Extractive separation of penicillin G by facilitated transport via carrier supported liquid membranes

The facilitated transport of penicillin G (Pen G), through a supported liquid membrane with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, were studied. The distribution coefficient was obtained from a batch extraction experiment. The effects of flow rate, carrier concentration, initial concentration of Pen G, and the pH of feed and stripping phases on the transport rate of Pen G through the supported liquid membrane were also investigated. The results are in agreement with theoretical predictions, and it is demonstrated that the transport of Pen G through the supported liquid membrane is controlled simultaneously by mass transfer across both aqueous and liquid membranes. (c) 1993 John Wiley & Sons, Inc.     

Effect of microorganisms on rate of liquid extraction of ethanol from fermentation broths

Liquid extraction is one means of removing metabolic products continuously during a fermentation and so reducing product inhibition. It is known that microbial organisms are attracted to liquid-liquid interfaces, and it is important for the design of extraction systems to establish if this has a detrimental effect on the rate of extraction. The extraction of ethanol from aqueous suspensions of yeast (Saccharomyces cerevisiae) using n- decanol is described in this paper. It was found that the presence of the yeast cells severely reduced the rate of ethanol extraction. The overall mass transfer coefficient was reduced from 5.0 x 10(-6) to 0.7 x 10(-6) m/s. This reduced overall mass transfer coefficient was unaffected by yeast concentration in the range 0.1-20 kg/m(3). The results are consistent with the yeast cells adsorbing to the interface in closely packed layers and preventing mass transfer by simply reducing the available interfacial area. Optical microscope observations confirmed that a yeast layer several cell diameters thick rapidly built up at the interface when a small decanol droplet was added to a yeast suspension.     

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.     

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.     

Transient response of encapsulated enzymes in hollow-fiber reactor

A mathematical model for the transient response of encapsulated enzymes is developed showing the effects of the outer boundary layer, the encapsulating membrane, the partition coefficient, and diffusion with reaction within the encapsulating medium. The model incorporates both first-order kinetics and Michaelis-Menten kinetics for the reaction rate. Using typical hollow-fiber or microcapsule parameters, the model shows that (a) the partition coefficient affects the overall rate only when the rate-limiting step is diffusion through the membrane, (b) the transient overall effectiveness factor rises sharply with time and approaches an asymptotic value for most situations, and (c) the first-order approximation to Michaelis-Menten kinetics is not valid when the initial outside bulk concentration is higher than the Michaelis constant and the overall rate is reaction limited. The model is compared with experimental data using uricase in a hollow-fiber enzyme reactor configuration. Batch assay and CSTUER (continuous-stirred ultrafiltration enzyme reactor) studies were conducted on the free enzyme to provide some of the parameters used in the model. The CSTUER data fit the case of substrate inhibition kinetics with the apparent Michaelis constant approaching zero. The hollow-fiber reactor was conducted with uricase dissolved in both a buffer solution and a concentrated hemoglobin solution. Diffusivities of the solute were measured in both solutions as was the osmotic pressure of the hemoglobin solution. While experimental data for uricase in buffer solution could easily be matched by the model, that in the concentrated hemoglobin solution could not.     

Development of a hollow-fiber system for large-scale culture of mammalian cells

A flat-bed hollow-fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge-type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless-steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm² of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.     

Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membrane

In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di‐n‐octylamine (DOA) + 30 vol % iso‐octanol + kerosene was used as liquid membrane phase, and Na₂CO₃ aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Kinetic study of a hollow-fiber enzyme reactor

Enzymes, such as urease and uricase, were entrapped in three kinds of hollow fibers. The apparent Michaelis–Menten constants K_m(app) obtained for these enzyme reactors were always larger than K_m of free enzyme because of the permeation resistance of substrate across the hollow-fiber membrane. K_m(app) increased with increasing degree of permeation resistance across the membrane by the increase in enzyme concentration. The half-life of the entrapped urease in the continuous reaction system was 60–80% of that of free enzyme. Activation energies of hollow-fiber enzyme reactors were always smaller than that of the free enzyme, because the activation energy of permeation was smaller than that of the enzyme reaction.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part II. Uniresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40 degrees C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped courrently through the shell side of a shell-and-tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four-parameter rate expression (which includes a two-parameter deactivation rate expression and a two-parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn-1,3 positions of the triglycerides can be released in the hollow-fiber reactor.     

A new coiled hollow-fiber module design for enhanced microfiltration performance in biotechnology.

The microfiltration performance of a novel membrane module design with helically wound hollow fibers is compared with that obtained with a standard commercial-type crossflow module containing linear hollow fibers. Cell suspensions (yeast, E. coli, and mammalian cell cultures) commonly clarified in the biotechnology industry are used for this comparison. The effect of variables such as transmembrane pressure, particle suspension concentration, and feed flow rate on membrane performance is evaluated. Normalized permeation fluxes versus flow rate or Dean number behave according to a heat transfer correlation obtained with centrifugal instabilities of the Taylor type. The microfiltration performance of this new module design, which uses secondary flows in helical tubes, is significantly better than an equivalent current commercial crossflow module when filtering suspensions relevant to the biotechnology industry. Flux and capacity improvements of up to 3.2-fold (constant transmembrane pressure operation) and 3.9-fold (constant flux operation), respectively, were obtained with the helical module over those for the linear module.     

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l⁻¹ h⁻¹) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l⁻¹ h⁻¹). Cell deactivation was observed after 10–12 h of operation.     

Transfer of carbon dioxide within cultures of microalgae: plain bubbling versus hollow-fiber modules

In attempts to improve the metabolic efficiency in closed photosynthetic reactors, availability of light and CO(2) are often considered as limiting factors, as they are difficult to control in a culture. The carbon source is usually provided via bubbling of CO(2)-enriched air into the culture medium; however, this procedure is not particularly effective in terms of mass transfer. Besides, it leads to considerable waste of that gas to the open atmosphere, which adds to operation costs. Increase in the interfacial area of contact available for gas exchange via use of membranes might be a useful alternative; microporous membranes, in hollow-fiber form, were tested accordingly. Two hollow-fiber modules, different in both hydrophilicity and outer surface area, were tested and duly compared, in terms of mass transfer, versus traditional plain bubbling. Overall volumetric coefficients (K(L)a) for CO(2) transfer were 1.48 x 10(-2) min(-1) for the hydrophobic membrane, 1.33 x 10(-2) min(-1) for the hydrophilic membrane, and 7.0 x 10(-3) min(-1) for plain bubbling. A model microalga, viz. Nannochloropsis sp., was cultivated using the two aforementioned membrane systems and plain bubbling. The produced data showed slight (but hardly significant) increases in biomass productivity when the hollow-fiber devices were used. However, hollow-fiber modules allow recirculation of unused CO(2), thus reducing feedstock costs. Furthermore, such indirect way of supplying CO(2) offers the additional possibility for use of lower gas pressures, as no need to counterbalance hydrostatic heads exists.     

High conversion in asymmetric hydrolysis during permeation through enzyme-multilayered porous hollow-fiber membranes

We describe a novel porous hollow-fiber support for immobilizing aminoacylase in multilayers. Epoxy-group-containing polymer chains were grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization of glycidyl methacrylate, and subsequently a diethylamino group as an anion-exchange group was introduced into the graft chain. Aminoacylase was adsorbed in multilayers by allowing the amioacylase buffer solution to permeate through the pores across the hollow fiber; the graft chains provided three-dimensional space for the enzymes because of their electrostatic repulsion. The adsorbed enzyme at a degree of multilayer binding of 15 was cross-linked with glutaraldehyde to prevent leakage. An acetyl-DL-methionine solution was allowed to permeate through the pores surrounded by the aminoacylase-immobilized graft chain. Production of L-methionine was observed at a 4.1 mol/h per L of the fiber for a space velocity of 200 h(-1), defined as the flow rate of the effluent penetrating the outside surface of the hollow fiber divided by the membrane volume including the lumen.     

Phenol biodegradation in hybrid hollow-fiber membrane bioreactors

Hollow-fiber membrane bioreactors were developed with granular activated carbon (GAC) for the biodegradation of phenol using Pseudomonas putida. Hollow fibers showed similar structure with/without GAC incorporated; while GAC hollow fiber had a stronger phenol adsorption capacity. In batch biotransformation experiments, complete depletion of 1000 mg phenol l⁻¹ (at which concentration free cells cannot grow) was accomplished in the reactor within 18 h in the hybrid bioreactor, comparing with 23 h in the GAC free bioreactor. Desorption and bioregeneration of the hollow-fiber membrane were believed to be the key for the enhancement of bioreactor performance. At continuous running, the GAC bioreactor showed its superiority over the GAC free bioreactor during start-up and elevated loading phase. More than 90% of the phenol was transformed in the GAC bioreactor when the phenol loading was <24 mg h⁻¹. The better bioreactor performance may be due to the enhanced mass transportation and adsorption capacity with the incorporation of GAC.     

A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase

A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358. The two-stage bioreactor system was successfully operated for 20 days. PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell. Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop. Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air. Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min. The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d. These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams.     

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.     

Development of a novel membrane aerated hollow-fiber microbioreactor

A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated.     

Development of a supported emulsion liquid membrane system for propionic acid separation in a microgravity environment

Perstractive fermentation is a good way to increase the productivity of bioreactors. UsingPropionibacteria as the model system, the feasibility of using supported emulsion liquid membrane (SELM) for perstractive fermentation is assessed in this study. Five industrial solvents were considered as the solvent for preparing the SELM. The more polar a solvent is, the higher the partition coefficient. However, toxicity of a solvent also increases with its polarity. CO-1055 (industrial decanol/octanol blend) has the highest partition coefficient toward propionic acid among the solvents that has no molecular toxicity towardPropionibacteria. A preliminary extraction study was conducted using tetradecane as solvent in a hydrophobic hollow fiber contactor. The result confirmed that SELM eliminates the equilibrium limitation of conventional liquid-liquid extraction, and allows the use of a non-toxic solvent with low partition coefficient.     

Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.     

Overloaded hollow-fiber liquid chromatography.

Liquid chromatography in hollow fibers can separate solutes like flavors and proteins by using a stationary phase of organic solvent, sometimes containing reversed micelles. Such separations, which have a much smaller pressure drop than equivalent separations in packed beds, show dispersion consistent with chromatographic theories at low flows and dilute feeds. These separations behave less predictably at high flows and concentrated feeds, which overload the hollow fibers. The results for flavors correlate well with the Graetz number, consistent with available theories of chromatography and adsorption. The results for proteins correlate poorly with the Graetz number but better with a dimensionless flux based on facilitated diffusion in the stationary phase.