bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of expression of the pet operon on intracellular metabolic fluxes of Escherichia coli
Authors: Diaz‐Ricci J. C. , Tsu M. , Bailey J. E. 
Published: December 2004

AbstractFermentation patterns of Escherichia coli HB101 carrying plasmids expressing cloned genes of Zymomonas mobilis pyruvate decarboxylase (PDC) and alcohol dehydrogenase li (ADH) were determined in glucose‐limited complex medium in pH‐controlled anaerobic batch cultivations. Time profiles of glucose, dry cell weight, succinate, formate, acetate, and ethanol were determined, as were the activities of ADH and PDC. Fluxes through the central carbon pathways were calculated for each construct utilizing exponential phase data on extracellular components and assuming quasi‐steady state for intermediate metabolites. Overall biomass yields were greatest for cells expressing both PDC and ADH activities. Yields of carbon catabolite end products were similar for all PDC‐expressing strains and different from those for other strains. Relative to its glucose uptake rate, the strain with greatest PDC and ADH activities produces formate and acetate more slowly and ethanol more rapidly than other strains. Strong influences of plasmid presence and metabolic coupling complicate detailed interpretations of the data.
Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase
Authors: Kim Dong‐Man , Kim Hak‐Sung 
Published: December 2004

AbstractGluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose‐fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross‐linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin‐immobilized inulinase in alginate beads. The optimum amounts of both chitin‐immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h−1 and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed‐bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h−1 and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed‐bed reactor without any loss of activity, while half‐life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.
A computer‐aided measuring system for the characterization of yeast populations combining 2D‐image analysis, electronic particle counter, and flow cytometry
Authors: Huls P. G. , Nanninga N. , van Spronsen E. A. , Valkenburg J. A. C. , Vishcer N. O. E. , Woldringh C. L. 
Published: December 2004

AbstractAn integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D‐image processing program for rapid and accurate determination of volume distributions of the different sub‐populations. The program combines the capacity of image processing and volume calculation by contour‐rotation, with the potential of visual evaluation of the cells. High‐contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D‐image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2‐D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.
A proposed biparticle fluidized‐bed for lactic acid fermentation and simultaneous adsorption
Authors: Davison Brian H. , Scott Charles D. 
Published: December 2004

AbstractA bioreactor configuration is proposed for simultaneous fermentation and separation of the desired product. The bioreactor consists of a columnar fluidized bed of immobilized microorganisms. Denser adsorbent particles are added to this column. These adsorbent particles fall through the bed, absorb the product, and are removed from the base of the columnar reactor. The system hydrodynamics and the separability of the two types of particles were confirmed for low‐density gel beads. The addition of the adsorbent, activated carbon, to a fermentation of Lactobacillus delbreuckii absorbed lactic acid. The addition of adsorbent enhanced the fermentation and controlled the pH.
Displacement effects in large‐scale chromatography?
Authors: Jungbauer Alois , Uhl Karola , Schulz Petra , Tauer Christa , Gruber Gerhard , Steindl Franz , Buchacher Andrea , Schoenhofer Wolfgang , Unterluggauer Florian 
Published: December 2004

AbstractDisplacement effects in large‐scale (total column volume vt = 150 L) and preparative ion‐exchange chromatography purifying human erythrocyte superoxide dismutase are described in the present article. The biomolecules are eluted in a very small peak elution volume (<0.2 vo) behind the salt wave using a step gradient. The theoretical peak width and retention behavior are calculated according to the model of Yamamoto. The theoretical values are then compared with the experimental data. There was a difference observed between the elution type I (also called fronting) and the experimentally obtained elution. Some instructions are given on how to achieve these phenomenona because a beneficial effect in respect to resolution and recovery of a biomolecule is observed.
Surface immobilization of anchorage‐dependent mammalian cells
Authors: Robert Joëlle , Côté Johanne , Archambault Jean 
Published: December 2004

AbstractAnchorage‐dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75–125 and 40 μm, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 μm, respectively, in 100‐mL spinner flasks and 2‐L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (≤2–3 h) attachment of inoculated cells (≥95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate ∼0.03–0.04 h−1, maximum cell density of 8 × 106–9 × 106 cells · mL−1, and yields of 0.4 × 108 cells · mM−1 on glucose and 2 × 108−3 × 108 cells · mM−1 on glutamine). Scale‐up of this culture technique in a 2‐L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 × 106 cells · mL−1. Two other anchorage‐dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.
Application of a new structured model to tobacco cell cultures
Authors: Hooker Brian S. , Lee James M. 
Published: December 2004

AbstractA new structured kinetic model has been formulated and applied to batch suspensions of Nicotiana tabacum. This model has been developed by representing culture interactions with pathways designated for structural component production, secondary metabolite synthesis, and cellular respiration. Additional provisions were made to distinguish growth‐competitive secondary metabolite production from non‐growth‐competitive secondary metabolite production. Parameters for kinetic rate expressions within the model were estimated based upon experimental observations utilized in conjunction with numerical optimization techniques. Using these parameters, culture growth, substrate uptake, cell respiration, and total phenolics production were all successfully correlated to experimenta data from shake flask cultures of N. tabacum.
Characterization of the growth and sporulation behavior of Penicillium roquefortii in solid substrate fermentation by material and bioenergetic balances
Authors: Larroche C. , Gros J. B. 
Published: December 2004

AbstractWhen Pencillium roquefortii is grown on two kinds of buckwheat, exhibiting a low [0.85 g water/g dry matter (DM), buckwheat A] and a high [1.5 g water/g initial dry matter (IDM), buckwheat B] water content, a marked difference in the mode of development of the fungus is observed. Material balances for buckwheat A show that growth does not stop because of nutrient exhaustion. Analysis of water balance shows that active growth proceeds with a permanent limitation by the turgor potential which disappears when the water activity of the substrate is close to 0.96, thus arresting growth. This limitation causes intensive water excretion from the system due to the lowering of the water activity of the substrate. The water content of the mycelium thus decreases from 79% at the beginning of the cultivation to 74% when the growth stops. This is linked to a substantial oxidative metabolism and a high sporulation efficiency, close to 0.85. The spores obtained have a low dry weight and a reduced nitrogen content. In the case of buckwheat B, the active growth is shown to stop because of available mineral nitrogen depletion. No significant decrease in the water activity of the substrate is found during the protein synthesis, and the turgor potential remains high at the end of this period. Culture proceeds with new wall synthesis; the sporulation efficiency remains high and the spores obtained exhibit a high dry weight and a high nitrogen content. The bioenergetic balances show that the P/O ratio varies with the kind of substrate used; its value is close to 1.56 for the low water medium and to 2.21 for the high one. The ATP yield Z is always close to 1, and fungal development occurs with limitations of both anabolism and catabolism on buckwheat B and only of anabolism and catabolism on buckwheat B and only of anabolism on buckwheat A.
Affinity purification of insoluble recombinant fusion proteins containing glutathione‐S‐transferase
Authors: Hartman John , Daram Paru , Frizzell Raymond A. , Rado Thomas , Benos Dale J. , Sorscher Eric J. 
Published: December 2004

AbstractProkaryotic expression of polypeptides as fusion proteins with glutathione‐S‐transferase has recently been reported as a one‐step means of purifying recombinant protein. The usefulness of the glutathione‐S‐transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione‐S‐transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one‐step purification utilizing this scheme requires proper folding of recombinant glutathione‐S‐transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione‐agarose affinity purification. Low‐temperature induction of fusion protein synthesis, but not incubation with anion‐exchange resins, led to improved one‐step purification of glutathione‐S‐transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity‐purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione‐S‐transferase.
Comparison of two experimental methods for the determination of Michaelis–Menten kinetics of an immobilized enzyme
Authors: Hooijmans C. M. , Stoop M. L. , Boon M. , Luyben K. C. A. M. 
Published: December 2004

AbstractFor the application of immobilized enzymes, the influence of immobilization on the activity of the enzyme should be Known. This influence can be obtained by determining the intrinsic kinetic parameters of the immobilized enzyme, and by comparing them with the kinetic parameters of the suspended enzyme. This article deals with the determination of the intrinsic kinetic parameters of an agarose‐gel bead immobilized oxygen‐consuming enzyme: L‐lactate 2‐monooxygenase. The reaction rate of the enzyme can be described by Michaelis–Menten kinetics. Batch conversion experiments using a biological oxygen monitor, as well as steady‐state profile measurements within the biocatalyst particles using an oxygen microsensor, were performed. Two different mathematical methods were used for the batch conversion experiments, both assuming a pseudosteady‐state situation with respect to the shape of the profile inside the bead. One of the methods used an approximate relation for the effectiveness factor for Michaelis–Menten kinetics which interpolates between the analytical solutions for zero‐ and first‐order kinetics. The other mathematical method was based on a numerical solution and combined a mass balance over the reactor with a mass balance over the bead. The main difference in the application of the two methods is the computer calculation time; the completely numerical calculation procedure was about 20 times slower than the other calculation procedure.The intrinsic kinetic parameters resulting from both experimental methods were compared to check the reliability of the methods. There was no significant difference in the intrinsic kinetic parameters obtained from the two experimental methods. By comparison of the kinetic parameters for the suspended enzyme with the intrinsic kinetic parameters for the immobilized enzyme, it appeared that immobilization caused a decrease in the value of Vm by a factor of 2, but there was no significant difference in the values obtained for Km.
Application of quantitative image analysis to a mammalian cell line grown on microcarriers
Authors: Pons Marie‐Noëlle , Wagner Anne , Vivier Harvé , Marc Annie 
Published: December 2004

AbstractQuantitative image analysis has been applied to the monitoring of cultures of a mammalian cell line on microcarriers. Procedures have been developed to investigate microcarrier colonization and cluster formation and to determine the eventual modification of cell size during cultivation using scanning electron microscopy (SEM) microphotography. The human kidney tumor cells (TCL 598) on which the procedures were tested underwent a slight size decrease during the development of the first cell layer on the microcarriers. The cluster size and the cell size remained constant during the culture stationary phase.
Oxygen transfer properties of bubbles in animal cell culture media
Authors: Zhang S. , Handa‐Corrigan A. , Spier R. E. 
Published: December 2004

AbstractOxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles (≈5 mm diameter) < intermediate bubbles (≈1 mm diameter) < micron‐sized bubbles (≈100 μm diameter). Under certain conditions, the micron‐sized bubbles were capable of achieving oxygen transfer rate up to 100 h−1, a 10–20‐fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new‐born calf serum (NBCS) and/or Pluronic F‐68 supplemented media. For the intermediate and micron‐sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F‐68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron‐sized bubbles in Pluronic F‐68 supplemented media containing antifoam agent and NBCS.
Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation
Authors: Thatipamala R. , Rohani S. , Hill G. A. 
Published: December 2004

AbstractIn ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.
A rapid, simple immunofluorometric assay: Development and characterization
Authors: Park Hongwoo , Wang Daniel I. C. , Yarmush Martin L. 
Published: December 2004

AbstractA novel two‐site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small‐sized beads (0.5 μm diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid‐phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti‐IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30‐minute incubation was 0.2 to 40 μg/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 μg/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 μg/mL of BSA and intra‐assay variability was 2.3% at 3.2 μg/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. © 1992 John Wiley & Sons, Inc.
Reaction and diffusion in a gel membrane reactor containing immobilized cells
Authors: De Backer L. , Devleminck S. , Willaert R. , Baron G. 
Published: December 2004

AbstractTo investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel‐immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well‐mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel‐containing immobilized cells. A mathematical model with time‐ and position‐dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel‐entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel‐immobilized cells. © 1992 John Wiley & Sons, Inc.
Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures
Authors: Linardos Theodor I. , Kalogerakis Nicolas , Behie Leo A. 
Published: December 2004

AbstractAs a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G1 phase of the cell cycle. The steady‐state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady‐state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. © 1992 John Wiley & Sons, Inc.