Redox-sensitive metal contaminants in subsurface environments can be reduced enzymatically or indirectly by microbial activity to convert them from soluble mobile (toxic) to comparatively insoluble, relatively immobile (less bioavailable) forms. The broad purpose of the research presented in this dissertation was to acquire a deep understanding of selenium and iron microbial reduction and immobilization in the subsurface and to characterize in detail the nature of the bioreduction products. To this end, biofilms formed during a biostimulation experiment in a metal-contaminated aquifer adjacent to the Colorado River in Colorado, USA were studied. Biofilms develop in a wide variety of natural settings and the aqueous chemical conditions within biofilms are strongly affected by the presence of extracellular polymers that potentially confer biofilm cells with a greater tolerance to heavy metals than planktonic cells.

This thesis integrates field and laboratory experimental methods to provide 2D and 3D ultrastructural information, 2D chemical speciation and community membership via metagenomics methods. In addition, physiological information was obtained via characterization of an isolated bacterium and insights related to the product structure and stability were achieved by chemical synthesis-based studies. In this dissertation, an apparatus permitting correlative cryogenic spectro-microscopy was developed (Appendix I) and applied to determine in detail the cell-mineral relationships and the speciation of selenium in the biofilms (Chapter 1). The research involved integration of both cryogenic electron microscopy and X-ray absorption spectroscopy datasets on the same sample region to document the size, structure and distribution of bioreduction products. Because many of the microbial species in the mine tailings-contaminated aquifer are novel and difficult to cultivate in the laboratory, part of the research involved phylogenic analyses of the biofilm organisms via analysis of 16S rRNA genes. A novel betaproteobacterium of the genus Dechloromonas (Dechloromonas selenatis strain RGW, Chapter 2) was isolated from the Rifle site and shown to be capable of reducing selenate to red amorphous elemental Se0. This isolate was also capable of reducing toxic arsenate. Chapter 3 investigates further the stability of elemental selenium colloids at ambient pressure as a function of temperature and particle size. The last chapter (Chapter 4) focuses on the distribution and speciation of iron in the Rifle aquifer during a biostimulation experiment. The combined results demonstrate the importance of both clays and cell-associated ferric iron oxyhydroxide aggregates for growth of planktonic iron-reducing bacteria.

These insights provide fundamental information about organisms that mediate selenium, iron and arsenic biogeochemical transformations in the subsurface and the nature of the product phases. The data may help to identify substrate amendment regimes for sustained Se remediation. Following short-term acetate addition to the aquifer, selenium remained immobile for at least one year, suggesting the acetate amendment approach has significant potential for bioremediation of selenium, in addition to uranium and vanadium as previously studied. Although focused on selenium and iron bio-reduction, the instrumentation and approaches developed here are generally applicable for accurate determination of cell-mineral interactions and metal speciation and can be further extended to constrain aquifer-scale reactive transport models in a wide range of environments.