Identification and quantification of metal-reducing bacteria that catalyze redox reactions in radionuclide-contaminated subsurface soils

Joel E. Kostka (1) and Joseph W. Stucki (2)

(1) Florida State University, Tallahassee, FL; (2) University of Illinois, Urbana, IL

Uranium bioremediation at contaminated sites managed by the U.S. Department of Energy (DOE) is currently focused towards stimulating indigenous subsurface microbial communities to bioimmobilize U(VI) by reducing it to U(IV).  Iron(III)-reducing prokaryotes (FeRP) and sulfate-reducing prokaryotes (SRP) are capable of rapidly catalyzing the reduction and immobilization of U(VI) in contaminated subsurface soils. However, the in situ mechanisms and controls of reductive immobilization are not well understood.  Our research group has been using a combination of cultivation-based and cultivation-independent approaches to study these metal-reducing microbial communities in contaminated subsurface soils from the U.S. DOE’s Oak Ridge Field Research Center (ORFRC) in Oak Ridge, Tennessee.

The objectives of ongoing research are to: 1) isolate and characterize novel metal-reducers from subsurface environments exposed to high levels of mixed contaminants (U(VI), nitrate, sulfate), 2) elucidate the diversity and distribution of metabolically active metal-reducing prokaryotes in subsurface soils, and 3) determine the biotic and abiotic mechanisms linking electron transport processes (nitrate, Fe(III), and sulfate reduction) to radionuclide reduction and immobilization.

The utility of transcripts for dissimilatory (bi)sulfite reductase (dsrA) genes and for Geobacteraceae citrate synthase (gltA) genes as molecular proxies for sulfate- and Fe(III)-reducing prokaryotes, respectively, was demonstrated in ORFRC subsurface sediments. Transcript abundance was correlated with the rate of the corresponding electron transport pathway in sediment microcosms.  Phylogenetic analysis of gltA mRNA clone sequences demonstrated that groups closely related to Geobacter uraniumreducens and Geobacter FRC-32 are active and abundant in microcosm sediments. In addition, phylogenetic analysis of sulfate reducer-specific 16S rRNA clone sequences retrieved from RNA isolated from in situ core samples indicated that populations closely related to the Desulfobacteraceae family are active and abundant in the ORFRC subsurface.

Novel iron(III)- and sulfate-reducing organisms were isolated from the contaminated ORFRC subsurface that shared high sequence identity (96 to 99%) to Geobacter bremensis and Desulfotomaculum ruminis, respectively.  The Desulfotomaculum-related isolate utilizes Fe(III) as well as sulfate as an electron acceptor.  The draft genome sequence of Geobacter strain FRC-32 has been completed by the Joint Genome Institute and annotation is currently underway.

Iron minerals were characterized in bioreduction experiments by Mossbauer spectroscopy over a wide range in temperature (4 to 298 K) in order to fully determine the form and speciation of Fe.  Spectra at room temperature (298 K) exhibited no sextet pattern, thus excluding the presence of hematite, magnetite, and maghemite.  At 77 K, the amount of Fe(II) doubled from 15 to 30 % in ethanol- and glucose-amended relative to unamended microcosm sediments, in parallel with wet chemical extractions and counts of Fe(III)-reducing bacteria.  Poorly ordered or Al-substituted goethite was identified and appeared to be dissolved by microbial activity.  However, silicate bound Fe(III) clearly predominated over the Fe minerals reduced.

Our results have the following implications for U(VI) bioremediation in the ORFRC subsurface: 1) the microbially-catalyzed mechanism of U(VI) reduction is electron donor dependent, 2) silicate bound Fe is an important oxidant that is transformed by indigenous microbial populations in the Area 2 subsurface, and 3) Geobacter sp. predominate over other Fe(III)-reducing bacteria during biostimulation with ethanol as an electron donor.