The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.

The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (De) in biofilms respiring on electrodes. Two model electrochemically active biofilms, Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1, were investigated. A novel nuclear magnetic resonance microimaging perfusion probe capable of simultaneous electrochemical and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) techniques was used. PFG-NMR allowed noninvasive, nondestructive, high spatial resolution in situ De measurements in living biofilms respiring on electrodes. The electrodes were polarized so that they would act as the sole terminal electron acceptor for microbial metabolism. We present our results as both two-dimensional De heat maps and surface-averaged relative effective diffusion coefficient (Drs) depth profiles. We found that 1) Drs decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; 2) Drs at a given location decreases with G. sulfurreducens biofilm age; 3) average De and Drs profiles in G. sulfurreducens biofilms are lower than those in S. oneidensis biofilms—the G. sulfurreducens biofilms studied here were on average 10 times denser than the S. oneidensis biofilms; and 4) halting the respiration of a G. sulfurreducens biofilm decreases the De values. Density, reflected by De, plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms.

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well-documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface-averaging methods are used, position-dependent measurements of the effective diffusion coefficient are currently: 1) invasive to the biofilm, 2) performed under unnatural conditions, 3) lethal to cells, and/or 4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time-dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: 1) measure the effective diffusion coefficient for water in live biofilms, 2) monitor how the effective diffusion coefficient changes over time under growth conditions, and 3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two-dimensional effective diffusion coefficient maps within Shewanella oneidensis MR-1 biofilms using pulsed-field gradient nuclear magnetic resonance methods, and used them to calculate surface-averaged relative effective diffusion coefficient (Drs) profiles. We found that 1) Drs decreased from the top of the biofilm to the bottom, 2) Drs profiles differed for biofilms of different ages, 3) Drs profiles changed over time and generally decreased with time, 4) all the biofilms showed very similar Drs profiles near the top of the biofilm, and 5) the Drs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm.

In this study, we quantified electron transfer rates, depth profiles of electron donor, and biofilm structure of Geobacter sulfurreducens biofilms using an electrochemical-nuclear magnetic resonance microimaging biofilm reactor. Our goal was to determine whether electron donor limitations existed in electron transfer processes of electrode-respiring G. sulfurreducens biofilms. Cells near the top of the biofilms consumed acetate and were metabolically active; however, acetate concentration decreased to below detection within the top 100 microns of the biofilms. Additionally, porosity in the biofilms fell below 10% near the electrode surface, exacerbating exclusion of acetate from the lower regions. The dense biofilm matrix in the acetate-depleted zone acted as an electrical conduit passing electrons generated at the top of the biofilm to the electrode. To verify the distribution of cell metabolic activity, we used uranium as a redox-active probe for localizing electron transfer activity and X-ray absorption spectroscopy to determine the uranium oxidation state. Cells near the top reduced UVI more actively than the cells near the base. High-resolution transmission electron microscopy images showed intact, healthy cells near the top and plasmolyzed cells near the base. Contrary to models proposed in the literature, which hypothesize that cells nearest the electrode surface are the most metabolically active because of a lower electron transfer resistance, our results suggest that electrical resistance through the biofilm does not restrict long-range electron transfer. Cells far from the electrode can respire across metabolically inactive cells, taking advantage of their extracellular infrastructure produced during the initial biofilm formation.

[1] Microscopic and spectroscopic analyses of uranium-contaminated sediments from select locations at the U.S. Department of Energy (DOE) Hanford site have revealed that sorbed uranium (U) often exists as uranyl precipitates associated with intragrain fractures of granitic clasts. The release of U to contacting fluids appears to be controlled by intragrain ion diffusion coupled with the dissolution kinetics of the precipitates that exist in the form of Na-boltwoodite. Here we present a coupled microscopic reactive diffusion model for the contaminated sediment on the basis of experimental measurements of intragrain diffusivity in the granitic lithic fragments and the dissolution kinetics of synthetic Na-boltwoodite. Nuclear magnetic resonance, pulse gradient spin echo measurements showed that the intragrain fractures of the granitic clasts isolated from the sediment contained two domains with distinct diffusivities. The fast diffusion domain had an apparent tortuosity of 1.5, while that of the slow region was two orders of magnitude larger. A two-domain diffusion model was assembled and used to infer the geochemical conditions that led to intragrain uranyl precipitation during waste-sediment interaction. Rapid precipitation of Na-boltwoodite was simulated with an alkaline U-containing, high-carbonate tank waste solution that diffused into intragrain fractures, which originally contained Si-rich pore water in equilibrium with feldspar grains in the lithic fragments. The model was also used to simulate uranyl dissolution and release from contaminated sediment to recharge waters. With independently characterized parameters for Na-boltwoodite dissolution, the model simulations demonstrated that diffusion could significantly decrease the rates of intragrain uranyl mineral dissolution due to diffusion-induced local solubility limitation with respect to Na-boltwoodite.