Long-range extracellular electron transport by dissimilatory metal-reducing bacteria across a physical separation

Nanopores in anaerobic sediments are stores of secondary minerals like Fe and Mn oxides that are often the most abundant electron acceptors for microbial respiration. The reduction of these minerals is catalyzed almost exclusively by dissimilatory metal-reducing bacteria (DMRB) that are defined by their ability to gain energy by coupling the oxidation of organic compounds and hydrogen to the reduction of metals. While DMRB are known to use a variety of electron transport mechanisms to reduce Fe and Mn minerals in contact with their outer membrane, little is known about their capacity to reduce sequestered minerals deep within pore spaces that are too small for cell passage. To study long-range extracellular electron transfer (LR-EET) to sequestered minerals, microfluidic reactors with the novel ability to separate DMRB from the insoluble Mn(IV) mineral birnessite were etched into silicon using photolithography or electron beam lithography. The central feature of the microfluidic reactors was a 1.5 μm thick wall containing an array of <200 nm deep pores that allowed the diffusion of solutes but not the passage of cells. The nanoporous wall bifurcated two parallel flow channels; one containing cells, and the other containing a mineral. Bacteria were completely prevented from crossing the wall as demonstrated using brightfield microscopy, fluorescent staining, and scanning electron microscopy (SEM). At the completion of an experiment, a novel method was used to debond the anodically bonded reactors for high-resolution imaging using SEM. Microfluidic reactors were reused by cleaning between experiments using a newly developed protocol. The first mechanism studied was reduction of birnessite by microbial nanowires using Geobacter sulfurreducens KN400. The nanoporous wall in these experiments was composed of an array of pillars separated by <200 gaps (i.e. nanopores) to provide nanowires access to the entire depth of deposited birnessite. Using optical microscopy and Raman spectroscopy, it was demonstrated that birnessite can be reduced up to 15 μm away from cell bodies, similar to the reported length of Geobacter nanowires. Inhibition of nanowire production showed that nanowires were essential for reducing birnessite across the nanoporous wall by LR-EET, but not for reducing birnessite by direct contact. In the latter case, birnessite reduction was likely the result of electron transfer from outer membrane c-Cyts. Reduction across the wall required reducing conditions, provided by Escherichia coli, and an exogenous supply of riboflavin. Riboflavin was found to act not as a diffusible electron shuttle, but as a bound redox cofactor. The high binding affinity of riboflavin reported for outer membrane c-type cytochromes (c-Cyts) suggests that riboflavin was bound to OmcS, a c-Cyt that decorates the nanowire surface. Upon addition of a soluble electron shuttle (i.e., AQDS), it was also demonstrated that reduction extends up to 40 μm into a layer of birnessite, well beyond the reported nanowire length of 15 μm. The second mechanism studied was the reduction of birnessite by electron shuttling using Shewanella oneidensis MR-1. The nanoporous wall in these experiments was composed of a series of highly uniform <200 nm slits along the top of a continuous wall in response to the increased ability of these bacteria to penetrate narrow pore spaces. It was demonstrated that birnessite reduction was driven by the endogenous production of riboflavin (RF) and flavin mononucleotide (FMN), which mediate redox reactions as diffusion-based shuttles or c-Cyt bound cofactors. Experiments with mutants that lacked flavin exporters showed that birnessite reduction is controlled by the concentration of flavin in the system. Addition of exogenous flavin to cells lacking flavin exporters restored birnessite reduction to wild-type rates in the microfluidic reactors. Experiments with mutants lacking conductive nanowires showed that nanowires were not responsible for birnessite reduction by LR-EET, and that the metal-reducing (Mtr) pathway, currently believed to be critical for efficient reduction of insoluble metal oxides, is not required for high rates of reduction by LR-EET. These results suggest the existence of alternative electron pathways for metal reduction, which may be investigated in future work. It was also demonstrated that S. oneidensis can decouple growth from metabolism, potentially expanding the conditions under which metal reduction can be possible in the natural environment. The results presented in this dissertation may lead to more accurate estimations of mineral redox cycling in anaerobic sediments, improved models of contaminant transport, and broadened understanding of carbon exchange between atmospheric and terrestrial ecosystems. Shewanella and Geobacter spp. are widely used in other applications such as energy production and wastewater treatment, and the results in my dissertation may aid in the design of more efficient bioelectrical systems

Gamma-ray and radio properties of six pulsars detected by the fermi large area telescope

We report the detection of pulsed γ-rays for PSRs J0631+1036, J0659+1414, J0742-2822, J1420-6048, J1509-5850, and J1718-3825 using the Large Area Telescope on board the Fermi Gamma-ray Space Telescope (formerly known as GLAST). Although these six pulsars are diverse in terms of their spin parameters, they share an important feature: their γ-ray light curves are (at least given the current count statistics) single peaked. For two pulsars, there are hints for a double-peaked structure in the light curves. The shapes of the observed light curves of this group of pulsars are discussed in the light of models for which the emission originates from high up in the magnetosphere. The observed phases of the γ-ray light curves are, in general, consistent with those predicted by high-altitude models, although we speculate that the γ-ray emission of PSR J0659+1414, possibly featuring the softest spectrum of all Fermi pulsars coupled with a very low efficiency, arises from relatively low down in the magnetosphere. High-quality radio polarization data are available showing that all but one have a high degree of linear polarization. This allows us to place some constraints on the viewing geometry and aids the comparison of the γ-ray light curves with high-energy beam models

Modeling the reversible and resistant components of munition constituent adsorption and desorption on soils

The reversible and resistant components of adsorption and desorption of munition constituents (MCs) on soils was studied to determine the environmental fate of these contaminants. The long-term desorption of MCs has applicability in formulating accurate risk assessments for operational military ranges. Batch experiments near 1:1 (w/v) soil-to-solution ratios reflecting field conditions using solutions containing mixtures of HMX, RDX, and nitroglycerine (NG) were conducted. The three soils used varied from 0.04 to 13.3 % organic matter. The experiment involved one adsorption step followed by four consecutive desorption steps. Adsorption times were 2, 5, 10, and 30 days. For each adsorption time, desorption times were carried out for 1, 12, 24 and 72 h and 30 days. The reversible/resistant component model was applied to the data. The model predicted the desorption concentrations of the MCs in the soil with root mean square errors of approximately 0.05 to 0.2 μg g soil . The extent of desorption hysteresis is not changed by the length of desorption time, irrespective of the initial adsorption time. -

Nanowires of <i>Geobacter sulfurreducens</i> Require Redox Cofactors to Reduce Metals in Pore Spaces Too Small for Cell Passage

Members of the Geobacteraceae family are ubiquitous metal reducers that utilize conductive “nanowires” to reduce Mn­(IV) and Fe­(III) oxides in anaerobic sediments. However, it is not currently known if and to what extent the Mn­(IV) and Fe­(III) oxides in soil grains and low permeability sediments that are sequestered in pore spaces too small for cell passage can be reduced by long-range extracellular electron transport via <i>Geobacter</i> nanowires, and what mechanisms control this reduction. We developed a microfluidic reactor that physically separates <i>Geobacter sulfurreducens</i> from the Mn­(IV) mineral birnessite by a 1.4 μm thick wall containing <200 nm pores. Using optical microscopy and Raman spectroscopy, we show that birnessite can be reduced up to 15 μm away from cell bodies, similar to the reported length of <i>Geobacter</i> nanowires. Reduction across the nanoporous wall required reducing conditions, provided by <i>Escherichia coli</i>, and an exogenous supply of riboflavin. Our results discount electron shuttling by dissolved flavins, and instead support their role as bound redox cofactors in electron transport from nanowires to metal oxides. We also show that upon addition of a soluble electron shuttle (i.e., AQDS), reduction extends beyond the reported nanowire length up to 40 μm into a layer of birnessite