Characterization of Co(III) EDTA-Reducing Bacteria in Metal- and Radionuclide-Contaminated Groundwater

Abstract

The Waste Area Grouping 5 (WAG5) site at Oak Ridge National Laboratory has a potential to be a field site for evaluating the effectiveness of various bioremediation approaches and strategies. The site has been well studied in terms of its geological and geochemical properties over the past decade. However, despite the importance of microorganisms in bioremediation processes, the microbiological populations at the WAG5 site and their potential in bioremediation have not been similarly evaluated. In this study, we initiated research to characterize the microbial populations in WAG5 groundwater. Approximately 100 isolates from WAG5 groundwater were isolated and selected based on colony morphology. Fifty-five unique isolates were identified by BOX-PCR and subjected to further characterization. 16S rRNA sequences indicated that these isolates belong to seventeen bacterial genera including Alcaligenes (1 isolate), Aquamonas (1), Aquaspirillum (1), Bacillus (10), Brevundimonas (5), Caulobacter (7), Dechloromonas (2), Janibacter (1), Janthinobacterium (2), Lactobacillus (1), Paenibacillus (4), Pseudomonas (9), Rhodoferax (1), Sphingomonas (1), Stenotrophomonas (6), Variovorax (2), and Zoogloea (1). Metal respiration assays identified several isolates, which phylogenically belong or are close to Caulobacter, Stenotrophomonas, Bacillus, Paenibacillus and Pseudomonas, capable of reducing Co(III)EDTA⁻ to Co(II)EDTA^{2 −} using the defined M1 medium under anaerobic conditions. In addition, using WAG5 groundwater directly as the inoculants, we found that organisms associated with WAG5 groundwater can reduce both Fe(III) and Co(III) under anaerobic conditions. Further assays were then performed to determine the optimal conditions for Co(III) reduction. These assays indicated that addition of various electron donors including ethanol, lactate, methanol, pyruvate, and acetate resulted in metal reduction. These experiments will provide useful background information for future bioremediation field experiments at the WAG5 site.

Keywords:

• bioremediation
• dissimilatory metal reduction
• groundwater
• metal reduction
• molecular ecology

We appreciate insightful comments and suggestions of anonymous reviewers on the improvement of this manuscript. This research was supported by The United States Department of Energy under the Laboratory Directed and Research Program at ORNL. Oak Ridge National Laboratory is managed by the University of Tennessee-Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725.

Notes

∗μ Ci/ml.

¹Direct from groundwater (GW) or enrichment with mR2A, NO₃ ⁻, or H₂ + Co(III) broth.

²Final agar used to isolate culture – nutrient agar (NA), mR2A, R2A, or trypticase soy agar (TSA).

³Based on partial 16S rDNA sequences. Percent similarity is indicated in parentheses.

⁴Isolates not tested are indicated by –.