Microbial growth rates and local external mass transfer coefficients in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity.

19 F nuclear magnetic resonance (NMR) oximetry and 1 H NMR velocimetry were used to noninvasively map oxygen concentrations and hydrodynamics in space and time in a model packed bed biofilm system in the presence and absence of flow. The development of a local oxygen sink associated with a single gel bead inoculated with respiring Escherichia coli was analyzed with a phenomenological model to determine the specific growth rate of the bacteria in situ, returning a value (0.66 hr-1 ) that was close to that measured independently in planktonic culture (0.62 hr-1 ). The decay of oxygen concentration in and around the microbiologically active bead was delayed and slower in experiments conducted under continuous flow in comparison to no-flow experiments. Concentration boundary layer thicknesses were determined and Sherwood numbers calculated to quantify external mass transfer resistance. Boundary layers were thicker in no-flow experiments compared to experiments with flow. Whereas the oxygen concentration profile across a reactive biofilm particle was symmetric in no-flow experiments, it was asymmetric with respect to flow direction in flow experiments with Sherwood numbers on the leading edge (Sh = 7) being larger than the trailing edge (Sh = 3.5). The magnitude of the experimental Sh was comparable to values predicted by a variety of correlations. These spatially resolved measurements of oxygen distribution in a geometrically complex model reveal in innovative detail the local coupling between microbial growth, oxygen consumption, and external mass transfer.

Heterogeneous diffusion in aerobic granular sludge.

Aerobic granular sludge (AGS) technology allows simultaneous nitrogen, phosphorus, and carbon removal in compact wastewater treatment processes. To operate, design, and model AGS reactors, it is essential to properly understand the diffusive transport within the granules. In this study, diffusive mass transfer within full-scale and lab-scale AGS was characterized with nuclear magnetic resonance (NMR) methods. Self-diffusion coefficients of water inside the granules were determined with pulsed-field gradient NMR, while the granule structure was visualized with NMR imaging. A reaction-diffusion granule-scale model was set up to evaluate the impact of heterogeneous diffusion on granule performance. The self-diffusion coefficient of water in AGS was ∼70% of the self-diffusion coefficient of free water. There was no significant difference between self-diffusion in AGS from full-scale treatment plants and from lab-scale reactors. The results of the model showed that diffusional heterogeneity did not lead to a major change of flux into the granule (<1%). This study shows that differences between granular sludges and heterogeneity within granules have little impact on the kinetic properties of AGS. Thus, a relatively simple approach is sufficient to describe mass transport by diffusion into the granules.

Characterizing the structure of aerobic granular sludge using ultra-high field magnetic resonance.

Despite aerobic granular sludge wastewater treatment plants operating around the world, our understanding of internal granule structure and its relation to treatment efficiency remains limited. This can be attributed in part to the drawbacks of time-consuming, labor-intensive, and invasive microscopy protocols which effectively restrict samples sizes and may introduce artefacts. Time-domain nuclear magnetic resonance (NMR) allows non-invasive measurements which describe internal structural features of opaque, complex materials like biofilms. NMR was used to image aerobic granules collected from five full-scale wastewater treatment plants in the Netherlands and United States, as well as laboratory granules and control beads. T1 and T2 relaxation-weighted images reveal heterogeneous structures that include high- and low-density biofilm regions, water-like voids, and solid-like inclusions. Channels larger than approximately 50 µm and connected to the bulk fluid were not visible. Both cluster and ring-like structures were observed with each granule source having a characteristic structural type. These structures, and their NMR relaxation behavior, were stable over several months of storage. These observations reveal the complex structures within aerobic granules from a range of sources and highlight the need for non-invasive characterization methods like NMR to be applied in the ongoing effort to correlate structure and function.

Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: Persistence of an oxygen sink despite prolonged antibiotic therapy.

PURPOSE: Oxygen availability is a critical determinant of microbial biofilm activity and antibiotic susceptibility. However, measuring oxygen gradients in these systems remains difficult, with the standard microelectrode approach being both invasive and limited to single-point measurement. The goal of the study was to develop a 19 F MRI approach for 2D oxygen mapping in biofilm systems and to visualize oxygen consumption behavior in real time during antibiotic therapy. METHODS: Oxygen-sensing beads were created by encapsulating an emulsion of oxygen-sensing fluorocarbon into alginate gel. Escherichia coli biofilms were grown in and on the alginate matrix, which was contained inside a packed bed column subjected to nutrient flow, mimicking the complex porous structure of human wound tissue, and subjected to antibiotic challenge. RESULTS: The linear relationship between 19 F spin-lattice relaxation rate R1 and local oxygen concentration permitted noninvasive spatial mapping of oxygen distribution in real time over the course of biofilm growth and subsequent antibiotic challenge. This technique was used to visualize persistence of microbial oxygen respiration during continuous gentamicin administration, providing a time series of complete spatial maps detailing the continued bacterial utilization of oxygen during prolonged chemotherapy in an in vitro biofilm model with complex spatial structure. CONCLUSIONS: Antibiotic exposure temporarily causes oxygen consumption to enter a pseudosteady state wherein oxygen distribution becomes fixed; oxygen sink expansion resumes quickly after antibiotic clearance. This technique may provide valuable information for future investigations of biofilms by permitting the study of complex geometries (typical of in vivo biofilms) and facilitating noninvasive oxygen measurement.

Glass Dynamics and Domain Size in a Solvent-Polymer Weak Gel Measured by Multidimensional Magnetic Resonance Relaxometry and Diffusometry.

Nuclear magnetic resonance measurements of rotational and translational molecular dynamics are applied to characterize the nanoscale dynamic heterogeneity of a physically cross-linked solvent-polymer system above and below the glass transition temperature. Measured rotational dynamics identify domains associated with regions of solidlike and liquidlike dynamics. Translational dynamics provide quantitative length and timescales of nanoscale heterogeneity due to polymer network cross-link density. Mean squared displacement measurements of the solvent provide microrheological characterization of the system and indicate glasslike caging dynamics both above and below the glass transition temperature.

NMR investigation of water diffusion in different biofilm structures.

Mass transfer in biofilms is determined by diffusion. Different mostly invasive approaches have been used to measure diffusion coefficients in biofilms, however, data on heterogeneous biomass under realistic conditions is still missing. To non-invasively elucidate fluid-structure interactions in complex multispecies biofilms pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was applied to measure the water diffusion in five different types of biomass aggregates: one type of sludge flocs, two types of biofilm, and two types of granules. Data analysis is an important issue when measuring heterogeneous systems and is shown to significantly influence the interpretation and understanding of water diffusion. With respect to numerical reproducibility and physico-chemical interpretation, different data processing methods were explored: (bi)-exponential data analysis and the Γ distribution model. Furthermore, the diffusion coefficient distribution in relation to relaxation was studied by D-T2 maps obtained by 2D inverse Laplace transform (2D ILT). The results show that the effective diffusion coefficients for all biofilm samples ranged from 0.36 to 0.96 relative to that of water. NMR diffusion was linked to biofilm structure (e.g., biomass density, organic and inorganic matter) as observed by magnetic resonance imaging and to traditional biofilm parameters: diffusion was most restricted in granules with compact structures, and fast diffusion was found in heterotrophic biofilms with fluffy structures. The effective diffusion coefficients in the biomass were found to be broadly distributed because of internal biomass heterogeneities, such as gas bubbles, precipitates, and locally changing biofilm densities. Thus, estimations based on biofilm bulk properties in multispecies systems can be overestimated and mean diffusion coefficients might not be sufficiently informative to describe mass transport in biofilms and the near bulk.

Impact of Mineral Precipitation on Flow and Mixing in Porous Media Determined by Microcomputed Tomography and MRI.

Precipitation reactions influence transport properties in porous media and can be coupled to advective and dispersive transport. For example, in subsurface environments, mixing of groundwater and injected solutions can induce mineral supersaturation of constituents and drive precipitation reactions. Magnetic resonance imaging (MRI) and microcomputed tomography (µ-CT) were employed as complementary techniques to evaluate advection, dispersion, and formation of precipitate in a 3D porous media flow cell. Two parallel fluids were flowed concentrically through packed glass beads under two relative flow rates with Na2CO3 and CaCl2 in the inner and outer fluids, respectively. CaCO3 became supersaturated and formed a precipitate at the mixing interface between the two solutions. Spatial maps of changing local velocity fields and dispersion in the flow cell were generated from MRI, while high resolution µ-CT imaging visualized the precipitate formed in the porous media. Formation of a precipitate minimized dispersive and advective transport between the two fluids and the shape of the precipitation front was influenced by the relative flow rates. This work demonstrates that the combined use of MRI and µ-CT can be highly complementary in the study of reactive transport processes in porous media.

Detecting Microbially Induced Calcite Precipitation in a Model Well-Bore Using Downhole Low-Field NMR.

Microbially induced calcite precipitation (MICP) has been widely researched recently due to its relevance for subsurface engineering applications including sealing leakage pathways and permeability modification. These applications of MICP are inherently difficult to monitor nondestructively in time and space. Nuclear magnetic resonance (NMR) can characterize the pore size distributions, porosity, and permeability of subsurface formations. This investigation used a low-field NMR well-logging probe to monitor MICP in a sand-filled bioreactor, measuring NMR signal amplitude and T2 relaxation over an 8 day experimental period. Following inoculation with the ureolytic bacteria, Sporosarcina pasteurii, and pulsed injections of urea and calcium substrate, the NMR measured water content in the reactor decreased to 76% of its initial value. T2 relaxation distributions bifurcated from a single mode centered about approximately 650 ms into a fast decaying population (T2 less than 10 ms) and a larger population with T2 greater than 1000 ms. The combination of changes in pore volume and surface minerology accounts for the changes in the T2 distributions. Destructive sampling confirmed final porosity was approximately 88% of the original value. These results indicate the low-field NMR well-logging probe is sensitive to the physical and chemical changes caused by MICP in a laboratory bioreactor.

In Situ Detection of Subsurface Biofilm Using Low-Field NMR: A Field Study.

Subsurface biofilms are central to bioremediation of chemical contaminants in soil and groundwater whereby micro-organisms degrade or sequester environmental pollutants like nitrate, hydrocarbons, chlorinated solvents and heavy metals. Current methods to monitor subsurface biofilm growth in situ are indirect. Previous laboratory research conducted at MSU has indicated that low-field nuclear magnetic resonance (NMR) is sensitive to biofilm growth in porous media, where biofilm contributes a polymer gel-like phase and enhances T2 relaxation. Here we show that a small diameter NMR well logging tool can detect biofilm accumulation in the subsurface using the change in T2 relaxation behavior over time. T2 relaxation distributions were measured over an 18 day experimental period by two NMR probes, operating at approximately 275 kHz and 400 kHz, installed in 10.2 cm wells in an engineered field testing site. The mean log T2 relaxation times were reduced by 62% and 43%, respectively, while biofilm was cultivated in the soil surrounding each well. Biofilm growth was confirmed by bleaching and flushing the wells and observing the NMR signal's return to baseline. This result provides a direct and noninvasive method to spatiotemporally monitor biofilm accumulation in the subsurface.

Permeability of a growing biofilm in a porous media fluid flow analyzed by magnetic resonance displacement-relaxation correlations.

Biofilm growth in porous media is difficult to study non-invasively due to the opaqueness and heterogeneity of the systems. Magnetic resonance is utilized to non-invasively study water dynamics within porous media. Displacement-relaxation correlation experiments were performed on fluid flow during biofilm growth in a model porous media of mono-dispersed polystyrene beads. The spin-spin T2 magnetic relaxation distinguishes between the biofilm phase and bulk fluid phase due to water-biopolymer interactions present in the biofilm, and the flow dynamics are measured using PGSE NMR experiments. By correlating these two measurements, the effects of biofilm growth on the fluid dynamics can be separated into a detailed analysis of both the biofilm phase and the fluid phase simultaneously within the same experiment. Within the displacement resolution of these experiments, no convective flow was measured through the biomass. An increased amount of longitudinal hydrodynamic dispersion indicates increased hydrodynamic mixing due to fluid channeling caused by biofilm growth. The effect of different biofilm growth conditions was measured by varying the strength of the bacterial growth medium.

Biofilm detection in natural unconsolidated porous media using a low-field magnetic resonance system.

The extent to which T(2) relaxation measurements can be used to determine biofouling in several natural geological sand media using a low-field (275 kHz, 6.5 mT) NMR system has been demonstrated. It has been previously shown that, at high laboratory strength fields (300 MHz, 7 T), T(2) techniques can be used as a bioassay to confirm the growth of biofilm inside opaque porous media with low magnetic susceptibilities such as borosilicate or soda lime glass beads. Additionally decreases in T(2) can be associated with intact biofilm as opposed to degraded biofilm material. However, in natural geological media, the strong susceptibility gradients generated at high fields dominated the T(2) relaxation time distributions and biofilm growth could not be reliably detected. Samples studied included Bacillus mojavensis biofilm in several sand types, as well as alginate solution and alginate gel in several sand types. One of the sand types was highly magnetic. Data was collected with a low-field (275 kHz, 6.5 mT) benchtop NMR system using a CPMG sequence with an echo time of 1.25 ms providing the ability to detect signals with T(2) greater than 1 ms. Data presented here clearly demonstrate that biofilm can be reliably detected and monitored in highly magnetically susceptible geological samples using a low-field NMR spectrometer indicating that low-field NMR could be viable as a biofilm sensor at bioremedation sites.

Detection of biological uranium reduction using magnetic resonance.

The conversion of soluble uranyl ions (UO2²âº) by bacterial reduction to sparingly soluble uraninite (UO2(s)) is being studied as a way of immobilizing subsurface uranium contamination. Under anaerobic conditions, several known types of bacteria including iron and sulfate reducing bacteria have been shown to reduce U (VI) to U (IV). Experiments using a suspension of uraninite (UO2(s)) particles produced by Shewanella putrefaciens CN32 bacteria show a dependence of both longitudinal (T1) and transverse (T2) magnetic resonance (MR) relaxation times on the oxidation state and solubility of the uranium. Gradient echo and spin echo MR images were compared to quantify the effect caused by the magnetic field fluctuations (T*2) of the uraninite particles and soluble uranyl ions. Since the precipitate studied was suspended in liquid water, the effects of concentration and particle aggregation were explored. A suspension of uraninite particles was injected into a polysaccharide gel, which simulates the precipitation environment of uraninite in the extracellular biofilm matrix. A reduction in the T2 of the gel surrounding the particles was observed. Tests done in situ using three bioreactors under different mixing conditions, continuously stirred, intermittently stirred, and not stirred, showed a quantifiable T2 magnetic relaxation effect over the extent of the reaction.

Magnetic resonance analysis of capillary formation reaction front dynamics in alginate gels.

The formation of heterogeneous structures in biopolymer gels is of current interest for biomedical applications and is of fundamental interest to understanding the molecular level origins of structures generated from disordered solutions by reactions. The cation-mediated physical gelation of alginate by calcium and copper is analyzed using magnetic resonance measurements of spatially resolved molecular dynamics during gel front propagation. Relaxation time and pulse-field gradient methods are applied to determine the impact of ion front motion on molecular translational dynamics. The formation of capillaries in alginate copper gels is correlated to changes in translational dynamics.

Dynamic length-scale characterization and nonequilibrium statistical mechanics of transport in open-cell foams.

Nuclear magnetic resonance measurements of scale dependent dynamics in a random solid open-cell foam reveal a characteristic length scale for transport processes in this novel type of porous medium. These measurements and lattice Boltzmann simulations for a model foam structure indicate dynamical behavior analogous to lower porosity consolidated granular porous media, despite extremely high porosity in solid cellular foams. Scaling by the measured characteristic length collapses data for different foam structures as well as consolidated granular media. The nonequilibrium statistical mechanics theory of preasymptotic dispersion, developed for hierarchical porous media, is shown to model the hydrodynamic dispersive transport in a foam structure.

Secondary flow mixing due to biofilm growth in capillaries of varying dimensions.

Using a magnetic resonance microscopy (MRM) technique, velocity perturbations due to biofouling in capillaries were detected in 3D velocity maps. The velocity images in each of the three square capillary sizes (2, 0.9, and 0.5 mm i.d.) tested indicate secondary flow in both the x- and y-directions for the biofouled capillaries. Similar flow maps generated in a clean square capillary show only an axial component. Investigation of these secondary flows and their geometric and dynamic similarity is the focus of this study. The results showed significant secondary flows present in the 0.9 mm i.d. capillary, on the scale of 20% of the bulk fluid flow. Since this is the "standard 1 mm" size capillary used in confocal microscopy laboratory bioreactors to investigate biofilm properties, it is important to understand how these enhanced flows impact bioreactor transport.

Probing diffusion dynamics during hydrate formation by high field NMR relaxometry and diffusometry.

High-field nuclear magnetic resonance (NMR) relaxometry and diffusometry along with magnetic resonance imaging were used to monitor phase transition molecular dynamics during hydrate formation occurring in water droplets dispersed in liquid cyclopentane. 1D T2 relaxation measurements indicate the extent of hydrate formation as well as a reduction in water droplet size with progression of hydrate growth. MRI intensity maps and T2 relaxation maps indicate spatially dependent hydrate formation rates due to the heterogeneity of the system. Spectrally resolved diffusion measurements indicate a reduction in the porosity of the hydrate agglomerate as the hydrate shell increases in thickness. A novel signal rise observed in two dimensional T1-T2 relaxation correlation experiments indicates complex diffusion dynamics due to coupling between regions with varying relaxation and diffusion. These results indicate the ability to monitor hydrate growth and phase transition molecular dynamics due to evolution of the porous hydrate agglomerate by means of high-field NMR.

A two-region transport model for interpreting T1-T2 measurements in complex systems.

A 1D two region coupled pore model with discrete pore coupling is developed to elucidate the eigenmode interactions in regions with different surface relaxivity. Numerical solution of the model and simulation of the correlation experiment for varying surface relaxivity, pore connectivity and pore size ratio indicate the role of negative eigenmodes and overlap of T1 and T2 eigenmodes in generating a time domain signal increase with inversion recovery time, t1. The eigenmodes and eigenfunctions are considered in detail providing connection between the mathematical model and the diffusion dynamics and spin physics of the system. Physical systems, i.e. a microporous glass bead pack, a cyclopentane/water hydrate former, and beeswax, showing experimentally measured T1-T2 time domain signal rise are considered within the limitations of the model.

Biopolymer and water dynamics in microbial biofilm extracellular polymeric substance.

Nuclear magnetic resonance (NMR) is a noninvasive and nondestructive tool able to access several observable quantities in biofilms such as chemical composition, diffusion, and macroscale structure and transport. Pulsed gradient spin echo (PGSE) NMR techniques were used to measure spectrally resolved biomacromolecular diffusion in biofilm biomass, extending previous research on spectrally resolved diffusion in biofilms. The dominant free water signal was nulled using an inversion recovery modification of the traditional PGSE technique in which the signal from free water is minimized in order to view the spectra of components such as the rotationally mobile carbohydrates, DNA, and proteins. Diffusion data for the major constituents obtained from each of these spectral peaks demonstrate that the biomass of the biofilm contains both a fast and slow diffusion component. The dependence of diffusion on antimicrobial and environmental challenges suggests the polymer molecular dynamics measured by NMR are a sensitive indicator of biofilm function.

Flow velocity maps measured by nuclear magnetic resonance in medical intravenous catheter needleless connectors.

This work explains the motivation, advantages, and novel approach of using velocity magnetic resonance imaging (MRI) for studying the hydrodynamics in a complicated structural biomedical device such as an intravenous catheter needleless connector (NC). MRI was applied as a non-invasive and non-destructive technique to evaluate the fluid dynamics associated with various internal designs of the NC. Spatial velocity maps of fluid flow at specific locations within these medical devices were acquired. Dynamic MRI is demonstrated as an effective method to quantify flow patterns and fluid dynamic dependence on structural features of NCs. These spatial velocity maps could be used as a basis for groundtruthing computational fluid dynamics (CFD) methods that could impact the design of NCs.