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.     

Evaluation of intrinsic immobilized kinetic parameters for factor X activation in a flow reactor

Summary Development of a double exponential model for determining the intrinsic kinetic parameters for factor X activation by tissue factor-factor VIIa (TF:VIIa) complex, during the complete course of the reaction in a flow reactor is described. The model data reveal that the factor X activation rate constant K₁ gradually increased from 0.0225 to 0.0456 min^-1 as the shear rate increased from 50 to 3000 sec^-1, whereas the factor Xa inhibition rate constant K₂ increased dramatically from 0.307 to 1.09 min^-1 for a similar increase in shear rate.     

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 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.     

Production of lipolyzed butteroil by a calf pregastric esterase immobilized in a hollow fiber reactor: III. Effect of glycerol

Lipolysis of butter oil in a hollow fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C, a pH of 6.0, and glycerol concentrations of 0, 150, and 500 g/L in the buffer solution. The concentrations of 10 fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography. The rate of loss of enzyme activity and the relative selectivities of this esterase depended on the glycerol concentration. By contrast, the overall rate of release of fatty acids was not affected by the glycerol concentration. Loss of enzyme activity was modeled using first-order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The model was successful in describing the rates of release of all 10 fatty acid species for a range of space times from 0 to 25 h. The parameters of the model were tested for dependence on glycerol concentration.     

Evidence of oxygen limitation in a hollow fibre reactor with immobilized E. coli

Summary A hollow fibre reactor (HFR) has been used to immobilize a regulatory mutant ofE. coli to produce phenylalanine. Fermentation performance in the HFR using both air and pure oxygen shows evidence of significant oxygen limitation. In particular, levels of volatile fatty acids (VFA) produced served as an indication of oxygen limitation.     

Recycle hollow fiber enzyme reactor with flow swing

The hollow fiber enzyme reactor with pulsation developed by Kim and Chang (1983) was operated in a differential mode by recycling a substrate solution, in order to assess the efficiency of ultrafiltration swing. The rates of lactose conversion by beta-galactosidase contained in the shell side of the reactor were measured to determine the effects of recirculation rate, pulsation period, and amplitude. The conversion increased with the increase of recirculation flow rate and the amplitude while variation in period affected the conversion relatively little. The maximum increase of 113% in the activity was observed in the reactor with pulsation as compared to that without pulsation. The two-compartment model well described the experimental data obtained in this study. Square-wave pulsation was theoretically more effective in increasing conversion than sine wave pulsation. However, in experimental operation the damping effect of the hollow fiber wall narrowed the difference between these two wave forms.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Continuous production of acrylamide byBrevibacterium sp. immobilized in a dual hollow fiber bioreactor

Summary Acrylamide was continuously produced from acrylonitrile usingBrevibacterium sp. CHl grown and immobilized in a dual hollow fiber bioreactor of 8.0 cm³. The biomass reached as high as 200 gm/L of the space available for the cell growth. The volumetric productivity of the reactor was 88 gm/L. h and the conversion of acrylonitrile varied with acrylonitrile concentration, pH and feed rate.     

Operation and pressure distribution of immobilized cell hollow fiber bioreactors

It has been cited in the literature on hollow fiber systems that pressure gradients persist, and the transmembrane flux of the hollow fiber system is dependent on the pattern of the pressure gradients. The pattern can be used to its advantage in immobilized enzyme systems. However, with immobilized living cell systems, the pressure gradients lead to a nonuniform environment within the hollow fiber cartridge and not necessarily favorable results. This article provides pertinent pressure-drop data on hollow fiber cartridges which are in flow configurations typical of immobilized cell culture work. The results illuminate operational problems that may arise in the culture of either anchorage dependent or independent cells. Possible solutions with crossflow systems are suggested.     

Consecutive immobilized enzymatic reactions in continuous stirred tank reactor systems

The performance characteristics of two-enzyme reaction in a continuous stirred tank reactor (CSTR) are analytical investigated in this work. A model is formulated to describe the substrate concentration variations by taking into account the external and internal diffusion resistances. It is found that the reaction system exhibits the characteristics of reaction control or diffusion control depending on the operating conditions. The single CSTR model is also extended to describe the multiple CSTR system. The latter model enables the prediction of the number of CSTRs in series required to achieve a prescribed substrate conversion.     

Design and performance study of a novel immobilized hollow fiber membrane bioreactor

A dual-layer coaxial hollow fiber (DLHF) bioreactor for cell immobilization developed to overcome nutrients transport limitation is presented. Cells were contained in the annular space between two coaxial hollow fibers, and nutrients were supplied by a forced convective transport from the shell side through the annular space to the lumen side. With judicious selection of the membrane materials, a low operating transmembrane pressure of 50 kPa, and using E. coli as the model organism, a high cell density of 10(11) cells/mL annular space volume and a high cell viability of (up to 80%) were obtained.     

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.     

Heparin removal from blood using poly(L-lysine) immobilized hollow fiber

Based on the negative charge density characteristics of heparin, an affinity adsorption technique has been developed for the removal of heparin from blood. Poly(L-lysine) . HBr (PLL . HBr), a polycation, was immobilized with the help of cyanogen bromide (BrCN) onto poly(ethylene-vinyl alcohol) (PEVAL) copolymer coated polyethylene (PE) hollow fibers. Heparin bound rapidly onto PLL . HBr imobilized surface in buffer, plasma, and blood. The heparin binding capacity of PLL immobilized surface increased sevenfold as compared to a non-PLL-treated control. When heparinized blood was recirculated through a PLL immobilized PEVAL hollow fiber cartridge, the anticoagulant activity of heparin decreased by 85% from initial activity in 25 min. Moreover, circulation of blood through PLL immobilized hollow fiber did not show any adverse effects; no hemolysis was observed and no significant loss of plasma proteins was noted during the heparin removal process. These results suggest that PLL immobilized surface may be utilized for rapid and effective removal of heparin from blood. (c) 1992 John Wiley & Sons, Inc.     

Biological conversion of rifamycin B by live Humicola sp. cells immobilized in a dual hollow fiber bioreactor

The biological transformation from rifamycin B to rifamycin S was carried out with the live whole cells of Humicola sp., ATCC 20620, immobilized in a dual hollow fiber bioreactor (DHFBR). Humicola sp., inoculated in the DHFBR, proliferated successfully to a high density cell mass within the space between an outer silicone tubing and three inner polypropylene hollow fiber membranes. In order to control the cell growth a nitrogen deficient medium was fed. Conversion of rifamycin B continued for more than 30 d, whereas that of immobilized rifamycin B oxidase lasted only for 3 d in comparable conditions.In the DHFBR the volumetric productivity of rifamycin S was 0.65–1.03 mmol/(dm³ · h) with 60% conversion, while that in the rotating packed disk reactor was 0.27 mmol/(dm³ · h) with 40% conversion at a residence time of 0.5–1.5 h.     

Studies on the kinetics of immobilized enzyme using a recycling enzyme reactor system

A method of measuring kinetic parameters of immobilized enzyme with a recycling enzyme reactor system is described. By analyzing the plot of the dimensionless variable Ln(1-X)/X versus the time needed for a unit conversion, t/X, the mechanism of enzymatic reaction can be recognized and then its basic parameters can be evaluated. On the basis of the experimental data measured by P.R. Coulet et al., it has been proposed that the successive degradation of maltodextrins by the collagen membrane-bound amyloglucosidase was a product glucose inhibition reaction and their corresponding constants have been found with this method.     

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized bed reactor

Degradation kinetics of phenol by free and agar-entrapped cells of Candida tropicalis was studied in batch cultures. The initial phenol degradation rate achieved with free cells was higher than that obtained with immobilized cells, when phenol concentrations up to 1000 mg l^–1 were used. However, at higher phenol concentrations, the behaviour was quite different. The initial degradation rate of the immobilized yeast cells was about 10 times higher than that of the free cells, at a phenol concentration of 3500 mg l^–1. The semicontinuous and continuous degradation of phenol by immobilized yeast cells was also investigated in a multi-stage fluidized bed reactor. The highest phenol removal efficiencies and degradation rates as well as the lowest values of residual phenol and chemical oxygen demand were obtained in the semicontinuous culture when phenol concentrations up to 1560 mg l^–1 were used.     

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.     

The design of stirred reactors with hollow fiber catalysts for Michaelis-Menten kinetics

A method of calculating the conversion in Rony's hollow fiber reactor is outlined. It, is assumed that, the kinetics are of Michaelis-Menten type and that diffusion within the hollow fiber, as well as through its wall, should be taken into account. The normalization of the Thiele modulus suffices to unify the treatment of internal diffusion and the pseudosteady state hypothesis is found to be valid under almost all conditions.