A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmentation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCE to cDCE when amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phylogenetic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64,000 L The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 microg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field date were rapid (half-lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene.

The environmental distribution of Dehalococcoides group organisms and their association with chloroethenecontaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.The chloroethenes tetrachloroethene (PCE) and trichloroethene (TCE) are commonly used organic solvents and degreasing agents. As a result of past disposal practices and spills, chloroethenes are now widely distributed in the environment and are found in many sediments, soils, groundwater aquifers, and subsurface environments throughout the world (13,21,28). Traditional approaches to groundwater remediation practices, such as pump and treat methods, have been shown to be ineffective and costly when applied to chlorinated solvent plumes (2a, 2c, 23). Therefore, there is a need for microbebased remediation approaches that could provide an inexpensive way to clean up chlorinated solvent contamination.Chloroethene solvents were previously believed to be resistant to degradation by microorganisms. There now is rapidly accumulating laboratory and field evidence that microorganisms can transform chloroethenes to nontoxic products under a variety of environmental conditions (3, 8-10, 12, 15, 20, 22, 27, 28, 30, 31, 35). Maymó-Gatell et al. have isolated an organism from an anaerobic dechlorinating laboratory culture inoculated with municipal sewage sludge, which fully dechlorinates chlorinated ethenes (25,26). This organism, Dehalococcoides ethenogenes strain 195, was shown to meet its energy needs, which are essential for growth, by a process known as dehalore...

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and B-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate coamplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, 3-Gal and hCG were respectively 1 x 1O.19, 1 x 10-17 and 1 x 10-17 mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.

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DuPont and BP have been working together to develop Microbial EOR targeted at viscous oil in the Schrader Bluff formation on the North Slope of Alaska. The goal of this program was a 5% increase in the recovery factor. Mechanisms to be assessed in the original agreement included Viscosity reduction of the oil by transformation or degradation of heavy components in the oil – thus improving the oil – water mobility ratio.Drastic reduction (to ~<0.01 dynes/cm) in the interfacial tension between water and the oil After extensive fundamental research we have learned many critical aspects of microbial EOR that made the application of these two mechanisms to the Schrader Bluff formation impractical. Instead, we have demonstrated two site appropriate mechanisms that achieved, in the lab, the targeted increase in the recovery factor. Improved flow conformance and increased sweep efficiency by preferential plugging of high permeable zones thereby forcing water to produce oil from previously unswept parts of the reservoir.Reduced oil / rock surface tension and a subsequent reduction in the oil "wetting" the rock. This results in changes in the relative permeability of the oil and the water and ultimately lower residual oil saturation. This paper describes the key laboratory tests used to evaluate these four mechanisms. The cornerstones of our work have been the detailed characterization of the waters, the oil, the formation matrix and the microbial community. In addition we describe our search for useful microbes isolated from a variety of environmental samples collected from the Milne Point Unit (MPU) of the Alaskan North Slope. These samples were taken over several years and included injection, production and power fluid waters. These samples were used to understand the temporal changes in the microbial populations and to provide inoculum for our enrichment cultures. Our ongoing research has provided many insights into the appropriate application of microbial EOR. The unique aspects of each production area, the nature of the oil, the water, the formation matrix, and the background microbial population and their complex interactions must all be assessed when considering the potential application of microbial EOR. The amount of work discribed below for assessing potential MEOR mechanisms is extensive. However, this process has been streamlined and we have been able to assess new target reservoirs for potential MEOR treatments in about 6 months.

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