Diversity and Distribution of Anaeromyxobacter Strains in a Uranium-Contaminated Subsurface Environment with a Nonuniform Groundwater Flow

Versaphilic Anaeromyxobacter dehalogenans strains implicated in hexavalent uranium reduction and immobilization are present in the fractured saprolite subsurface environment at the U. S. Department of Energy Integrated Field-Scale Subsurface Research Challenge (IFC) site near Oak Ridge, TN. To provide insight into the in situ distribution of Anaeromyxobacter strains in this system with a nonuniform groundwater flow, 16S rRNA gene-targeted primers and linear hybridization (TaqMan) probes were designed for Oak Ridge IFC Anaeromyxobacter isolates FRC-D1 and FRC-W, along with an Anaeromyxobacter genus-targeted probe and primer set. Multiplex quantitative real-time PCR (mqPCR) was applied to samples collected from Oak Ridge IFC site areas 1 and 3, which are not connected by the primary groundwater flow paths; however, transport between them through cross-plane fractures is hypothesized. Strain FRC-W accounted for more than 10% of the total quantifiable Anaeromyxobacter community in area 1 soils, while strain FRC-D1 was not detected. In FeOOH-amended enrichment cultures derived from area 1 site materials, strain FRC-D1 accounted for 30 to 90% of the total Anaeromyxobacter community, demonstrating that this strain was present in situ in area 1. The area 3 total Anaeromyxobacter abundance exceeded that of area 1 by 3 to 5 orders of magnitude, but neither strain FRC-W-nor FRC-D1-like sequences were quantifiable in any of the 33 area 3 groundwater or sediment samples tested. The Anaeromyxobacter community in area 3 increased from <10(5) cells/g sediment outside the ethanol biostimulation treatment zone to 10(8) cells/g sediment near the injection well, and 16S rRNA gene clone library analysis revealed that representatives of a novel phylogenetic cluster dominated the area 3 Anaeromyxobacter community inside the treatment loop. The combined applications of genus- and strain-level mqPCR approaches along with clone libraries provided novel information on patterns of microbial variability within a bacterial group relevant to uranium bioremediation.