Microbiological-enhanced mixing across scales during in-situ bioreduction of metals and radionuclides at Department of Energy Sites

TITLE: Microbiological-enhanced mixing across scales during in-situ bioreduction of metals and radionuclides at Department of Energy Sites ABSTRACT: Bioremediation is being investigated as an effective strategy for long-term management of DOE sites contaminated by metals and radionuclides. Bioremediation typically requires injection of chemicals into the subsurface which mix at varying scales with the contaminant to stimulate the growth of dissimilatory metal reducing bacteria (DMRB). These bacteria couple the oxidation of injected chemicals to the reduction of contaminants as they mix in the groundwater. Syntrophic interactions with other bacterial species may also be exploited to supply DMRB with higher quality electron donors such as H2 that are otherwise difficult to deliver to bacteria. Evidence from DOE field experiments suggests that mixing limitations of substrates at all scales may affect biological growth and activity for reduction. Bioremediation is being investigated as an effective strategy for long-term management of DOE sites contaminated by metals and radionuclides. Bioremediation typically requires injection of chemicals into the subsurface which mix at varying scales with the contaminant to stimulate the growth of dissimilatory metal reducing bacteria (DMRB). These bacteria couple the oxidation of injected chemicals to the reduction of contaminants as they mix in the groundwater. Syntrophic interactions with other bacterial species may also be exploited to supply DMRB with higher quality electron donors such as H2 that are otherwise difficult to deliver to bacteria. Evidence from DOE field experiments suggests that mixing limitations of substrates at all scales may affect biological growth and activity for reduction. In order to elaborate and investigate the energy transfer from an obligate symbiont, syntroph, to a partnering metal-reducing organism, we set up a two-species culture ofSyntrophobacter wolinii DB and Geobacter sulfurreducens. Two strains of Geobacter sulfurreducens were successful partners: PCA (type strain) and KN400 (strain producing more nanowires than average). In contrast, a G. sulfurreducens PCA hydrogenase mutant could not serve as a partner. Therefore, hydrogen likely served as an electron shuttle between S. wolinii DB and G. sulfurreducens. We successfully fabricated nanofluidic reactors designed to investigate the ability of bacterially produced conductive pili, or ‘nanowires’, to enhance the zone of mixing beyond what is possible by advection and dispersion alone. Our microfluidic experiments have identified realistic flow parameters and growth conditions amenable for growth and attachment inside these reactors that will be used in nanofluidic experiments. Selenite was chosen as a representative metal and was reduced to an insoluble precipitate inside the microfluidic reactor by Anaeromyxobacter dehalogens. Precipitates were analyzed by Raman spectroscopy and energy dispersive spectroscopy to confirm the presence of reduced selenium. The density of bacteria appeared greatest on the edges of the red precipitates, suggesting that microenvironments favorable for selenite reduction and growth occur near existing crystals. Microfluidic experiments were modeled in 2-D to predict biomass distribution and chemical mixing at the pore scale in homogenous soils. Electron donor and acceptor were delivered through the inlets and mixed occurred along the centerline by transverse diffusion. Results indicate that microbes grow along the entire centerline where mixing occurs, that shear stress mitigates growth in pore throats but promotes growth in pore bodies, and that literature parameters for growth kinetics obtained from batch reactors can be used to simulate reactive transport at the pore scale. Our future work focuses on evaluating electron transfer across a nanoporous barrier using the twospecies syntroph / Geobacter culture in order to elucidate electron transfer mechanisms, and on modeling this process in more realistic pore-scale geometries.