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.