Non-invasive mass transfer measurements in complex biofilm-coated structures

We demonstrate a novel application of (13)C pulsed field gradient (PFG) NMR to monitor mass transfer, due to both flow and diffusion, in a 3D complex porous support structure modified by biofilm growth. This enables timescales an order of magnitude larger than previously possible to be accessed with respect to displacement probability distribution (propagator) measurements. The evolution in the propagator shape with observation time to the Gaussian asymptote (constant dispersion coefficient) is consequently well resolved. We also simulated the measured displacement propagators with good agreement between experiment and prediction. The methodology has significant potential for the selective characterization of the transport of nutrients, metabolic products, pollutants and biocides in such complex biofilm-containing structures.     

Mathematical modelling of biofilm structures

The morphology of biofilms received much attention in the last years. Several concepts to explain the development of biofilm structures have been proposed. We believe that biofilm structure formation depends on physical as well as general and specific biological factors. The physical factors (e.g. governing substrate transport) as well as general biological factors such as growth yield and substrate conversion rates are the basic factors governing structure formation. Specific strain dependent factors will modify these, giving a further variation between different biofilm systems. Biofilm formation seems to be primarily dependent on the interaction between mass transport and conversion processes. When a biofilm is strongly diffusion limited it will tend to become a heterogeneous and porous structure. When the conversion is the rate-limiting step, the biofilm will tend to become homogenous and compact. On top of these two processes, detachment processes play a significant role. In systems with a high detachment (or shear) force, detachment will be in the form of erosion, giving smoother biofilms. Systems with a low detachment force tend to give a more porous biofilm and detachment occurs mainly by sloughing. Biofilm structure results from the interplay between these interactions (mass transfer, conversion rates, detachment forces) making it difficult to study systems taking only one of these factors into account. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interaction of ferulic acid derivatives with human erythrocytes monitored by pulse field gradient NMR diffusion and NMR relaxation studies

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.     

Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR

We have previously shown that ¹H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile method for monitoring proteinself-association and can be used, albeit with some caveats, to measure theapparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol.NMR, 6, 321–328]. In this paper we show that, for¹⁵N-labelled proteins, selection of¹H-¹⁵N multiple-quantum (MQ) coherences in PFGdiffusion experiments provides several advantages over monitoring¹H single-quantum (SQ) magnetization. First, the use of agradient-selected MQ filter provides a convenient means of suppressingresonances from both the solvent and unlabelled solutes. Second,¹H-¹⁵N zero-quantum coherence dephases morerapidly than ¹H SQ coherence under the influence of a PFG.This allows the diffusion coefficients of larger proteins to be measuredmore readily. Alternatively, the gradient length and/or the diffusion delaymay be decreased, thereby reducing signal losses from relaxation. In orderto extend the size of macromolecules to which these experiments can beapplied, we have developed a new MQ PFG diffusion experiment in which themagnetization is stored as longitudinal two-spin order for most of thediffusion period, thus minimizing sensitivity losses due to transverserelaxation and J-coupling evolution.     

An experimental study of mass diffusion and reaction rate in an anaerobic biofilm

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 mum was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 mum. Deviation from linearity appeared in biofilm thicknesses greater than 800 mum. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.     

Analysis of peptide rotational diffusion by homonuclear NMR

The analysis of the rotational diffusion of a molecule using homonuclear NMR is investigated. The homonuclear longitudinal and transverse cross-relaxation rates, which can be quantitatively measured using off-Resonance Rotating frame nuclear Overhauser Effect Spectroscopy (ROESY), are used to build a distribution, which exhibits a solid-state-like pattern characteristic of the diffusion tensor. The distributions of the antimicrobial peptide ranalexin in water and in 30% of trifluoracetic acid (TFE) are compared, and the peptide rotational diffusion is shown to be more isotropic in water than in 30% TFE. This difference is further supported by the analysis of NMR ranalexin conformers in 30% TFE, and by the analysis of a molecular dynamics simulation of peptide in water.     

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (ΔH) with the value of ? 4.03 kcal mol^?1, while it is opposed by the entropic term (?TΔS) by approximately 1.28 kcal mol^?1 with a small negative change in heat capacity (ΔC_p). The enthalpy‐driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

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. Biotechnol. Bioeng. 2009;103: 353–360. © 2009 Wiley Periodicals, Inc.     

New insights into the interaction of proteins and disaccharides—The effect of pH and concentration

To gain new insights into the interaction of proteins and disaccharides, we investigated the hydrodynamic radii, , of lysozyme molecules in solution and in a ternary protein‐sugar‐water system by PFG‐NMR. Our approach is based on the assumption that the anhydrobiotic properties of disaccharides like trehalose are based on aggregation of sugar molecules to the proteins, i.e., accumulation of sugar molecules close to the protein, and that this process can be investigated by the experimentally detectable value of the protein. The R_h values are calculated from the experimentally determined diffusion coefficients and the application of a viscosity correction using the inert molecule dioxane as an internal viscosity reference. The experiments were performed as a function of sugar concentration, the overall particle concentration and the pH value. We investigated the disaccharides trehalose and sucrose, mainly for the reason that trehalose has well know cryptobiotic properties while sucrose, which is similar in size and structure, lacks these properties. The results show the formation of a protective sugar shell around the proteins over a wider range of concentrations and pH values in the case of trehalose.     

Flux balancing of light and nutrients in a biofilm photobioreactor for maximizing photosynthetic productivity

This article reports a combined experimental and numerical study on the efficient operation of Porous Substrate Bioreactors. A comprehensive model integrating light transport, mass transport, and algal growth kinetics was used to understand the productivity of photosynthetic biofilms in response to delivery rates of photons and nutrients. The reactor under consideration was an evaporation driven Porous Substrate Bioreactor (PSBR) cultivating the cyanobacteria Anabaena variabilis as a biofilm on a porous substrate which delivers water and nutrients to the cells. In an unoptimized experimental case, this reactor was operated with a photosynthetic efficiency of 2.3%, competitive with conventional photobioreactors. Moreover, through a scaling analysis, the location at which the phosphate delivery rate decreased the growth rate to half of its uninhibited value was predicted as a function of microorganism and bioreactor properties. The numerical model along with the flux balancing techniques presented herein can serve as tools for designing and selecting operating parameters of biofilm based cultivation systems for maximum productivity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:348–359, 2014     

Magnetic resonance microscopy analysis of advective transport in a biofilm reactor

In this article we present magnetic resonance microscopy (MRM) characterization of the advective transport in a biofilm capillary reactor. The biofilm generates non-axial flows that are up to 20% of the maximum axial velocity. The presence of secondary velocities of this magnitude alters the mass transport in the bioreactor relative to non-biofilm fouled reactors and questions the applicability of empirical mass transfer coefficient approaches. The data are discussed in the context of simulations and models of biofilm transport and conceptual aspects of transport modeling in complex flows are also discussed. The variation in the residence time distribution due to biofilm growth is calculated from the measured propagator of the motion. Dynamical systems methods applied to model fluid mixing in complex flows are indicated as a template for extending mass transport theory to quantitatively incorporate microscale data on the advection field into macroscale mass transfer models.     

Tracer measurements reveal experimental evidence of biofilm consolidation

The ability to simultaneously measure both biofilm thickness and the mass transfer coefficient of an inert tracer through it provides a powerful method to study biofilm development. In this communication previously published data has been collated to interpret global trends in biofilm structure during the transition towards steady-state. It appears that sudden changes in biofilm structure (directly related to the rate of change of biofilm mass transfer resistance) may occur following transitions in rate of biomass production. These observations are consistent with the concept of consolidation, recently introduced into spatially structured biofilm mathematical models to account for structural realignment of the biofilm under dynamic conditions.     

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Potentials of radio-frequency field gradient NMR microscopy in environmental science

An understanding of transport, flow, diffusivity and mass transfer processes is of central importance in many fields of environmental biotechnology such as biofilm, bioreactor and membrane engineering, soil and groundwater bioremediation, and wastewater treatment. Owing to its remarkable sensitivity to molecular displacements and to its noninvasive and nondestructive character, pulsed field gradient (PFG) nuclear magnetic resonance (NMR) can be a valuable tool for investigating such processes. In conventional NMR microscopy, spatial encoding is achieved by using static magnetic field gradients (B ₀ gradients). However, an interesting alternative is to use radio-frequency magnetic field gradients (RF or B ₁ gradients). Although the latter are less versatile than the former, RF field gradient microscopy is particularly suitable for dealing with heterogeneous systems such as porous media because of its quasi-immunity to background static magnetic field gradients arising from magnetic susceptibility inhomogeneities, unlike the B ₀ gradients microscopy. Here, we present an overview of basic principles and the main features of this technique, which is still relatively unused. Different examples of diffusion imaging illustrate the potentialities of the method in both micro-imaging and the measurement of global or local diffusion coefficients within membranes and at liquid–solid interfaces. These examples suggest that a number of environmental problems could benefit from this technique. Different future prospects of application of B ₁ gradient NMR microscopy in environmental biotechnology are considered. Journal of Industrial Microbiology & Biotechnology (2001) 26, 53–61. Received 09 February 2000/ Accepted in revised form 07 August 2000     

Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements

Defining the self-association state of a molecule in solution can be an important step in NMR-based structure determination. This is particularly true of peptides, where there can be a relatively small number of long-range interactions and misinterpretation of an intermolecular NOE as an intramolecular contact can have a dramatic influence on the final calculated structure. In this paper, we have investigated the use of translational self-diffusion coefficient measurements to detect self-association in aqueous trifluoroethanol of three peptides which are analogues of the C-terminal region of human neuropeptide Y. Experimentally measured diffusion coefficients were extrapolated to D⁰, the limiting value as the peptide concentration approaches zero, and then converted to D_20,w, the diffusion coefficient after correction for temperature and the viscosity of the solvent. A decrease in D_20,w of about 16% was found for all three peptides in aqueous TFE (30% by volume) compared with water, which is in reasonable agreement with the expected decrease upon dimerisation, the presence of which was indicated by sedimentation equilibrium measurements. Apparent molecular masses of these peptides in both solutions were also calculated from their diffusion coefficients and similar results were obtained. Several potential internal standards, including acetone, acetonitrile, dimethylsulfoxide and dioxane, were assessed as monitors of solution viscosity over a range of trifluoroethanol concentrations. Compared with independent measurements of viscosity, acetonitrile was the most accurate standard among these four. The practical limitations of a quantitative assessment of peptide self-association from translational diffusion coefficients measured by PFGNMR, including the calculation of apparent molecular mass, are also discussed.     

Pore‐network modeling of biofilm evolution in porous media

The influence of bacterial biomass on hydraulic properties of porous media (bioclogging) has been explored as a viable means for optimizing subsurface bioremediation and microbial enhanced oil recovery. In this study, we present a pore network simulator for modeling biofilm evolution in porous media including hydrodynamics and nutrient transport based on coupling of advection transport with Fickian diffusion and a reaction term to account for nutrient consumption. Biofilm has non‐zero permeability permitting liquid flow and transport through the biofilm itself. To handle simultaneous mass transfer in both liquid and biofilm in a pore element, a dual‐diffusion mass transfer model is introduced. The influence of nutrient limitation on predicted results is explored. Nutrient concentration in the network is affected by diffusion coefficient for nutrient transfer across biofilm (compared to water/water diffusion coefficient) under advection dominated transport, represented by mass transport Péclet number >1. The model correctly predicts a dependence of rate of biomass accumulation on inlet concentration. Poor network connectivity shows a significantly large reduction of permeability, for a small biomass pore volume. Biotechnol. Bioeng. 2011;108: 2413–2423. © 2011 Wiley Periodicals, Inc.     

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. Biotechnol. Bioeng. 2013; 110: 1366–1375. © 2012 Wiley Periodicals, Inc.     

TASSER-based refinement of NMR structures

The TASSER structure prediction algorithm is employed to investigate whether NMR structures can be moved closer to their corresponding X-ray counterparts by automatic refinement procedures. The benchmark protein dataset includes 61 nonhomologous proteins whose structures have been determined by both NMR and X-ray experiments. Interestingly, by starting from NMR structures, the majority (79%) of TASSER refined models show a structural shift toward their X-ray structures. On average, the TASSER refined models have a root-mean-square-deviation (RMSD) from the X-ray structure of 1.785 A (1.556 A) over the entire chain (aligned region), while the average RMSD between NMR and X-ray structures (RMSD(NMR_X-ray)) is 2.080 A (1.731 A). For all proteins having a RMSD(NMR_X-ray) >2 A, the TASSER refined structures show consistent improvement. However, for the 34 proteins with a RMSD(NMR_X-ray) <2 A, there are only 21 cases (60%) where the TASSER model is closer to the X-ray structure than NMR, which may be due to the inherent resolution of TASSER. We also compare the TASSER models with 12 NMR models in the RECOORD database that have been recalculated recently by Nederveen et al. from original NMR restraints using the newest molecular dynamics tools. In 8 of 12 cases, TASSER models show a smaller RMSD to X-ray structures; in 3 of 12 cases, where RMSD(NMR_X-ray) <1 A, RECOORD does better than TASSER. These results suggest that TASSER can be a useful tool to improve the quality of NMR structures.