Whole-cell hollow-fiber reactor: Effectiveness factors

Analytical expressions, which allow the generation of effectiveness factor graphs for a reactor system employing immobilized whole cells a biocatalyst, are presented. In particular hollow-fiber devices (such as dialysis or ultrafiltration units) are considered. Such devices are analogs to a shell-and-tube heat exchanger. Whole cells are entrapped on the shell side: a nutrient solution is circulated through the tubes, substrate diffuses from the tube side, across the fiber, and into the cell mass on the shell side, where it irreversibly reacts to form product. The product back-diffuses into the circulating nutrient solution. The overall substrate mass-transfer process is hypothesized to be either diffusion limited in the hollow-fiber tube wall and/or the shell-side cell suspension and/or reaction limited at the enzyme sites within the whole cells. The first- and zero-order limits of the Michaelis-Menten rate law are used in generating effectiveness factor expressions. The effectiveness factor is a function of reaction order, Thiele modulus, diffusion coefficient ratio (defined as the effective substrate diffusivity in the hollow-fiber membrane wall divided by the effective substrate diffusivity in the cell suspension), partition coefficient, volume of the cell suspension, and hollow-fiber width. Equations for the effectiveness factor are also detailed when the hollow-fiber mass-transfer resistance is far greater than the cell suspension mass-transfer resistance. An effectiveness factor chart is presented specifically for the commercially available C-DAK 4 dialyzer (Cordis Dow Co., Miami, Florida). In general terms the effectiveness factor expressions are applicable for characterizing diffusion and reaction within a catalytically active cylindrical annulus, Whose inner surface offers a diffusional resistance and whose outer surface is impermeable to reactants. Some generalization of the Thiele modulus is undertaken which serves to draw the asymptotes on the effectiveness factor charts together. Comment is made on the variation of the slope of the effectiveness factor graph and its relation to the change in the observed reaction activation energy. Possible application of the model to the catalytic tube wall reactor is discussed.     

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).     

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.     

Performance of batch membrane reactor: Glycerol-3-phosphate synthesis coupled with adenosine triphosphate regeneration.

Glycerol-3-phosphate (G3P) was synthesized from glycerol using glycerol kinase (GK). This reaction requires adenosine triphosphate (ATP) and was coupled with the ATP regeneration reaction using acetate kinase (AK) in a batch-operated ultrafiltration hollow-fiber reactor. By taking into consideration the dynamic nature of the bioreactor performance under non-steady-state conditions, a model for the performance of a batch membrane reactor for G3P synthesis coupled with ATP regeneration was developed and studied. The simulation results showed good agreement with the experimental results. The simulation studies have provided some insight into the process dynamics of the coupled reactions in the reactor system studied. For the reactor operational model used, in which the enzymes are retained in the shell side and the substrates are also initially placed in the shell side, it was found that the substrate concentration in the lumen side increased to a level higher than that in the shell side, and a backdiffusion occurred from the lumen side to the shell side during reactor operation. The ratio of the reaction rate to diffusion rate goes through a sharp peak during the time that the direction of diffusion is reversed. For another reactor operational model, in which the substrates were initially placed in the lumen side and enzymes were retained in the shell side, it was found that the rate-controlling step between the reaction and diffusion was switched during the reactor operation. Initially, the reaction rate increased while the diffusion rate was high and the substrate concentrations increased in the shell side. The ratio of reaction rate to diffusion rate increased to a maximum and remained at a constant level as the diffusion rate decreased to a low level due to the nonlinear characteristics of mass transfer process. This study provides information that is useful for optimization of batch membrane enzyme reactor operation and for a fed-batch-type process with an intermittent feeding strategy for efficient use of substrates.     

Mathematical model of microporous hollow-fiber membrane extractive fermentor

A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize product inhibition.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

Invertase embedded-PVC tubing as a flow-through reactor aimed at conversion of sucrose into inverted sugar

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the inner surface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group on the surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highly reactive nitrene, which can insert to any C–H bond. CCl₄ lacking C–H bond is taken as a solvent for loading FNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction. Invertase is immobilized in the activated PVC tube at 50 °C in 45 min. Invertase embedded-PVC tube is used as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor converted sucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycles with 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier of biomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can be used for any enzymatic conversion in a flow-through system.     

Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis

The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde. Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general.     

Invertase embedded-PVC tubing as a flow-through reactor aimed at conversion of sucrose into inverted sugar

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the inner surface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group on the surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highly reactive nitrene, which can insert to any C–H bond. CCl₄ lacking C–H bond is taken as a solvent for loading FNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction. Invertase is immobilized in the activated PVC tube at 50 °C in 45 min. Invertase embedded-PVC tube is used as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor converted sucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycles with 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier of biomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can be used for any enzymatic conversion in a flow-through system.     

Hollow-fibre fermenter using ultrafiltration

Summary A novel bioreactor with hollow fibres was developed to facilitate substrate transfer across membrane walls as well as to retain a continuous cell growth in the shell side. Ultrafiltration was induced through membrane by pressurizing feed solution to the inside of a hollow fibre with inlet and outlet pumps. The ultrafiltrate accumulated outside the hollow fibres was recirculated through a reservoir where a part of solution containing cells and substrate was removed to keep the level of reservoir solution constant. Ethanol production by Saccharomyces cerevisiae was carried out to test the feasibility of this reactor. The productivity of this reactor was compared with that of a continuous stirred tank reactor (CSTR).     

A New Method for Carbon Addition in an Anoxic Denitrification Bioreactor

A new method for carbon addition was developed in a batch denitrification system by feeding methanol through a silicon tube. The methanol then diffuses across the membrane to the other side where the biofilm is formed. The results show that the residual COD could be controlled to less than 50 mg/L during the denitrification period with denitrification rates higher than 4,500 mg NO₃ ^--N/M²·d. Once the denitrification is completed, the COD breakthrough occurs. The advantages and disadvantages of this system are discussed.     

A kinetic model for the activated sludge process which considers diffusion and reaction in the microbial floc

A mathematical model has been developed which describes substrate removal, oxygen utilization, and biomass production in an aggregated microbial suspension containing the substrate as a soluble biodegradable material and a uniform floc size. It is applicable to both steady-state and transient conditions. The model, consisting of three partial differential equations and two ordinary differential equations, takes into account the flow pattern in the reactor, intraparticle mass transport of oxygen and substrate, and biochemical reaction by individual cells embedded in the floc. Efficient numerical solution of the coupled nonlinear equations is obtained using an implicit finite difference approach for both the reactor and floc equations. A convergent solution is realized through block interation utilizing the tridiagonal algorithm. Results indicate that a unifying theory of activated sludge dynamics will have to consider coupling between floc chemical kinetics and changes in the bulk liquid characteristics. Floc size emerges as an important influence on system performance. It appears necessary to distinguish between a system response caused by diffuslonal resistances and nutrient limitations within the floc and a response caused by physiological adaption when analyzing the transient behavior of an activated sludge process. Future research should be devoted to rigorous laboratory determinations of model parameters along with extensions to include limitations of nutrients other than orgabnic carbon and oxygen.     

Prediction of optimal biofilm thickness for membrane-attached biofilms growing in an extractive membrane bioreactor

This article presents a mathematical model of membrane-attached biofilm (MAB) behavior in a single-tube extractive membrane bioreactor (STEMB). MABs can be used for treatment of wastewaters containing VOCs, treatment of saline wastewaters, and nitrification processes. Extractive membrane bioreactors (EMBs) are employed to prevent the direct contact between a toxic volatile pollutant and the aerated gas by allowing counterdiffusion of substrates; i.e., pollutant diffuses from the tube side into the biofilm, whereas oxygen diffuses from the shell side into the biofilm. This reduces the air stripping problems usually found in conventional bioreactors. In this study, the biodegradation of a toxic VOC (1,2-dichloroethane, DCE) present in a synthetic wastewater has been investigated. An unstructured model is used to describe cell growth and cell decay in the MAB. The model has been verified by comparing model predicted trends with experimental data collected over 5 to 20-day periods, and has subsequently been used to model steady states in biofilm behavior over longer time scales. The model is capable of predicting the correct trends in system variables such as biofilm thickness, DCE flux across the membrane, carbon dioxide evolution, and suspended biomass. Steady states (constant biofilm thickness and DCE flux) are predicted, and factors that affect these steady states, i.e., cell endogeneous decay rate, and biofilm attrition, are investigated. Biofilm attrition does not have a great influence on biofilm behavior at low values of detachment coefficient close to those typically reported in the literature. Steady-state biofilm thickness is found to be an important variable; a thin biofilm results in a high DCE flux across the membrane, but with the penalty of a high loss of DCE via air stripping. The optimal biofilm thickness at steady state can be determined by trading off the decrease in air stripping (desirable) and the decrease in DCE flux (undesirable) which occur simultaneously as the thickness increases. (c) 1996 John Wiley & Sons, Inc.     

The Sugar Model: Catalytic Flow Reactor Dynamics of Pyruvaldehyde Synthesis from Triose Catalyzed by Poly-L-Lysine Contained in a Dialyzer

The formation of pyruvaldehyde from triose sugars was catalyzedby poly-L-lysine contained in a small dialyzer with a 100molecular weight cut off (100 MWCO) suspended in a much largertriose substrate reservoir at pH 5.5 and 40 °C. Thepolylysine confined in the dialyzer functioned as a catalyticflow reactor that constantly brought in triose from thesubstrate reservoir by diffusion to offset the drop in trioseconcentration within the reactor caused by its conversion topyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35mL) within the dialyzer generated pyruvaldehyde with a syntheticintensity (rate/volume) that was 3400 times greater than that ofthe triose substrate solution (12 mM, 120 mL) outside thedialyzer. Under the given conditions the final yield ofpyruvaldehyde was greater than twice the weight of thepolylysine catalyst. During the reaction the polylysine catalystwas poisoned presumably by reaction of its amino groups withaldehyde reactants and products. Similar results were obtainedusing a dialyzer with a 500 MWCO. The dialyzer method ofcatalyst containment was selected because it provides a simpleand easily manipulated experimental system forstudying the dynamics and evolutionary development of confinedautocatalytic processes related to the origin of life underanaerobic conditions.     

Theoretical analysis of G6P production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

The performance of an ultrafiltration hollow-fiber reactor, in which the enzymatic synthesis of glucose 6-phosphate from glucose and cofactor ATP and the enzymatic regeneration of ATP from ADP and acetyl phosphate are performed simultaneously, was analyzed theoretically. A simple analytical model in which the liquid flowing in the fiber tubes is assumed to be plug flow, and the radial concentration gradients in the tube and shell sides are both neglected, could simulate the reactor performance with satisfactory accuracy. The simulation elucidated the effects of the reactor configurations and various operational conditions on glucose conversion, ATP recycle number, and space-time yield. If the fiber tubes, through which the permeability of the relevant components such as substrates is high, were packed as much as possible in the reactor, good reactor performance could be expected. Furthermore, with a sufficiently high enzyme concentration, low ATP concentration in the feed solution, and appropriate space velocity, good space-time yield with high glucose conversion and with very high ATP recycle number is theoretically possible.     

Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

Effects of reactant heterogeneity and mixing on catabolite repression in cultures of Saccharomyces cerevisiae

An experimental study was conducted into the effect of reactant heterogeneity on glucose-fed continuous cultures of S. cerevisiae, The heterogeneity was altered by varying mixing intensity in the nutrient entry region within a static mixing device. Experimental results confirm simulation predictions based upon a simple growth model, showing that mixing in the entry region can govern macroscopic culture behavior. Specifically, at high dilution rates, the biomass concentration was reduced by mixing patterns that increased the size of regions where glucose exceeded the threshold for catabolite repression. Because the size of such repressive regions is not uniquely determined by reactant segregation, the authors argue that in biological systems (and others involving a threshold response) an alternative measure of mixing quality should be used. Conclusions are drawn concerning the simulation of biological reactors for design purposes, and the importance of nutrient delivery systems to reactor performance.     

Ultrastructure of sea urchin tube feet

Summary An analysis of the ultrastructure of the tube feet of three species of sea urchins (Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus) revealed that the smooth muscle, although known to be cholinoceptive, receives no motor innervation.The muscle fibers are attached to a double layer of circular and longitudinal connective tissue which surrounds the muscle layer and contains numerous bundles of collagen fibers. On its outside, the connective tissue cylinder is invested by a basal lamina of the outer epithelium to which numerous nerve terminals are attached. These are part of a nerve plexus which surrounds the connective tissue cylinder. The plexus itself is an extension of a longitudinal nerve that extends the whole length of the tube foot. It is composed of axons, but nerve cell bodies and synapses are conspicuously lacking, suggesting that the axons and terminals derive from cells of the radial nerve. Processes of the epithelial cells penetrate the nerve plexus and attach to the basal lamina. There is no evidence that the epithelial cells function as sensory cells.On the basis of supporting evidence it is suggested that the transmitter released by the nerve terminals diffuses to the muscle cells over a distance of several microns and in doing so affects the mechanical properties of the connective tissue.Supported by the Sonderforschungsbereich 138 of the Deutsche Forschungsgemeinschaft     

Continuous production of ethanol by yeast "immobilized" in a membrane-contained fermentor

A dialysate-feed, immobilized-cell dialysis continuous fermentation system was investigated as a method of relieving product inhibition in the conversion of glucose to ethanol by cells of Saccharomyces cerevisiae ATCC 4126. The substrate was fed into a continuous dialysate circuit and then into a batch fermentor circuit via diffusion through the microporous membranes of an intermediate dialyzer. Simultaneously, product was withdrawn from the fermentor circuit through the dialyzer membranes into the dialysate circuit and out in the effluent. Since the fermentor was operated without an effluent, the cells essentially were immobilized and converted substrate to product by maintenance metabolism. Contrary to prior results with this novel system for the continuous fermentation of lactose to lactate by lactobacillus cells, a steady state of yeast cells in the fermentor did not occur initially but was obtained by the depletion of medium nitrogen and the prevention of cell breakage, although the substrate and product concentrations then became unsteady. The inherent advantages of the system was offset in the ethanol fermentation by relatively low productivity, which appeared to be limited by membrane permeability.