bluuebunny/crossref_metadata_2025_split huggingface dataset:
High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor
Authors: Lee Youl Lark , Chang Ho Nam 
Published: December 2004

AbstractA recombinant Escherichia coli HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle fermentor. The strain was very stable throughout the whole experiment. The main inhibitory by‐product was acetic acid, and cell growth ceased when its concentration was above 14 g/L Cell density could be increased up to 145 g/L dry weight without experiencing by‐product inhibition by regulating glucose concentration in the fermentor and by using total membrane recycle. Acetic acid formation was negligible not only when cells were cultured in medium containing no glucose but also when glucose was limited. Dissolved oxygen control as well as glucose limitation was an indispensable condition for minimizing acetic acid formation when the medium contained glucose. Low concentrations of accumulated acetic acid were reused when glucose was limited. Use of highly concentrated medium reduced the membrane surface area required for cell recycle greatly. The recycle fermentor could be operated in various operational modes including partial bleed and repeated recycle culture to give high productivity. Productivity of a repeated recycle system was over 10 times higher than that of a simple batch system.
Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: III. Hydrodynamic aspects
Authors: Vos Henk J. , van Houwelingen Cees , Zomerdijk Max , Luyben Karel Ch. A. M. 
Published: December 2004

AbstractIn Parts I and II of this series we described the modelling, design, and operation of a multistage fluidized bed reactor (MFBR) for immobilized biocatalysts. This article deals with those aspects of the MFBR which are different from single‐stage fluidized beds which are operated in batch mode with respect to the solids. The semicontinuous transport of the particles requires perfect mixing of the particles in the reactor compartments, because particles are mainly transported from the bottom of these compartments. A large spread in the physical properties of the biocatalyst particles, especially of both size and density, may cause the particles to segregate into layers with different diameter and/or density. This affects the efficient use of the biocatalyst. The properties of the particles are dependent on the immobilization method. The suitability of different methods for possible future application in the MFBR is therefore compared. Because of segregation, successful use of a biofilm catalyst with a nonuniform thickness of the biofilm is doubtful. Experiments in a small scale reactor (± 0.1 m diameter) demonstrated that perfect particle mixing is possible using commercially available biocatalyst particles of uniform density. Co‐immobilization of the biocatalyst with glass powder in a gel is a simple and effective method of increasing gel density. High density particles allow high liquid flow rates, and thus an improved external mass transfer can be achieved.The distributor plates, which separate the reactor compartments, must allow unhindered transport of particles. Therefore, the holes in these plates must have a diameter of at least 4.5 times that of the largest particles which are present in the particle mixture used. Furthermore, the plates must be designed such that, when scaling‐up the reactor, a uniform liquid distribution over the cross‐sectional area of the reactor occurs. Large‐scale experiments were not carried out, but published correlations, indicate that particle mixing and a uniform liquid distribution can be accomplished in a large‐scale reactor under similar flow conditions.
Immobilization of the serine protease from Thermomonospora fusca YX on porous glass
Authors: Gusek Todd W. , Tyn Myo T. , Kinsella John E. 
Published: December 2004

AbstractAs much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86°C) and pH optimum (9.4) for activity compared to soluble YX‐protease (80°C and pH 9.0, respectively). Immobilization improved enzyme thermo‐stability above 90°C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12°C). A continuous‐flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.
Diffusional limitations of immobilized Escherichia coli in hollow‐fiber reactors: Influence on 31P NMR spectroscopy
Authors: Briasco Catherine A. , Karel Steven F. , Robertson Channing R. 
Published: December 2004

AbstractEscherichia coli cells were immobilized and grown in hollow‐fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using 35S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 °C were shown to be severe, with regions of starved cells occupying up to 80% of the cell‐containing region. Phosphorus‐31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume‐averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur‐35 autoradiographs of a reactor operated at 16°C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near‐normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.
Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors
Authors: Piret James M. , Cooney Charles L. 
Published: December 2004

AbstractThe heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high‐molecular‐weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell‐side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 × 104 D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell‐side convective flow and its influence on the inoculum and high‐molecular‐weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.
The long‐term effects of ethanol on immobilized cell reactor performance using K. fragilis
Authors: Chen C. , Dale M. C. , Okos M. R. 
Published: December 2004

AbstractThe effects of ethanol on reactor performance were studied in a small, 5‐cm packed height, “differential” type immobilized cell reactor. Lactose utilizing yeast cells, Kluyveromyces fragilis, were absorbed to a porous adsorbant sponge matrix in a gas continuous reactor. Step changes in the feed ethanol concentration to the column (10–130 g/L) were used to test the reactor response over extended periods of time (about 30–50 h per dosage level) followed by a return to basal zero inlet ethanol feed. Effluent cell density and effluent cell viability were measured at intervals. An inhibitory response in ethanol productivity to feed dosage ethanol levels above 20 g/L was detected almost immediately, with a near steady state response noted within 2.5 h of initiating the dosage. Feed ethanol levels above 50 g/L resulted in a subsequent gradual decrease in reactor productivity over time, which was associated with a decrease in the fraction of viable shed cells in the reactor effluent. The reactor response to a step removal of the ethanol inhibition was also monitored. Quick and complete rebounding of the fermentation rate to the original basal rate was noted following dosage concentrations of under 50 g/L ethanol. Recovery rates slowed following ethanol dosage levels above 50 g/L. Viable shed cell density improved overtime during the slow recovery periods. Growth rates (as determined by shed cell density) were more strongly inhibited than productivity. Growth responded more slowly to changes in ethanol environment as growth rates at 30 h fell to about 40% of the rates measured 7.5 h after initiation of a dosage level. It is concluded that ethanol contributions to cell injury and death (and consequent ICR performance degradation) may be more important than ethanol inhibition of productivity rates in the long‐term operation of immobilized cell reactors at ethanol concentrations over 50 g/L.
Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: Hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture
Authors: Grosz Ron , Stephanopoulos Gregory 
Published: December 2004

AbstractThe growth and metabolism of Saccharomyces cerevisiae was studied in steady‐state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady‐state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady‐state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro‐aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.
On‐line prediction of fermentation variables using neural networks
Authors: Thibault Jules , Van Breusegem Vincent , Chéruy Arlette 
Published: December 2004

AbstractThis article presents an introduction to the use of neural network computational algorithms for the dynamic modeling of bioprocesses. The dynamic neural model is used for the prediction of key fermentation variables. This relatively hew method is compared with a more traditional prediction technique to judge its performance for prediction. Illustrative simulation results of a continuous stirred tank fermentor are used for this comparison. It is shown that neural network models are accurate with a certain degree of noise immunity. They offer the distinctive ability over more traditional methods to learn very naturally complex relationships without requiring the knowledge of the model structure.
Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers
Authors: Agerkvist Iréne , Enfors Sven‐Olof 
Published: December 2004

AbstractThe protein releases, the particle size distribution and the viscosity of disrupted E. coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M‐8TA and Microfluidizer M‐110 were determined. The effects of these parameters on separation of the cell debris from the protein solution by centrifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably different physical properties of the cell disintegrates which influences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disruption in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, increasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high‐pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disintegrate both in filtration and in centrifugation.
Effects of solvent molecular toxicity and microenvironment composition on the Δ1 dehydrogenataon activity of Arthrobacter simplex cells
Authors: Pinheiro H. M. , Cabral J. M. S. 
Published: December 2004

AbstractSolvent selection tests were carried out for the Δ1 dehydrogenation of 6‐α‐methylhydrocortisone‐21‐acetate by Arthrobacter simplex cells in the presence of organic solvents. Solubility limits were determined for substrate and product in dry and water‐saturated solvents and solvent mixtures. Molecular toxicity levels were estimated by measuring the dehydrogenation activity decay of freely suspended cells incubated in solvent‐saturated aqueous media. Chloroform and n‐decan‐1‐ol were the best choice of solvent, for both solubility and catalytic stability. High concentrations of water‐soluble additives, such as monosodium glutamate, were found to greatly improve the retention of activity in chloroform‐saturated media.
Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta
Authors: Caldwell Steven R. , Raushel Frank M. 
Published: December 2004

AbstractA purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 μmol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10‐unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1‐unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent Km for the immobilized enzyme was 3–4 times greater than the Km (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH–rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.
Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: Modeling studies of stationary and oscillatory states
Authors: Aon Miguel A. , Cortassa Sonia 
Published: December 2004

AbstractThermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S1), in the presence of a second substrate, formate (S2), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S2 allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S1 which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S1) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (η) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S2/S1 ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of η was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high η predicting that higher biomass yields may be obtained through a variable, high, η by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.
Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum
Authors: Chun G.‐T. , Agathos S. N. 
Published: December 2004

AbstractProcess improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L‐valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L‐valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L‐valine during or after exponential growth. In contrast to the free cell cultures, the addition of L‐valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.
Recovery of copper and cobalt by biopolymer gels
Authors: Jang L. K. , Lopez S. L. , Eastman S. L. , Pryfogle P. 
Published: December 2004

AbstractThe recovery of copper from synthetic aqueous media circulating in a loop fluidized bed reactor operated batchwise was investigated by using the following biopolymer systems: (1) a viscous solution of sodium alginate (from kelp) dispensed directly into the reactor fluid containing dissolved copper (sulfate salt) at initial concentrations of 60–200 ppm, (2) partially coagulated calcium alginate spheres for absorbing dissolved copper at initial concentrations of 10–40 ppm, and (3) a mixture of green algae Microcystis and sodium alginate dispensed directly into the reactor fluid. The recovery of copper and cobalt, a strategic metal, from cobalt ore leachate was achieved by a two‐step approach: direct dispensing of sodium alginate to absorb the bulk of metals followed by the addition of partially coagulated calcium alginate spheres to “polish” the leachate. Metal binding capacity and conditional stability constant of each biopolymer system as well as the effective diffusivity of cupric ion in the matrix of biopolymer gels are reported.