Wolf R. Vieth

Mass transfer and biochemical reaction in enzyme membrane reactor systems—I. Single enzyme reactions

Abstract The effective use of an enzyme as a biological catalyst for process scale conversion is greatly enhanced by immobilizing the enzyme within the matrix of a membrane whose walls are permeable to the substrate and product. The enzyme is thus stabilized for reuse, but the apparent activity is reduced compared to that obtained with the free enzyme because of transport resistances in the bulk fluid near the membrane and within the membrane itself. To account for this reduction, solutions to the differential equations describing the mass transport-kinetic models can be obtained for certain important cases. The reaction is considered to be first-order and isothermal, and to occur in one of two modes of operation: fixed-bed or batch (limited volume). Irreversible and reversible reactions and plane sheet and spherical geometries of the membrane are considered. An example of the application of these solutions to actual data obtained with a laboratory scale immobilized enzyme reactor is presented.

Characterization of glucose oxidase immobilized on collagen

SummaryThe enzyme glucose oxidase (E.C. 1.1.3.4) was immobilized on collagen — a proteinaceous material found in biological systems as a structural material for a wide variety of cells and membranes. The novel technique of electrocodeposition, which utilizes the principles of electrophoresis, was used to deposit the enzyme-collagen complex on stainless steel helical supports. This technique has been developed in our laboratory. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds and van der Waals interactions.As a first step toward examining its feasible technical use, the kinetic behavior of the collagen-supported glucose oxidase was studied in a batch recycle type reactor and was compared with that for the soluble form. A novel reactor configuration consisting of multiple concentric electrocodeposited helical coils was used. The reactor was found to attain a stable level of activity which was maintained for several months under cyclic testing. The optimum levels of pH and temperature for the immobilized form of the enzyme were the same as those of the soluble enzyme, but the immobilized enzyme was more active than the soluble form at higher temperatures and pH. The values of the Michaelis-Menten parameters indicate that the overall reaction rate of the immobilized enzyme may be partially restricted by bulk and matrix diffusion.

Chemical Engineering Analysis of Immobilized-Cell Systems

Publisher Summary This chapter reviews the chemical engineering analysis of immobilized cells. With the advent of immobilized-cell reactors mediating more complex biocatalytic processes, a thorough understanding of reactor dynamics and stability becomes even more important. The choice of reactor type for a particular process would depend on the process requirements and conditions. Much of the literature on the design and performance analysis of immobilized-cell reactors focuses on single-enzyme kinetics. Detailed chemical engineering analyses for such systems are at present well documented for a variety of reactor configurations. The analysis of an immobilized-whole-cell reactor would depend primarily on the type of immobilized-cell process to be used. When the cells are nonviable and only a single intracellular enzyme is of interest, it can be treated essentially as a bound-single-enzyme system. The analysis of such a system involves the application of enzyme kinetics to the appropriate reactor performance equations and the development of appropriate quantitative indices to account for external and internal mass-transfer resistances. Several reactor configurations are available for use for the immobilized-whole-cell processes.

Biophotolytic membranes: Simplified kinetic model of photosynthetic electron transport☆

Abstract The ability to isolate and stabilize thylakoid membranes from photosynthetic organisms (cyanobacteria, green algae, or higher plants) facilitates their study as photocatalysts in processes for the production of hydrogen from water using solar energy (biophotolysis). The feasibility of immobilizing photosynthetic membranes with retention of their electron transport capability was explored in this study. The method of immobilizing chloroplast thylakoid membranes which was observed to best preserve their photochemical activity involved the following steps: 1. (1) mixing an aqueous suspension of thylakoid membranes (isolated from Spinacia oleracea ) with an aqueous suspension of 1.5% (w/v) collagen; 2. (2) adding glutaraldehyde to a concentration of 0.01% (w/v) and cross-linking for 20 min at 0 °C; 3. (3) casting the collagen-thylakoid mixture on a flat surface; and 4. (4) freeze-drying the cast mixture to form a macroporous collagen-thylakoid film composite. Films prepared by this immobilization technique had a very open, sponge-like fibrous structure with a bulk density of 0.049 g/cm 3 of film. Photosynthethic electron transport activities of free and immobilized thylakoid membranes were determined by measuring the photoproduction of oxygen with a Clark-type polarographic probe using potassium ferricyanide as the electron acceptor (Hill reaction). A simplified kinetic model of the photosynthetic electron transport system (PETS) was formulated based on a reaction scheme which is consistent with current concepts of photosynthesis. The kinetic model relating electron transport rate (O 2 evolution) to incident light intensity is a rectangular hyperbolic function, in agreement with the observed behavior of both immobilized The photocatalytic properties of the collagen-thylakoid film were characterized with respect to electron transport kinetics. A simplified kinetic model of the photosynthetic electron transport system was formulated based on a reaction scheme which is consistent with the current concept of photosynthesis. The model accounts for the significant features of photosynthetic electron transport and yet has sufficient simplicity to be useful in the eprocess engineering of photosynbetic reactor systems. The dependence of initial reaction rates on incident light intensity predicted by the kinetic model was experimentally verified for both free and immobilized thylakoid membranes. Initial-rate data were used to evaluate the parameters of the kinetic model. Future kinetic studies might fruitfully focus on dissolved O 2 catalyst-poisoning effects, as well as photoinactivation and/or photoinhibition effects.

Synthesis of Organic Acids and Modification of Steroids by Immobilized Whole Microbial Cells

As part of our continued efforts to develop a technology based on collagen immobilized enzymes and whole microbial cells, we have investigated a number of reaction schemes catalyzed by collagen-whole cell complexes. We initiated our studies on immobilized whole cell systems with simple systems such as glucose isomerization which involve only a single enzymatic reaction. The encouraging results obtained in this case (production of high fructose syrup by immobilized Streptomyces venezuelae) (1,2) prompted us to examine other systems listed in Table 1.

Membrane-water structural interactions in reverse osmosis transport

Abstract Reverse-osmotic water permeabilities, equilibrium water sorption levels, and rates of approach to sorption equilibrium were measured for a series of polymers, including hydroxyethyl methacrylate (HEMA), copolymers of HEMA and ethyl methacrylate (EMA), cellulose acetate, cellulose nitrate, and poly(urethans). Pronounced equilibrium solvent clustering behavior was observed for these systems as vapor saturation was approached in sorption experiments. However, clustering tendency was not found to be a function of total membrane water content at saturation but rather appears to be a function of the chemical nature of the polymer in question. Moreover, clustering of water molecules in (relatively) hydrophobic membranes resulted in low effective diffusivities (reverse osmotic permeability divided by equilibrium water content) whereas clustering in hydrophilic membranes led to higher effective water diffusivities. Clustering tendency was not as strong in the case of the weakly interacting membranes (i.e....

Mass transfer and biochemical reaction in enzyme membrane reactor systems—II: Expanded analysis for single enzyme systems: effects of enzyme intermediates, denaturation and elution

Abstract A detailed study of the dynamics of a packed-bed reactor containing immobilized enzyme particles is presented. The analysis consists of (i) transient state behavior; (ii) models for interphase and interfacial mass transfer between fluid and solid phases and intraphase mass transfer for the solid phase; (iii) detailed reaction rate model for the Bodenstein intermediates; (iv) mass balances for substrates, Bodenstein intermediates, unoccupied enzyme active sites, and products; and (v) models for enzyme denaturation and elution. The general reactor model consists of a set of nonlinear, coupled, partial differential equations. Numerical solutions of the system equations were obtained, using the discrete-space, continuous-time method of lines and realistic parameter values. A generalized map of the range of validity of the Steady-State Hypothesis was established under conditions where multiple mass transfer gradients were present within the reactor. A detailed analysis of the computational errors was performed. It was conclusively shown that the computer simulation solutions obtained in the analyses were not disguised to any significant degree as a result of employing finite difference approximations to the spatial derivatives. It was shown that the level of “error” involved in invoking the Steady-State Hypothesis depends on the relative magnitude of the kinetic parameters and also on the level of “disturbance” at the reactor inlet (i.e. per cent change in substrate inlet concentration). The “error”, however, did appear to be strikingly insensitive to the magnitude of the resistances to mass transfer, as characterized by the Modified Sherwood Number. It was concluded that, given any complete set of kinetic parameters, a transient, heterogeneous, isothermal reactor model based on the Steady-State Hypothesis may be used for predicting time-varying concentration profiles for minor (i.e., less than 5 per cent change in substrate inlet concentration) “disturbances” at the reactor inlet. The corresponding “errors” would be at an acceptable level (i.e., less than 2 per cent in the concentration and less than 10 per cent in the time lag) under these conditions. Further, various mechanisms for enzyme denaturation and elution were incorporated in the general reactor model. Numerical solutions of the resulting system of partial differential equations were obtained, using hypothetical parameter values. Through extensive simulation research, it was shown that the loss in activity of immobilized enzyme reactors cannot be uniquely ascribed to any one particular set of mechanistic deactivation modes.

Design and Analysis of Immobilized-Enzyme Flow Reactors

Publisher Summary The overall efficiency of an enzyme reactor design would be determined by myriad factors. Although some general analyses and design procedures for enzyme reactors are known, the dearth of experimental information—particularly on a pilot-plant scale—renders the task of designing and scaling-up an enzyme reactor difficult. A designer, contemplating the design of a given enzymic reaction system, might be posed with several alternative approaches—none of them having a clear advantage or design precedence. If comparative information was available on different alternative design schemes, it would facilitate the choice of a particular system. This chapter presents the comparison between the relative efficiencies of several reactor configurations, based on research done in the laboratory on free and immobilized enzymes. The data was obtained under conditions that permit their meaningful comparison. The most important factors governing the overall reactor efficiency include the enzyme loading factor, carrier loading factor, operational stability of enzyme, external and internal diffusional efficiency, and residence time distribution.

Wet poly(vinyl chloride) membrane as a support: Sorption and transport of low-molecular-weight organic compounds and proteins

Abstract Characteristics of sorption and transport behavior of “wet” (PVC) membranes prepared by a poly(vinyl chloride) casting method were studied. It was found that wet PVC membranes adsorbed enzymes and proteins, whilst they did not adsorb low-molecular-weight compounds. The diffusivity of acetylcholine iodide as a penetrant through the wet PVC membrane was approximately 10 −7 cm 2 /sec, although it depended strongly on membrane thickness and slightly on PVC concentrations in the casting solution. Activation energy of acetylcholine iodide diffusivity was 2.6 kcal/mol. These results were explained by hydrophobic interaction and minute pore dimensions. Wet PVC membrane was suggested as a support for immobilization and was compared with collagen on the basis of transport behavior.

Design and Operating Strategies for Immobilized Living Cell Reactor Systems Part I. Biosynthesis of Candicidina

Several immobilized living cell processes for the production of biochemicals as a viable alternative to traditional “free cell” fermentations have been extensively reported.’” A majority of these reports are on ethanol fermentation. This process is a particularly good model system due to its anaerobic nature and simplicity of operation. However, more complex fermentations such as secondary metabolites production and a cell culture with higher organisms present unique problems and have not been fully addressed in the earlier research. There are only a few reports in the recent literature on antibiotic production by immobilized living cells.c6 In this paper, we examine some key features of secondary metabolite synthesis by immobilized living cells and suggest operating strategies to gain maximum productivity from such systems. Candicidin synthesis by immobilized Streptomyces griseus is chosen as a model for studying these processes. Secondary metabolite fermentations are characterized by the fact that significant product formation begins only after cell growth has slowed down or stopped. This suggests that by controlling the physiology of the cell, one might be able to optimize antibiotic productivity and also achieve significant economies of substrate consumption. In other words, it might be possible to divert the substrate consumption from cell growth to product formation, thus increasing the carbon conversion efficiency and overall productivity of the system. To this end, we have studied the carbon conversion efficiency of Streptomyces griseus under various growth conditions and shown that by proper administration of the nutrients, the substrate can be channeled into metabolite formation by suppressing growth. The growth versus product synthesis conditions can be effectively controlled by the phosphate level in the input medium.’