Environmental contamination with radionuclides and heavy metals is a global problem resulting from nuclear weapons production during the Cold War Era. Microbial activity may limit the mobility of contaminants through natural attenuation or bioremediation where organisms, such as fungi, plants and bacteria, naturally reduce, eliminate, or contain hazardous particles. These processes differ, as bioremediation increases the activity of organisms via the addition of nutrients to an environment, whereas attenuation is a naturally occurring process. My research has focused on both bioremediation and natural attenuation of uranium and heavy metals by naturally occurring microorganisms, which can immobilize contaminants via direct biological reduction (e.g., reduction of soluble U(VI) to insoluble U(IV)) or by indirect pathways (e.g., biogenic mineral precipitation).
I assessed the potential for bioremediation at two different locations heavily contaminated with uranium and heavy metals: the subsurface of the U.S. Department of Energy’s Oak Ridge Field Research Center (ORFRC), in Oak Ridge, TN and surficial soils of the Gessen Creek within the former Ronneburg uranium-mining district in Germany. The ORFRC site was the focus of my dissertation research and is contaminated with waste products from uranium enrichment processes, which were collected and stored in three unlined ponds until 1988 when the ponds were pumped and capped by a parking lot. Subsurface groundwater flow created a contaminant plume originating from the pond site, that currently extends approximately 7 km east and west of the ponds to a depth of >150 m. The composition, distribution, and metabolic potential of ORFRC communities varied with subsurface geochemistry (Akob et al. 2007) and biostimulation via carbon addition (e.g., ethanol) promoted complete nitrate reduction and U(VI) precipitation with U(VI) reduction overlapping with sulfate or Fe(III) reduction (Akob et al., 2008 and in press).
Uranium mining near Ronneburg, Germany during the Cold War Era resulted in significant, widespread environmental impacts at the mining site. Extensive remediation efforts began soon after closing the mines and, by focusing on massive physical remediation, i.e., the removal and stabilization of tons of impacted topsoil, the environmental legacy of mining in this area has mostly been erased. Nonetheless, groundwater is still contaminated and a threat to nearby ecosystems. Therefore, during my Ph.D. and post-doc I investigated bank soils of the Gessen Creek with members of Dr. Kirsten Küsel’s lab group. These soils are moderately acidic and contain high concentrations of U and heavy metals. Addition of carbon substrates stimulated activity in Gessen Creek bank soils under Fe(III)- and sulfate-reducing conditions and caused immobilization of some metals (e.g., Cu, Ni, and Co) and mobilization of Co, Ni, Zn, As, and surprisingly U (Burkhardt et al., 2010, Sitte et al., 2010). Microbial community characterization showed that there was some overlap in the metabolically active microbial communities at the two sites (Akob et al. 2008, Burkhardt et al. 2010, 2011) and that Gessen Creek iron-reducing bacteria were very tolerant to heavy metals (Burkhardt et al. 2011).
The most surprising result of this work was that microorganisms at the two contaminated sites greatly differed in their ability to mediate U immobilization. The findings that bioremediation via stimulation of reductive processes negatively effected uranium and heavy metal mobility at Gessen Creek led me to speculate that natural attenuation, via the formation of Fe and Mn oxides, is a more important process at this site. Therefore, my post-doc research investigates how natural attenuation at Ronneburg by is linked to iron (Fe) and manganese (Mn) cycling. I focus on Fe and Mn as they are the two most abundant redox active metals on Earth and are important trace metals for all organisms. Microbial redox cycling determines the availability of oxidized and reduced Fe and Mn species, with oxidation typically resulting in precipitation and immobilization of Fe or Mn oxides. At contaminated sites, such as Ronneburg, Fe(II)- (FeOB) and Mn(II)- (MOB) oxidizing bacteria can affect the fate and transport of metal-contaminants by co-precipitation along with Fe(III) or Mn(II) oxides or by adsorption of metals to biogenic oxides. However, little is known about FeOB in environments with circumneutral pH and Mn oxidation at pH 5.5 has never been shown before in literature as it is predicted to be thermodynamically unfavorable. Therefore, I was involved in isolating three novel species of aerobic FeOB (Fabisch et al. in prep) and a new MOB at pH 5.5 (Akob et al. in prep). These new strains either share less than 97% sequence similarity with their closest fully characterized isolate or exhibit unique physiology compared to their closest fully characterized relative, suggesting that they are new species or ecotypes.
With these new FeOB and MOB strains I am using a polyphasic approach to study their physiology, the mechanisms involved in Fe(II) and Mn(II) oxidation, the effects of heavy metals on their activity, and the mineralogy of Fe and Mn oxides formed. In collaboration with Dr. Martial Taillefert (Georgia Tech), I have used voltammetric techniques to understand how soluble Fe(III)-complexes, i.e., colloidal or chelated Fe(III), are involved in oxidation of Fe(II) by the new FeOB. We are expanding this work to investigate how Fe(III)-complexes play a role as intermediates and affect the transport of Fe(III) from oxic to anoxic zones in the environment.
I am currently working with the Joint Genome Institute (JGI) to sequence the genomes of these four strains in order to assess the genetic components involved in Mn and Fe oxidation and heavy metal tolerance. This will be performed in conjunction with proteomics to investigate proteins involved in Fe/Mn oxidation or metal resistance due to differential expression during growth on ferrous iron or reduced Mn with and without heavy metals. Once sequences coding for such proteins/genes are identified I will develop a quantitative PCR assay to evaluate the activity and abundance of these strains in both in controlled laboratory manipulation experiments and in environmental samples over different spatial and temporal scales. Such molecular proxy for monitoring activity of FeOB and MOB could then be used as a method for monitoring on-going natural attenuation in contaminated sites.
Another facet of my work is to identify the biogenic minerals formed by the new FeOB and MOB strains using a combination of electron microscopy (SEM, TEM) and energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analysis. Preliminary results suggest that mainly amorphous or nanocrystalline material was formed by the new isolates. For FeOB we could identify the minerals lepidocrocite, goethite and magnetite or maghemite, whereas, MOB minerals were tentatively identified as birnessite. More detailed mineralogical investigations are currently on going in cooperation with Carol Johnson and Dr. Michael Hochella (Virginia Tech). The goal of this work is to estimate the biological contribution to in situ mineralogy and to evaluate how these organisms affect the mobility of heavy metals in radionuclide and heavy metal contaminated sites.
- Kirsten Küsel, Friedrich Schiller University Jena
- Joel Kostka, Georgia Institute of Technology
- Stefan J. Green, University of Illinois at Chicago
- Martial Taillefert, Georgia Institute of Technology
- Kuk-Jeong (Kuki) Chin, Georgia State University
- Franziska Schäffner, Dirk Merten, and Georg Büchel, Institute of Earth Sciences, Friedrich Schiller University Jena, Germany
- Matthias Händel and Kai-Uwe Totsche, Institute for Geoscience, Friedrich Schiller University Jena, Germany
- Carol Johnson and Michael Hochella, Jr., Virginia Tech, USA
Akob, D. M.*, S. H. Lee*, M. Sheth, K. Küsel, D. B. Watson, A.V. Palumbo, J.E. Kostka, and K.-J. Chin. 2012. Gene expression correlates with process rates quantified for sulfate- and Fe(III)-reducing bacteria in U(VI)-contaminated sediments. Frontiers in Terrestrial Microbiology, 3:280. *Equal contribution.
Akob, D. M., L. Kerkhof, K. Küsel, D. B. Watson, A. V. Palumbo, and J. E. Kostka. 2011. Linking specific heterotrophic bacterial populations to bioreduction of uranium and nitrate using stable isotope probing in contaminated subsurface sediments. Applied and Environmental Microbiology. 77(22):8197-8200
Burkhardt, E.-M., S. Bischoff, D. M. Akob, G. Büchel, and K. Küsel. 2011. Heavy metal tolerance of Fe(III)-reducing microbial communities in a contaminated creek bank soil. Applied and Environmental Microbiology. 77(9): 3132-3136.
Lu, S., S. Gischkat, M. Reiche, D. M. Akob, K. B. Hallberg, and K. Küsel. 2010. Ecophysiology of Fe-cycling Bacteria in Acidic Sediments. Applied and Environmental Microbiology 76 (24): 8174-8183.
Vishnivetskaya, T. A., C. C. Brandt, A. S. Madden, M. S. Drake, J. E. Kostka, D. M. Akob, K. Küsel, and A. V. Palumbo. 2010. Microbial Community Changes in Response to Ethanol or Methanol Amendments for U(VI) Reduction. Applied and Environmental Microbiology 76(17): 5728-5735.
Sitte, J., D. M. Akob, C. Kaufmann, K. Finster, D. Banerjee, E.-M. Burkhardt, J. E. Kostka, A. Scheinost, Georg Büchel and K. Küsel. 2010. Microbial Links between Sulfate Reduction and Metal Retention in Uranium and Heavy Metal-contaminated Soil. Applied and Environmental Microbiology 76(10): 3143–3152.
Green, S. J., O. Prakash, T. M. Gihring, D. M. Akob, P. Jasrotia, P. M. Jardine, D. B. Watson, S. D. Brown, A. V. Palumbo and J. E. Kostka. 2010. Denitrifying bacteria from the terrestrial subsurface exposed to mixed waste contamination. Applied and Environmental Microbiology 76(10): 3244–3254.
Burkhardt, E.-M., D. M. Akob, S. Bischoff, J. Sitte, J. E. Kostka, D. Banerjee, A. C. Scheinost, K. Küsel. 2010. Impact of Biostimulated Redox Processes on Metal Dynamics in an Iron-rich Creek Soil of a former Uranium Mining Area. Environmental Science & Technology 44(1): 177-183.
O. Prakash, S. J. Green, D. M. Akob, T. M. Gihring, P. Jardine, D. B. Watson, and J. E. Kostka. 2010. Novel denitrifying bacteria isolated from the terrestrial subsurface exposed to mixed waste contamination. Environmental Microbiology 76, 3244-3254.
O. Prakash, T.M. Gihring, D.D. Dalton, K.-J. Chin, S.J. Green, D.M. Akob, G. Wanger, J.E. Kostka. 2010. Geobacter daltonii sp. nov., an iron(III)- and uranium(VI)-reducing bacterium isolated from the shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination. International Journal of Systematic and Evolutionary Microbiology 60: 546-553.
D. M. Akob, H. J. Mills, T. M. Gihring, L. Kerkhof, J. W. Stucki, Kuk-Jeong Chin, Kirsten Kuesel, Anthony V. Palumbo, David B. Watson, and Joel E. Kostka. 2008. Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide contaminated subsurface sediments. Applied and Environmental Microbiology 74: 3159-3170.
D. M. Akob, H. J. Mills, D. L. Swofford, J. E. Kostka. 2007. Metabolically-Active Microbial Communities in Uranium-Contaminated Subsurface Sediments. FEMS Microbiology Ecology 59: 95-107.