James R. Brainard

Solubilization of plutonium hydrous oxide by iron-reducing bacteria

The removal of plutonium from soils id challenging because of its strong sorption to soils and limited solubility, Microbial reduction of metals is known to affect the speciation and solubility of sparingly soluble metals in the environment, notably iron and manganese. The similarity in reduction potential for [alpha]-FeOOH(s) and hydrous PuO[sub 2](s) suggests that iron-reducing bacteria may also reduce and solubilize plutonium. Bacillus strains were used to demonstrate that iron-reducing bacteria mediate the solubilization of hydrous PuO[sub 2](s) under anaerobic conditions. Up to [approximately]90% of the PuO[sub 2] was biosolubilized in the presence of nitrilotriacetic acid (NTA) within 6-7 days. Biosolubilization occurred to a lesser extent ([approximately] 40%) in the absence of NTA. Little PuO[sub 2] solubilization occurred in sterile culture media or in the presence of a non-iron-reducing Escherichia coli. These observations suggest a potentially attractive, environmentally benign strategy for the remediation of Pu-contaminated soils. 26 refs., 5 figs., 2 tabs.

Actinide Binding and Solubilization by Microbial Siderophores

Accurate predictions of actinide and fission product migration in the geosphere are critically dependent on identification of the biological, chemical and physical processes which affect actinide mobility in soil and water. Siderophores are low molecular weight iron chelators produced by microbes in response to low availability of soluble iron. Because of the similarities between iron(III) and tetravalent actinides, and the prevalence of siderophore-producing microbes in soil, there is strong likelihood that siderophores may also bind actinides, thereby influencing their mobility in the environment. In order to begin to assess the potential importance of siderophore-mediated actinide mobility, we have determined rate constants for solubilization of hydrous plutonium oxide by the siderophores enterobactin and desferrioxamine Β and selected carboxylate, amino polycarboxylate, and catecholate ligands. The measured rate constants for solubilization of insoluble actinide oxides show that siderophores are extremely effective in solubilizing actinides; on a per molecule basis, enterobactin is ~ 1 0 3 times more effective than the other chelators tested in increasing the rate of solubilization of hydrous plutonium oxide. Notably, ferric-siderophore complexes are more effective in solubilizing actinide oxides than the siderophores in the absence of iron. These results suggest that siderophores have the potential to mobilize actinides in the environment.

13C Nuclear Magnetic Resonance Evidence for γ‐Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

Abstract: The compartmentation of amino acid metabolism is an active and important area of brain research. I3C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of γ‐aminobutyric acid (GABA) and glutamate in rat brain. [l‐l3C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the l3C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This l3C labeling pattern occurs when [1‐l3Cjglucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (>80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.

Use of multiple 13C-labeling strategies and 13C NMR to detect low levels of exogenous metabolites in the presence of large endogenous pools: Measurement of glucose turnover in a human subject

A significant problem which may be encountered in 13C NMR studies of metabolism is the contribution that background levels of 13C may make to the observed spectra when low or tracer levels of the 13C label are used. We propose that the introduction of two or more labeled sites in the same tracer molecule is an effective strategy for eliminating or reducing this difficulty and demonstrate its feasibility in an isotope dilution study of glucose turnover in a human volunteer. This approach has two significant advantages over the more common use of a singly enriched labeling strategy: (i) as a consequence of the scalar coupling interactions, multiple-labeled metabolites will yield spectra distinct from those containing natural abundance 13C, and (ii) at a 99% level of enrichment for the precursor, concentration levels which are approximately 1% of the endogenous pools can be detected with approximately equal sensitivity. As a demonstration of this strategy, glucose production in a human subject was determined by continuous infusion of tracer levels of [U-13C6]glucose over a 4-h period and subsequent analysis of plasma levels of the tracer in vitro by NMR. Mass spectroscopy was used on the same samples to provide a basis for comparison of the precision and accuracy of the NMR technique. The results demonstrate the feasibility of the multiply labeled approach for detection by NMR of tracer amounts of label in the presence of a much larger endogenous pool of glucose. The NMR and mass spectrometric data gave quantitatively identical results for the glucose production rate demonstrating that equivalent data may be obtained by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Biodegradation of paint stripper solvents in a modified gas lift loop bioreactor

Paint stripping wastes generated during the decontamination and decommissioning of former nuclear facilities contain paint stripping organics (dichloromethane, 2-propanol, and methanol) and bulk materials containing paint pigments. It is desirable to degrade the organic residues as part of an integrated chemical-biological treatment system. We have developed a modified gas lift loop bioreactor employing a defined consortium of Rhodococcus rhodochrous strain OFS and Hyphomicrobium sp. DM-2 that degrades paint stripper organics. Mass transfer coefficients and kinetic constants for biodegradation in the system were determined. It was found that transfer of organic substrates from surrogate waste into the air and further into the liquid medium in the bioreactor were rapid processes, occurring within minutes. Monod kinetics was employed to model the biodegradation of paint stripping organics. Analysis of the bioreactor process was accomplished with BIOLAB, a mathematical code that simulates coupled mass transfer and biodegradation processes. This code was used to fit experimental data to Monod kinetics and to determine kinetic parameters. The BIOLAB code was also employed to compare activities in the bioreactor of individual microbial cultures to the activities of combined cultures in the bioreactor. This code is of benefit for further optimization and scale-up of the bioreactor for treatment of paint stripping and other volatile organic wastes in bulk materials.

In vivo screening of haloalkane dehalogenase mutants

Haloalkane dehalogenase (Dh1A) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type Dh1A is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify Dh1A mutant with improved catalytic activity. After screening 50,000 colonies, we identified a Dh1A mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of Dh1A. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.

REPLY FROM DRS. BRAINARD AND ROSENBERG

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