Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.     

Wax ester synthesis by lipase-catalyzed esterification with fungal cells immobilized on cellulose biomass support particles

Esterification reactions between long-chain alcohol and oleic acid were performed for producing wax esters. The reaction can be catalyzed efficiently by cell-bound lipase of Rhizopus niveous fungal cells immobilized within cellulose biomass support particles. Carrying out the reaction in a solvent-free system is feasible by adding a molecular sieve for dehydration purposes. To optimize the yield, addition of a molecular sieve should be performed gradually during the whole course starting from the beginning of the reaction. The influence of reaction conditions such as temperature and substrate concentrations on reaction rates and yields were investigated; however, this reaction system is under the influence of both internal and external mass transfer resistance. Conducting the reaction in an organic solvent system with hexane or heptane as the solvent can eliminate diffusional effects. Reaction kinetics were subjected to detailed study in this system. The kinetics of the reaction can be represented satisfactorily by a Ping-Pong Bi Bi mechanism with deadend inhibition by alcohol.     

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.     

Membrane adsorption characteristics determine the kinetics of flow-through transductions

Retrovirus-mediated gene transfer is currently limited by random Brownian motion of the retrovirus. This limitation can be overcome by flowing the retrovirus solution through a porous membrane that supports the target cells, leading to a significant increase in the transduction efficiency. This procedure is termed "flow-through transduction." In this study, we characterized the effects of the fluid flowrate and the influence that membrane characteristics have on the flow-through transduction procedure. The transduction efficiencies increased with flowrate until a plateau was reached at average flow velocities exceeding 0.3 cm/h for flow times of 3 to 4 h, using a collagen-coated depth (COL) membrane. A correlation between the optimal time for maximal gene transfer using flow-through transductions and the optimal time for maximal virus activity on the membrane was found, suggesting that the membrane adsorption capacity for virus determined the amount of gene transfer that could occur.Membrane adsorption characteristics were further investigated using two different membrane types: a tracketched polyester screen (PE) membrane and the COL membrane. Flow-through transductions using the PE and COL membranes showed that a high level of gene transfer could be attained using the COL membrane while the PE membrane gave much lower transduction efficiencies. The addition of the polycation polybrene (PB) changed these results markedly, making transductions achieved on the PE membrane similar in number to those obtained on the COL membrane. Since PB is believed to influence the virus adsorption to PE membrane, these results further support the conclusion that the increase in gene transfer achieved by the flow-through transduction procedure is due to virus adsorption to the membrane. The flow-through transduction procedure thus leads to co-localization of the viral vector and the target cell that in turn leads to a high transduction efficiency. (c) 1996 John Wiley & Sons, Inc.     

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.     

A combined phenolic extraction and fermentation reactor engineering model for multiphase red wine fermentation

Red wine production begins with a simultaneous fermentation and solid-phase extraction process. Red wine color and mouthfeel is the result of the extraction of phenolics from grape skins and seeds during fermentation, where extraction is a strong function of temperature and ethanol concentration. During fermentation, grape solids form a porous “cap” at the top of the fermentor, resulting in a heterogeneous fermentation system with significant temperature and concentration gradients. In this work, we present a spatial, time-variant reactor engineering model for phenolic extraction during red wine fermentation, incorporating fermentation kinetics, mass transfer, heat transfer, compressible fluid flow, and phenolic extraction kinetics. The temperature and ethanol concentration profiles predicted by this model allow for the calculation of phenolic extraction rates over the course of fermentation. Phenolic extraction predictions were validated against prior experimental data to good agreement and compared to a well-mixed model's predictions to show the utility of a spatial model over well-mixed models.     

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.     

Immobilized glucoamylase on porous glass

A study was made to determine the controlling mass transfer resistance in the overall reaction rate for conversion of maltose to glucose, catalyzed by glucoamylase immobilized onto porous glass. For normal operation of a packed column and air-stirred batch reactor, the rate controlling step was found to be the internal resistance of simultaneous pore diffusion and chemical reaction. Experimental effectiveness factors were determined and are compared with those derived from a theoretical diffusion model based on Michaelis-Menten kinetics. Also given are temperature and pH relationships for the free and immobilized glucoamylase.     

Displacement of equilibrium in electroenzymatic reactor for acetaldehyde production using yeast alcohol dehydrogenase

Electrochemical regeneration of NAD was performed in a bench scale reactor in which yeast alcohol dehydrogenase catalyzed the oxidation of ethanol. By recycling one of the products of the reaction, it was possible to displace the equilibrium and favor the production of acetaldehyde. The flow-through electrode was made of graphite felt and had a specific area of 275 cm(-1). A mathematical model taking into account the enzymatic and electrochemical reaction rates as well as the mass transfer to the electrode was used to analyze the results. The limiting steps in the reactor are the electrochemical reaction for low potentials and the cofactor mass transfer for high potentials.     

Enzyme immobilized in a packed-bed reactor: kinetic parameters and mass transfer effects.

To describe axial dispersion, particle film mass transfer, intraparticle diffusion, and the chemical reaction of the substrate for enzymes immobilized in porous particles in packed columns, we have developed mathematical models for first- and zero-order limits of Michaelis-Menten kinetics. Steady-state solutions were derived for both long and short column boundary conditions and for plug flow. Theory was compared to experiments by hydrolysis of sucrose catalyzed by invertase bound to porous glass particles. Steady-state conversions were measured for a range of flow rates. Pulse response experiments with inert packing were used to determine values of bed void fraction and particle porosity.     

Mass-transfer effects on the rate of isomerization of D-glucose into D-fructose, catalyzed by whole-cell immobilized glucose isomerase.

The investigated catalyst system consists of immobilized Arthrobacter cells containing the enzyme glucose isomerase, which catalyzes the isomerization of glucose into fructose. The internal structure of the catalyst was determined from electrom microscope photographs of replicas of freeze-etched catalyst. On the basis of the photographs a model for the internal structure of the catalyst was proposed. This structure was subsequently used to describe the reaction including mass-transfer effects. It appeared that under normal operating conditions the external mass-transfer rate does not influence the overall rate of reaction. The effect of internal mass-transfer resistances on the overall reaction rate can well be accounted for by the so-called porous sphere model. The intrinsic kinetics of the isomerization catalyzed by the present catalyst system can be represented by a modified Michaelis-Menten equation for a reversible one-substrate reaction.     

Oxygen supply to immobilized cells: 5. Theoretical calculations and experimental data for the oxidation of glycerol by immobilized Gluconobacter oxydans cells with oxygen or p-benzoquinone as electron acceptor

Theoretical calculations of reaction kinetics were done for one-step reactions catalyzed by cells immobilized in spherical beads. The reactions catalyzed by free cells were assumed to obey Michaelis-Menten kinetics for a one-substrate reaction. Both external (outside the beads) and internal (inside the beads) mass transfer of the substrate were considered for the immobilized preparations. The theoretical calculations were compared with experimental data for the oxidation of glycerol to dihydroxyacetone by Gluconobacter oxydans cells immobilized in calcium alginate gel. Glycerol was present in excess so that the reaction rate was limited by oxygen. The correlation between experimental data and theoretical calculations was quite good. The calculations showed how the overall effectiveness factor was influenced by, for example, the particle size and the cell density in the beads. In most cases the reaction rate was mainly limited by internal mass transfer of the substrate (oxygen). As shown previously, p-benzoquinone can replace oxygen as the electron acceptor in this reaction. The same equations for reaction kinetics and mass transfer were used with p-benzoquinone as the rate-limiting substrate. Parameters such as diffusivity, maximal reaction rate, and K were, of course, different. In this case also, the correlation between the model and the experimental results was quite good. Much higher production rates were obtained with p-benzoquinone as the electron acceptor compared to when oxygen was used. The reasons for this fact were that p-benzoquinone gave a higher maximal reaction rate for free cells and the solubility of p-benzoquinone was higher than for oxygen. Different methods of increasing the rate of microbial oxidation reactions are discussed.     

Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support.

Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.     

Investigation of behavior of an enzyme in a biphasic system: soybean lipoxygenase-1

Soybean lipoxygenase-1 (EC 1.13.11.12) reaction with linoleic acid as substrate was used to study the biocatalysis in a biphasic system when the reactants have surface-active properties. The poorly water-soluble substrate was initially dissolved in an apolar solvent (octane). The hydroperoxide produced was water soluble and remained in the aqueous phase (borate buffer). The bioreactor was a modified Lewis cell with a well-defined interfacial area between the two phases. Two phenomena were studied separately: the reactant transfer between the two phases and the biocatalyzed reaction in an aqueous medium. This allowed determination of the transfer and the reaction constants. Substrate transfer was found to be affected by the progress of the reaction, because linoleic acid and the hydroperoxy acid have an influence on the interfacial tension. Inactivation of the biocatalyst at the interface was observed in the bioreactor. These results indicate that it is impossible to analyze the system behavior with the method proposed in the literature, which is based on the sequential study of the substrate transfer to the aqueous phase and its biocatalysis by lipoxygenase. The interaction between transfer phenomena and reaction kinetics was studied in the biphasic system. The kinetics were different from those obtained in the aqueous medium. Catalysis and transfer influence each other reciprocally. In this compartmentalized system, cooperativity phenomena were obtained using a nonallosteric enzyme. The evolution of the system was modeled (Runge-Kutta algorithm). The curves obtained were very close to those determined experimentally.     

Influence of intraparticle diffusional limitation on the observed kinetics of immobilized enzymes and on catalyst design

Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes. These solutions have also been compared with experimental data for enzymes immobilized to DEAE-cellulose particles. The influence of particle size and enzyme content on catalyst design is discussed.     

Modeling multiphase fluid flow,mass transfer,and chemical reactions in bioreactors using large-eddy simulation

We present a transient large eddy simulation (LES) modeling approach for simulating the interlinked physics describing free surface hydrodynamics, multiphase mixing, reaction kinetics, and mass transport in bioreactor systems. Presented case-studies include non-reacting and reacting bioreactor systems, modeled through the inclusion of uniform reaction rates and more complex biochemical reactions described using Contois type kinetics. It is shown that the presence of reactions can result in a non-uniform spatially varying species concentration field, the magnitude and extent of which is directly related to the reaction rates and the underlying variations in the local volumetric mass transfer coefficient.     

Mass Transfer and Cholesterol Oxidase Kinetics in a Liquid-Liquid Two-Phase System

The kinetics of cholesterol oxidase and the effect of mass transfer in an aqueous-organic dispersion have been investigated. Cholesterol oxidase exhibits Michaelis-Menten behaviour in terms of substrate and enzyme concentrations. However, the observed rate of reaction depends on the nature and concentration of surfactants added, agitation rate, and organic phase composition. Analysis of the experimental data in terms of simultaneous mass transfer with reaction indicates that the system may operate in the slow reaction-diffusional regime.     

Model-based optimization of a conductive matrix enzyme electrode

A mathematical model has been developed to describe the mechanism for internal mass transfer and enzyme reaction kinetics of an amperometric conductive matrix enzyme electrode. The model is simplified and solved analytically to arrive at a representation for the response slope in the linear range as well as for the response time. This is the first time that the response time of an enzyme electrode is described by a mathematical model. Simulations give information on how the design parameters influence the performance of the electrode for a glucose oxidase catalyzed sensing reaction process. Based on this information, several designs were constructed and tested showing suitable agreement with theoretical predictions. Finally, an optimized electrode was designed and validated.