Paul D. Majors

Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization.

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.

Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy

Bacterial biofilms are complex, three-dimensional communities found nearly everywhere in nature and are also associated with many human diseases. Detailed metabolic information is critical to understand and exploit beneficial biofilms as well as combat antibiotic-resistant, disease-associated forms. However, most current techniques used to measure temporal and spatial metabolite profiles in these delicate structures are invasive or destructive. Here, we describe imaging, transport and metabolite measurement methods and their correlation for live, non-invasive monitoring of biofilm processes. This novel combination of measurements is enabled by the use of an integrated nuclear magnetic resonance (NMR) and confocal laser scanning microscope (CLSM). NMR methods provide macroscopic structure, metabolic pathway and rate data, spatially resolved metabolite concentrations and water diffusion profiles within the biofilm. In particular, current depth-resolved spectroscopy methods are applied to detect metabolites in 140–190 nl volumes within biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1 and the oral bacterium implicated in caries disease, Streptococcus mutans strain UA159. The perfused sample chamber also contains a transparent optical window allowing for the collection of complementary fluorescence information using a unique, in-magnet CLSM. In this example, the entire three-dimensional biofilm structure was imaged using magnetic resonance imaging. This was then correlated to a fluorescent CLSM image by employing a green fluorescent protein reporter construct of S. oneidensis. Non-invasive techniques such as described here, which enable measurements of dynamic metabolic processes, especially in a depth-resolved fashion, are expected to advance our understanding of processes occurring within biofilm communities.

Investigations of structure and metabolism within Shewanella oneidensis MR-1 biofilms

Biofilms possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments that may ultimately drive species diversity, determine biofilm structure and the spatial distribution of the community members. Using non-invasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated the kinetics and stratification of anaerobic metabolism within live biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1. Biofilms were pre-grown using a defined minimal medium in a constant-depth film bioreactor and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. Biofilms generated in this manner were subjected to changing substrate/electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate) and the metabolic responses measured. Localized NMR spectroscopy was used to non-invasively measure hydrogen-containing metabolites at high temporal resolution (4.5 min) under O(2)-limited conditions. Reduction of electron acceptor under anaerobic conditions was immediately observed upon switching feed solutions indicating that no gene induction (transcriptional response) was needed for MR-1 to switch metabolism from O(2) to fumarate, dimethyl sulfoxide or nitrate. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate changes in population response to the availability of electron acceptor and to probe metabolic competition under O(2)-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how these factors relate to environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described herein.

Diffusion in biofilms respiring on electrodes

The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (D(e)) 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 D(e) 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 D(e) heat maps and surface-averaged relative effective diffusion coefficient (D(rs)) depth profiles. We found that 1) D(rs) decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; 2) D(rs) at a given location decreases with G. sulfurreducens biofilm age; 3) average D(e) and D(rs) 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 D(e) values. Density, reflected by D(e), plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms.

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

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. Biotechnol. Bioeng. 2010;106: 928–937.

Metabolic spatial variability in electrode-respiring Geobacter sulfurreducens biofilms

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.

Biofilm shows spatially stratified metabolic responses to contaminant exposure

Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR-1 biofilms to U(VI) (uranyl, UO(2)(2+)) and Cr(VI) (chromate, CrO(4) (2-)) using non-invasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm-contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant-induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom-developed based on local microenvironments.

Identifying Low pH Active and Lactate-Utilizing Taxa within Oral Microbiome Communities from Healthy Children Using Stable Isotope Probing Techniques

Background Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species. Methodology/Principal Findings Supragingival plaque samples from caries-free children incubated with 13C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by Lactobacillus and Propionibacterium species, both of which have been previously found within carious lesions from children. Conclusions/Significance Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.

NMR bioreactor development for live in-situ microbial functional analysis.

A live, in-situ metabolomics capability was developed for prokaryotic cultures under controlled growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuel production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study also showed that E. aggregans produces lactate end product in significant concentrations-a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics/systems biology methods are discussed.

Localized in vivo isotropic-anisotropic correlation 1H NMR spectroscopy using ultraslow magic angle spinning†

In a previous work ( 1 ), the susceptibility broadening in the 1H NMR metabolite spectrum obtained in a live mouse was separated from the isotropic information, which significantly increased the spectral resolution. This was achieved using ultraslow magic angle spinning (MAS) of the animal combined with a modified phase‐corrected magic angle turning (PHORMAT) pulse sequence. However, PHORMAT cannot be used for spatially selective spectroscopy. This article introduces a modified sequence called localized magic angle turning (LOCMAT) that makes this possible. Proton LOCMAT spectra were obtained from the liver and heart of a live mouse while the animal was spun at a speed of 4 Hz in a 2 Tesla field. It was found that even in this relatively low field, LOCMAT provided isotropic line widths that were a factor of 4–10 times smaller than those obtained in a stationary animal. Furthermore, the susceptibility broadening of the heart metabolites showed unusual features that are not observed in dead animals. The limitations of LOCMAT and possible ways to improve the technique are discussed. It is concluded that in vivo LOCMAT can significantly enhance the utility of NMR spectroscopy for biomedical research. Magn Reson Med, 2006. Published 2005 Wiley‐Liss, Inc.