Kinetics studies in a continuous steady state hollow fiber membrane enzyme reactor

Porous hollow cellulose fibers have been used to separate a nonflowing enzyme solution of alkaline phosphatase from a continuous flow of substrate. The porosity of the hollow fiber membrane allows the substrate and product to diffuse freely through the membrane while restricting the permeation of the enzyme. The resulting “immobilized” enzyme system has been shown to behave as a continuous reactor—converting p-nitrophenylphosphate to p-nitrophenol. By varying the concentrations, flow rate, etc., either diffusion or enzyme kinetics can be studied. The continual influx of product and removal of substrate at steady state allows the study of kinetics of relatively short half-life enzymes and unstable systems.     

Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.     

Kinetics of a hollow fiber dehydrogenase reactor

A hollow fiber module was used as a reactor for conversion of ethanol to acetaldehyde in the presence of horse liver alcohol dehydrogenase as catalyst. Mass transport rates for NAD⁺, the overall acetaldehyde generation rate, catalyst effectiveness factors, and the overall order of the reaction with respect to NAD⁺ concentration were measured. A coupled-substrate reactor with continuous in situ regeneration of cofactor was also examined. Two substrates of opposite redox state were added simultaneously to the feed stream. NADH and acetaldehyde concentrations were monitored in the effluent stream. The cofactor recycle number, or ratio of moles of product to moles of NADH produced, exceeded 10,000 under certain conditions. While decreasing the NAD⁺ concentration in the feed stream decreased reactor productivity somewhat, it greatly enhanced the ratio of product formed per mole of NAD⁺ fed to the reactor. It is suggested that high cofactor costs in dehydrogenase reactors may be overcome with efficient in situ regeneration and secondary recovery and recycling of cofactor from the process stream.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Urocanic acid production using whole cells immobilized in a hollow fiber reactor

The feasibility of using hollow fiber membrane dialyzers (C-DAK) for immobilization of microbial whole cells was investigated. The cells are located on the shell side of the dialyzer, while substrates and products are free to diffuse across the hollow fiber membranes. The biochemical reaction studied was the conversion of L -histidine to urocanic acid and catalyzed by L -histidine ammonia-lyase. C-DAK dialyzers containing a heat-treated suspension of Pseudomonas fluorescens ATCC 11299b (with L -histidine ammonia–lyase activity) were incorporated into constant volume recycle reactor systems for continuous product formation. A simple model successfully correlated the data and predicted performance. It was found that the reaction was not likely to be diffusion limited, and such a cell immobilization scheme is convenient and workable for continuous production of biochemicals.     

Cross flow filtration of RNA extracts by hollow fiber membrane

The feasibility of using ultrafiltration to concentrate RNA was investigated. Ultrafiltration flux and solute retentivity were affected by membrane cut-off and inlet pressure. The RNA can be concentrated by selecting a hydraulically permeable membrane (PM 30) and then operating the system at a sufficiently high pressure (2.07 · 10⁵ Nm⁻²) and a high recirculation rate (150 cm⁻¹). The use of this procedure was limited to a high-flux regime. However, a very high retentivity of macrosolute was achieved. The effect of transmembrane pressure on the apparent mass transfer coefficient in the “gel polarization model” was discussed and related to ultrafiltration flux.     

Improving the continuous production of lipolyzed butteroil with pregastric esterases immobilized in a hollow fiber reactor

Summary Kid and calf pregastric esterases were semi-purified by micro- and ultrafiltration of a crude tissue preparation. When the resulting solutions were utilized to immobilize these enzymes in a polypropylene hollow fiber reactor, the activities obtained when both techniques were employed were greater than those observed when only microfiltration was performed.     

Kinetics of CH4 production from H2 and CO2 in a hollow fiber reactor by plug flow reaction model

For microbial production of CH₄ from H₂ and CO₂, a hollow fiber reactor had been developed to increase an interfacial area between liquid and gas phases. The CH₄ production with the hollow fiber reactor was analyzed by applying a plug flow reaction model of a tubular reactor. It was possible to apply the model to the reaction of CH₄ production. The relationships between influent gas velocity, length of reactor and reaction yield were simulated by the reaction model. The plug flow reaction model was useful to design a hollow fiber bioreactor for the biomethanation of H₂ and CO₂.     

Novel reactor for aqueous-organic two-phase biocatalysis: A packed bed hollow fiber membrane bioreactor

Summary Hollow fiber membranes were potted in a tubular shell with a particulate, microporous, enzyme bearing support packed in the shell space. A bicontinuous system was thus formed with the reactants, supplied through the shell and the fiber lumen, forming an interface at the surface of the particles. Acid production rates, without any reactor optimization, up to four times greater than with membrane reactors were obtained during the lipase catalyzed hydrolysis of ethyl laurate and olive oil.     

Analysis of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities

Mathematical analyses of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities are presented. An analytical solution to the mathematical model with a first order substrate consumption rate is used to show that the procedure of Webster and Shuler(1) is incorrect. A rigorous analysis that requires numerical solution is also outlined for any form of the substrate consumption rate. These analyses allow for more accurate estimations of effective substrate diffusivities since they should be used in conjunction with integral reactor behavior.     

Iontophoretic release and transport of alkaloids fromCatharanthus roseus cells in a ceramic hollow fiber reactor

Summary An iontophoretic device with a configuration similar to that of a single hollow fiber reactor was found to enhance the release and transport of intracellular alkaloids fromCatharanthus roseus cells. As the applied current increased from 1 to 2 milliamperes, the rate of release and transport of alkaloids almost doubled. Pretreatment of the cells with DMSO further enhanced the production.     

A forced flow membrane enzyme reactor for sucrose inversion using molasses

We have developed a bioreactor which uses enzyme immobilized within a ceramic membrane support (1 mm thickness). Substrate is forced through the membrane by cross-flow filtration with the reaction taking place during the process of crossing the membrane. The bioreactor is termed forced-flow membrane enzyme reactor, FFMER. Invertase, which uses sucrose to form glucose and fructose, was tested in this system. The immobilized invertase membrane converted 100% of the sucrose in a feed stream made up of a 50% molasses solution. Because molasses contains many substances besides sucrose, this method is applicable to processes using substrates present in impure feeds.     

Dual hollow fiber membrane bioreactor for whole cell enzyme immobilization of Streptomyces griseus with glucose isomerase activity

A new process using the dual hollow fiber bioreactor (DHFBR) system for whole cell enzyme immobilization was developed. This method allows Streptomyces griseus with glucose isomerase activity to proliferate in DHFBR to a desired density and convert glucose to fructose with high productivity. After 6 days the dry cell mass amounted to 140 g/l based on the space volume available for cell growth. The volumetric productivity of fructose by DHFBR was 22.5 g/l·h (based on 34% glucose conversion and the inner silicone tube volume), which correspond to a 12-fold increase over that of the batch method (1.8 g/l·h, based on 44% glucose conversion).     

Chitosan hollow fiber membranes

Chitosan hollow fibers were produced by wet spinning, taking advantage of the unique rheological properties of highly viscous chitosan solutions in acetic acid. The mechanical and separation properties of hollow fibers were tested. The mechanical properties were determined by measuring tensile force, tensile stress, elongation, and initial elasticity module. The separation properties were specified by determining retention coefficients of particular blood components and determining cut-off of the membrane by the analysis of dextran molecular weight distribution in the feed and permeate using a technique of gel chromatography (GPC)-Shimadzu gel chromatograph.     

Microbial hollow fiber bioreactors

Hollow fiber membranes have been used for more than a decade to culture mammalian cells and immobilize enzymes. More recently, hollow fiber bioreactors have shown encouraging potential for culturing microbes but many of the practical aspects of their operation have not been explored.     

A radial flow hollow fiber bioreactor for the large-scale culture of mammalian cells

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization of fiber surface for cell growth while eliminating nutrient and metabolic gradients inherent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube surrounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and CO(2) is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 1150 cm(2) was developed and H1 cells were grown to a density of 7.3 x 10(6) cells/cm(2).