral results from the SIMS analyses showed marked differences in spectral features. Comparing the SIMS results to the fatty acid profiles generated by MIS, many of the fatty acids were identified on the basis of specific anions observed in the SIMS data. By applying principal component analysis to the SIMS data, microbes having similar phospholipid compositions could be statistically grouped. A second approach for microbe identification is to utilize mass spectrometry/mass spectrometry (MS/MS) to detect specific biomarker molecules which are contained in the cell membrane. Results from the early stages of this research will be reported as part of this presentation. Currently, we are investigating detection limits and how specific microorganisms can be typed (groups, species, subspecies) by SIMS. We are also investigating isolates collected from the Shiprock UMTRA site, and plan to discuss those results as part of this presentation. Funding from DOE-OBER NABIR is gratefully acknowledged. [Back to Award Recipients Page]