bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of population dynamic for a recombinant bacterium during continuous cultures: Appliction of data filtering and smoothing
Authors: Nancib Nabil , Mosrati Ridha , Boudrant Joseph
Published: December 2004

A numerical method to process experimental data concerning plasmid stability of a recombinant bacteria during continuous cultures with nonselective media is proposed here. This method differs from previous ones in that it uses the derivatve form of the state equation of the Imanaka‐Aiba model for recombinant cultures. The methodology proposed here allows one to estimate values for the two model parameters without forcing them to be constant. Until now, this could not be done using classical analytical techniques because these parameters have been considered invariable because of the integration used in the evaluation of the model. These parameters are (1) the difference in the specific growth rates between plasmid‐carrying cells and plasmid‐free cells (δμ), and (2) the probability of plasmid loss by plasmid‐containing cells (ρr μ+). The derivative technique used here is completed by mathematical treatments involving data filtering and smoothing. The values of the two parameters are in agreement with those already publised. The current technique does not impose preconditions and permit us to further study related phenomena.
Modeling the population dynamics of baculovirus‐infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields
Authors: Licari P. , Bailey J. E.
Published: December 2004

The insect cell‐baculovirus model presented here is capable of simulating cell population dynamics, extracellular virion densities, and heterologous product titers in reasonable agreement with experimental data for a wide rang of multiplicities of infection (MOI) and times of infection. The model accounts for the infection of a single cell by multiple virions and the consequences on the time course of infection. The probability of infection by more than one virion was approximated using the Poisson distribution, which proved to be a refinement over second‐order kinetics. The model tracks initiation and duration of important events in the progression of infected cell development (virus replication, recombinant protein synthesis, and cell lysis) for subpopulations delineated by the time and extent of their initial infection. The model suggests infection strategies, weighing the importance of MOI and infection time. Maximum product titers result from infection in the early exponential growth phase with low MOI.
New approach for selective separation of dilute products from simulated clostridial fermentation broths using cyclodextrins
Authors: Shity Halla , Bar Raphael
Published: December 2004

The ability of cyclodextrins (CD) to form crystalline insoluble complexes with organics was explored in this study in view of a selective separation of dilute products obtainable from three clostridial fermentation systems. To this purpose, a product or a product mixture at a concentration of 0.150 mM each were treated with α‐CD or β‐CD (0.150 mM) in aqueous solutions as well as in a nutrient broth as a simulated fermentation medium. In the acetone‐butanol‐ethanol system, and in the butanol‐iso‐propanol system, α‐CD was found to precipitate selectively 48% and 46% butanol after 1 h agitation at 30°C. However, β‐CD was found to be superior for the butyric acid‐acetic acid system because it selectively precipitated 100% butyric acid under the same conditions. Cooling the three product system with α‐CD to 4°C for 24 h increased significantly the crop of precipitates but decreased the selectivity for either butanol or butyric acid. Cyclodextrins were thus shown to offer potentially a new exciting possibility for downstream processing of low‐concentration fermentation products.
Gas phase transesterification reactions catalyzed by lipolytic enzymes
Authors: Parvaresh Firooze , Robert Hervé , Thomas Daniel , Legoy Marie‐Dominique
Published: December 2004

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed‐bed gas‐solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (Aw) of the enzyme preparation.
Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles
Authors: Sarcar Shraboni , Jain Tanoj Kumar , Maitra Amarnath
Published: December 2004

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w0, pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w0 = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.
Knowledge‐based control of fermentation processes
Authors: Konstantinov Konstantin B. , Yoshida Toshiomi
Published: December 2004

A decade has passed since the first applications of a knowledge‐based approach to the control of bioprocesses were reported. During this period, both the development and application of intelligent control in biotechnology have undergone remarkable evolution in terms of concepts, objectives, and tools. Stimulated by rapid progress in the field of real‐time expert systems, knowledge‐based methodology for the control of fermentation processes has now reached a more mature phase. A growing interest among the biotechnology community and intensive, realistic, and fruitful research being undertaken both in universities and in industry suggest that large‐scale application of knowledge‐based systems for the control of bioprocesses is inevitable. This article provides a concise summary of the main achievements in this new area and discusses recent trends, porblems, and perspectives.
Epoxidation of propene by microbial cells immobilized on inorhanic supports
Authors: Kovalenko G. A. , Sokolovskii V. D.
Published: December 2004

Immobilization of gas‐utilizing microorganism strains (Mycobacteria, Rhodococcus, methane‐utilizers) on inorganic supports based on alumina, silicates, and carbon was carried out to develop heterogeneous biocatalysts for the biotechnologic processes, including the process of propene epoxidation. Adsorption ability of these microorganisms, biocatalytic properties of resting and immobilized bacterial cells, and effect of immobilization tehniques on biocatalysis were studied. An approach of double immobilization using inorganic materials (supports and gel) was proposed as simple, universal, and available methopd to immobilize bacterial cells, resulting in a higher retention (up to 100%) of cells' enzymatic activity and enhanced stability.
Protein chromatography in neat organic solvents
Authors: Chang Nancy , Klibanov Alexander M.
Published: December 2004

Pure formamide and ethylene glycol are used instead of water as processing media for protein chromatography. A number of common proteins are freely soluble in these solvents and most do not undergo irrersible inactivation in them. Batch adsorption studies reveal that proteins readily adsorbed to various ion‐exchangers in formamide and ethyline glycol and subsequently can be completely desorbed by adding inorganic salts (LiCl and NH4NO3) to the solvents. The idea of protein separations in formamide and ethylene glycol is illustrated by column chromatography and preparative separation of mixtures of (i) oxidized A and B chains of insulin and (ii) lysozyme and ribonuclease on the anion‐exchanger triethylaminoethycellulose and the cation‐exchanger phosphocellulose, respectively.
Kinetic analysis of hybridoma growth and monoclonal antibody production in semicontinuous culture
Authors: Leno M. , Merten O.‐W. , Hache J.
Published: December 2004

Hybridoma I.13.17 was grown in semicontinuous culture in an attempt to investigate the steady‐state concentrations of key components of monoclonal antibody (MAb) synthesis (e.g., intracellular MAb, IgG messenger RNAs) at different dilution rates between 0.008 and 0.055 h−1. There was a general trend of increasing steady‐state levels of total cytoplasmic RNA, total cell‐associated MAb or cytoplasmic MAb, DNA synthesis rate, cellular metabolic activity, heavy (H‐) and light (L‐) chain IgG mRNAs with the increase in dilution rates. Increase in the half‐lives of H‐ and L‐chain mRNAs with increase in dilution rates may be sufficient to account for their increasing levels found under the same conditions. The specific growth rate was profoundly affected by the dilution rate, particularly near the lower end of the dilution rate range. Linear relationships were observed between the steady‐state amounts of total cell‐associated MAb and the relative levels of H‐ and L‐chain mRNAs. Material balances on intracellular MAb demonstrated an increasing percentage of antibody not released into the growth medium (e.g., stored within the cell or anchored to the cell membrane) with increasing dilution rate. The MAb production rate per cell decreased significantly with the increase in dilution rates. No correlation was found between the relative levels of H‐ or L‐chain mRNAs and the specific MAb production rate. Possible implications of rate‐limiting steps in MAb synthesis and secretion are discussed.
Mathematical modeling of induced foreign protein production by recombinant bacteria
Authors: Lee Jongdae , Ramirez W. Fred
Published: December 2004

A new generalized mathematical model for recombinant bacteria which includes inducer effects on cell growth and foreign protein production is developed. The model equation set was applied to a host‐vector system, Escherichi coli D1210 and plasmid pSD8. Batch experiments were designed and performed in shake flasks to verify the model. A parameter estimation method was developed and proven to be efficient. Although simple, the model can effectively describe the dynamics of the production of foreign protein in recombinant bacteria and can be used for optimization and control studies to maximize foreign protein production.
Hydrolysis of butteroil by immobilized lipase using a hollow‐fiber reactor: Part I. Lipase adsorption studies
Authors: Malcata F. Xavier , Garcia Hugo S. , Hill Charles G. , Amundson Clyde H.
Published: December 2004

Adsorption of proteins from a crude preparation containing a lipase from Aspergillus niger on microporous polypropylene hollow fibers was studied at six different temperatures. Langmuir isotherms accurately describe the overall adsorption equilibria. Lipase is selectively adsorbed relative to the other proteins in the crude preparation. Hence, immobilization also provides further purification of the lipase. The predictions of the Langmuir model for the change in the specific activity of lipase upon adsorption are consistent with experimental results. The loading capacity of the hollow fibers decreases and the adsorption constant increases as temperature is increased. This effect is more significant in the case of lipolytic activity than it is for the total amount of adsorbed protein. Small, positive enthalpy changes are associated with the adsorption of lipase on these hydrophobic membranes.
Metabolic activity of CHO fibroblasts in HEMA–MMA microcapsules
Authors: Uludag Hasan , Sefton Michael V.
Published: December 2004

Chinese hamster ovary (CHO) fibroblast cells were microencapsulated in polyacrylate membranes (HEMA‐MMA: 75% HEMA) via an interfacial precipitation process. The CHO cells were observed to grow in large aggregates, attached to each other instead of to the capsule wall. When CHO cells were encapsulated at high density (4 × 106 cells/mL), the initial metabolic activity in microcapsules, as determined by the MTT assay, correlated with the polymer‐cell extrusion ratio, presumably because of the dependence of encapsulation efficiency on the relative flow rates. However, there was a large variation in the metabolic activity among individual microcapsules throughout the present study. Capsules with low encapsulation efficiency (at a “seeding” density of 4 × 106 cells/mL) exhibited a rapid increase in the metabolic activity during the following week. When CHO cells were encapsulated at low density (4 × 105 cells/mL), there was only a small increase in the metabolic activity. Only a small fraction (∼5%) of the capsules exhibited a high level of metabolic activity and 40% of the capsules exhibited undetectable metabolic activity even after 2 weeks. We conclude that CHO cells, which served as model cells, survive the encapsulation process and retain an active metabolic state once enclosed by the HEMA‐MMA membranes. However, the resultant microcapsules are extremely heterogeneous in the amount of retained metabolic activity.
Determination of total and active immobilized enzyme distribution in porous solid supports
Authors: Scharer Rolf , Hossain Md. M. , Do D. D.
Published: December 2004

The total and active immobilized enzyme (IME) distributions in porous supports are studied both theoretically and experimentally. In order to determine experimentally the enzyme distribution profiles within a single particle, we construct a diffusion cell containing controlled‐pore glass particles such that the cell would mimic a large pellet support. Our purpose is to study the interplay between the diffusion process within the interparticle void space and immobilization process in the controlled‐pore glass particles onto the evolution of the (total and active) enzyme distributions. A mathematical model is developed to describe the interaction of various processes within the diffusion cell. The immobilized enzymes are determined for a system of trypsin and controlled‐pore glass particles. The total amount of enzymes are determined by the amino acid analysis, and the active fraction is obtained by an active‐site titration. The experimentally measured total IME profiles compare very well with that predicted by the model. The determined active enzyme profile is found to be nonuniform one, and it represents about 40% of the total enzyme immobilized in the support particles.
Ion‐exchange and affinity chromatography costs in α‐galactosidase purification
Authors: Porter Jill E. , Ladisch Michael R.
Published: December 2004

The purification of α‐galactosidase from soybean seeds is a five to six‐step procedure consisting of cryoprecipitation, acid precipitation and ammonium sulfate fractionation followed by two or three chromatography steps. The procedures, while not optimized, were carried out in a manner that resulted in 414–515‐fold purification, as reported previously. The costs of two purification sequences were compared. In the best case, the preparative‐scale costs of stationary phase, reagents, and hardware were $790 per million enzyme units, excluding labor. Stationary phase costs predominated over extraction, chromatography reagent, and eluent costs when the stationary phase is replaced after 10–40 cycles of use. However, if stationary phase life exceeds 50–200 cycles, stationary phase costs become similar in magnitude to eluent and reagent costs. Labor costs, which are process‐specific and difficult to estimate, exceed all other costs by a factor of 10–50 at a small scale of operation and constitute a major cost, regardless of scale. This case study provides equations and a frame‐work for carrying out a first comparison of costs for multistep purification sequences. Column life, throughput, and scale of operation were found to determine not only the magnitude, but also the relative contributions, of the different components that make up purification costs. This analysis shows that there are major opportunities for reducing purification costs through the development of less expensive stationary phases and the implementation of intelligent process control and automation for process scale chromatography.
Irreversible enzyme‐shuttle immunoassay
Authors: Paek Se‐Hwan , Schramm Willfried
Published: December 2004

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte‐enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab1, and the enzyme component binds to a spatially separated immobilized antibody, Ab2. The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab1. With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab2. The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual‐antibody systems. The theoretical predictions are supported by experimental results. The dual‐antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.
Characterization of mixing in shaker table containers
Authors: Gardner Jeannie , Tatterson Gary
Published: December 2004

Mixing in shaker table beakers is studied using dye dispersion to measure mixing times. Experimental conditions range from the laminar regime into the turbulent mixing. Different flow patterns occurring in the beakers are reported for the mixing. The transition Reynolds number is determined. Rotational speed of the table, volume of material to be mixed, and viscosity of the material are studied as to their effects on mixing time. A graphical mixing time correlation is provided which is useful for the translation of mixing from laboratory scale to pilot scale.
Cellulase production in continuous culture by Trichoderma reesei on xylose‐based media
Authors: Schafner D. W. , Toledo R. T.
Published: December 2004

Trichoderma reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m3 occurred at pH 4.0 and a dilution rate of 0.0391 h−1 where residual substrate was 0.43 kg/m3, but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at pH 3.5 and a dilution rate of 0.0110 h−1, where cell mass production was 2.56 kg/m3 and residual substrate was 0.15 kg/m3. Monod kinetic constants, corrected for endogenous metabolism, were 0.091 h−1, 0.469 kg/m3, 0.00923 h−1, and 0.470 kg cells/kg xylose at pH 3.5, for the maximum specific growth rate, Michaelis–Menten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing pH.
Effect of serum concentration on hybridoma viable cell density and production of monoclonal antibodies in cstrs and on shear sensitivity in air‐lift loop reactors
Authors: Martens D. E. , Beuvery E. C. , De Gooijer C. D. , Tramper J.
Published: December 2004

The death rate of hybridoma cells, grown in a continuous culture, has been studied in a small air‐lift loop reactor as a function of reactor height and injected gas flow rate. The first‐order death‐rate constant was found to be proportional to the reciprocal height and to the gas flow rate, in accordance with the hypothetical killing volume model for insect cells in bubble columns. Furthermore, the effect of the serum concentration on viable cell concentration and cell productivity has been investigated in a continuous culture. A serum component became growth limiting when the serum concentration was decreased from 2% to 1%. No effect of the serum concentration on specific cell productivity could be measured. Samples from this culture were also studied in the air‐lift loop reactor to determine the effect of serum concentration on the shear sensitivity. The cells' shear sensitivity increased with decreasing serum concentration. The protective effect of serum was found to be physical as well as physiological.
Quantification of the intragranular porosity formed in bioleaching of pyrite by Thiobacillus ferroxidans
Authors: Mustin C. , de Donato Ph. , Berthelin J.
Published: December 2004

During the bacterial oxidation of a pure pyrite by Thiobacillus ferrooxidans, a great number of corrosion tunnels appear that are easily revealed by scanning electron microscopy observations. This involves an increase in the surface area without significant granulometric reduction of mineral grains. Thus, the evaluation of intragranular porosity, determined by elution front analysis, allows one to estimate accurately the fraction of oxidized sulphide, because of the development of deep holes (propagating pore mechanism). After 60 days of bioleaching, the intragranular porosity represents about 34% of the initial sulphide volume, which corresponds to 25 km of tunnels (2 μm i.d.) per gram of pyrite. On other hand, the granulometric reduction (≈7%) is responsible for a 23% decrease of the initial sulphide volume. The elution front analysis appears as a nondestructive method for measuring the intragranular porosity of the bioleached pyrite.
An experimental study of mass diffusion and reaction rate in an anaerobic biofilm
Authors: Kitsos H. M. , Roberts R. S. , Jones W. J. , Tornabene T. G.
Published: December 2004

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 μm was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 μm. Deviation from linearity appeared in biofilm thicknesses greater than 800 μm. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.
Effect of hydration on the morphology of enzyme powder
Authors: Roziewski Kristin , Russell Alan J.
Published: December 2004

We report the first direct images of the hydration of protein powders. Using an environmental scanning electron microscope (ESEM) we have taken a series of micrographs of a region of the enzyme (subtilisin) power whilst hydrating the sample. In addition, the sample has been viewed during exposure to toluene vapors. The ESEM is a remarkable new instrument that will have wide applicability in imaging of biological materials in their native environments.
Hybridoma perfusion systems: A comparison study
Authors: de la Broise Denis , Noiseux Manon , Massie Bernard , Lemieux Réal
Published: December 2004

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long‐term perfusion cultures. The results showed that steady‐state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactro, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a percell basis. This result is well in agreement with the previously presented model of “grow or die” cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.
Dye‐ligand membranes as selective adsorbents for rapid purification of enzymes: A case study
Authors: Champluvier B. , Kula M.‐R.
Published: December 2004

A new adsorbent for the selective binding of enzymes, in the form of microporous membranes carrying triazine dyes as pseudo‐affinity ligand, has been implemented in the recovery of glucose‐6‐phosphate dehydrogenase from yeast. A detailed investigation of the process parameters has been performed. In the adsorption step, the contact time for binding G6PDH could be reduced down to 0.25 s without significant decrease of the capture efficiency. Hence, fast filtration allowed to isolate G6PDH from a dilute extract (1.6 μg G6PDH · mL−1), where the enzyme accounted for 1% of the proteins. The yield of the selective elution step using NADP was only 70% at best. It could be improved to near 100% by supplementing the eluent with ethylene glycol, without loss of selectivity. A Scale‐up of the cross‐section of the membrane by a factor of 40 allowed to purify 1140 U from 0.6 L extract from 1% to 57% purity with 82% yield, within 10 minutes. The case study presented here demonstrates the applicability of general‐purpose membrane adsorbents for the purification of enzymes.
Recovery of canola meal proteins by precipitation
Authors: Chen M. , Rohani S.
Published: December 2004

Recovery of rapeseed proteins from defatted canola meal by precipitation was investigated. The ability of different precipitating agents, such as sodium hexametaphosphate (HMP), carboxymethylcellulose (CMC), ammonium sulphate, and isoelectric precipitation using HCl, were evaluated based on the yield and mean size of protein aggregates. Almost 94% of dissolved protein was precipitated in the presence of 2.7M ammonium sulphate, while the largest mean protein particle size (32 μm) was obtained in the presence of HMP at pH 3.3.
Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells
Authors: Pendse Girish J. , Karkare Subhash , Bailey James E.
Published: December 2004

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster overy (CHO) cell line producing recombinant hepatits B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gane copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autroadiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse‐chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5‐h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.
Thermostability of soluble and immobilized α‐amylase from Bacillus licheniformis
Authors: De Cordt S. , Vanhoof K. , Hu J. , Maesmans G. , Hendrickx M. , Tobback P.
Published: December 2004

In view of a possible application of the α‐amylase from Bacillus licheniformis as a time–temperature integrator for evaluation of heat processes,11 thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90–108°C. The D‐values (95°C) for inactivation of α‐amylase, dissolved in tris‐HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca2+ and enzyme concentration. The z‐value fluctuated between 6.2 and 7.6°C. On immobilization of the α‐amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D‐values (95°C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z‐value fluctuated between 8.1 and 12.9°C. In each case of biphasic inactivation, the z‐value of the stable fraction (with the higher D‐values) was lower than the z‐value of the labile fraction. © 1992 John Wiley & Sons, Inc.
Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth
Authors: Park Chang‐Ho , Martinez Blanca C.
Published: December 2004

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. © 1992 John Wiley & Sons, Inc.
Properties of microfiltration membranes: The effects of adsorption and shear on the recovery of an enzyme
Authors: Bowen W. Richard , Gan Quan
Published: December 2004

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low‐shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. © 1992 John Wiley & Sons, Inc.
Biofilm reactors: An experimental and modeling study of wastewater denitrification in fluidized‐bed reactors of activated carbon particles
Authors: Coelhoso Isabel , Boaventura Rui , Rodrigues Alírio
Published: December 2004

A fluidized‐bed biofilm reactor using activated carbon particles of 1.69 mm diameter as the support for biomass growth and molasses as the carbon source is used for wastewater denitrification.

The start‐up of the reactor was successfully achieved in 1 week by using a liquor from garden soil leaching as the inoculum and a superficial velocity u0 = 5umf. Typical biofilm thickness is 800 μm; therefore covered activated carbon particles have 3.3 mm in diameter.

Reactor hydrodynamics was studied by tracer (KCl solution) experiments. The analysis based on residence time distribution theory involved a model with axial dispersion flow and tracer diffusion with linear adsorption inside the biofilm. Peclet numbers higher than 100 were found, allowing the plug flow assumption for the reactor model.

Experimental profiles of nitrate and nitrite species were explained by a kinetic model of two consecutive zero‐order reactions coupled with substrate diffusion inside the biofilm. Under the operating conditons used thick biofilms were obtained working in a diffusion‐controlled regime.

Comparison is made with results obtained in the same reactor with sand particles as the support for biomass growth. Activated carbon as the support has the following advantages: good adsorptive characteristics, homogeneous biofilm thickness along the reactor, and easy restart‐up of the reactor. © 1992 John Wiley & Sons, Inc.
Estimation of disruption of animal cells by laminar shear stress
Authors: Born C. , Zhang Z. , Al‐Rubeai M. , Thomas C. R.
Published: December 2004

Using mechanical cell properties measured by micromanipulation, and a model of cell distortion in laminar flow fields, a method has been developed for predicting disruption of animal cells by laminar shear stresses. Predictions of the model were compared with measured losses of cell number and viability of TB/C3 murine hybridomas sheared in a cone and plate viscometer at shear rates up to 3950 s−1, and shear stresses up to 600 Nm−2, achieved by enhancement of viscosity with dextran. In all cases, the experimental, results and predictions were within 30%. Such excellent agreement suggests it might be possible to use micromanipulation measurements of animal cell mechanical properties to predict cell damage in more complex flow fields, such as those in bioreactors. © 1992 John Wiley & Sons, Inc.
An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics
Authors: Ogden Kimberly L. , Davis Robert H. , Taylor Autin L.
Published: December 2004

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth‐rate differential between plasmid‐bearing and plasmid‐free cells, and thereby affects the culture dynamics of a plasmid‐containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid‐bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild‐type levels, and thus have more of a metabolic burden than do the plasmid‐bearng cells that overproduce CAT. However, when the level of the plasmid‐directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid‐bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid‐containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. © 1992 John Wiley & Sons, Inc.
The extended serial subculture of human diploid fibroblasts on microcarriers using a new medium supplement formulation
Authors: Forestell Sean P. , Kalogerakis Nicolas , Behie Leo A.
Published: December 2004

Human diploid fibroblasts serially passaged on microcarriers exhibit a decrease in their proliferative capacity with each transfer from microcarrier‐to‐microcarrier. This phenomenon, which does not occur in the same time scale with cells cultured in T‐flasks, has been a serious barrier to the systematic utilization of microcarriers in the scale‐up of anchorage‐dependent human diploid cell cultures. This decreases in cell growth with each passage is shown to be related to the serum content of the medium, with high serum concentrations resulting in a more rapid decrease in cell growth with each serial transfer. As a result, methods for reducing the serum requirement of the cells were investigated. A new medium supplement mixture, PPRF92, has been developed, which allows the serial passaging of MRC5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier‐to‐microcarrier tranfers, and at a serum levels as low as 1%, with no decrease in the proliferative capacity of the cells until they approach their reported population doubling limit. This new supplement mixture is a significant improvement to microcarrier technology in that it enables the use of microcarriers in the early stages of inocculum build‐up for the production purposes. © 1992 John Wiley & Sons, Inc.
Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements
Authors: Smith Craig G. , Greenfield Paul F.
Published: December 2004

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS‐supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum‐free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein‐free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient‐rich basal media, in addition to the considerations of gas bubble effects described by other workers. © 1992 John Wiley & Sons, Inc.
Activity and stability of an entrapped‐cell system for the Δ1‐dehydrogenation of steroids in organic media
Authors: Pinheiro H. M. , Cabral J. M. S.
Published: December 2004

Whole Arthrobacter simplex cells entrapped in κ‐carra‐geenan or in two types of polyurethane foam, or adsorbed on silanized Celite, were tested for the Δ1‐dehydrogenation of hydrocortisone and its derivatives in organic media. Catalytic activity and stability levels were evaluated for the immobilized cells in buffer with 2.5% (vol/vol) methanol, and in three buffer‐saturated solvents (n‐octan‐1‐ol, n‐decan‐1‐ol, and chloroform). The addition of glutamate to the immobilization support stabilized the activity of the immobilized cells in the tested organic media. The system with polyurethane (HYPOL6100)‐entrapped cells (with coimmobilized glutamate) in n‐decan‐1‐ol provided the highest long‐term activity levels. Several factors involved in the polyurethane‐entrapment procedure were also studied. © 1992 John Wiley & Sons, Inc.
Amino acid supplementation decreases plasmid retention in Escherichia coli
Authors: Shu J. , Shuler M. L.
Published: December 2004

The effect of amino acid supplementation on plasmid stability in Escherichia coli B/r was tested experimentally. Comparisons of experimental results to computer‐predicted values were made using a detailed, structured single‐cell model. The plasmid, pDW17 (a pBR322 derivative with a mutated tac promoter controlling the β‐lactamase gene), was used. In chemostat cultures, the amino acid supplemented cultures were always less stable than those grown in minimal medium. This effect was not a growth rate effect, as increasing growth rate imsproves stability for both cultures in minimal medium and in amino acid supplemented medium. The computer model also predicted a decrease in stability due to amino acid supplementation. The model also predicts that amino acid supplementation, combined with moderately strong plasmid‐encoded protein expresion, results in a depletion of low‐molecular‐weight organics compared with plasmid‐free cells. In minimal medium the same level of plasmid‐encoded protein synthesis results in a strong reduction in amino acid pools compared with plasmid‐free cells. With amino acid supplementation the growth differential between plasmid‐bearing and plasmid‐free cells may be due to an “energy limitation,” while in minimal medium the size of the growth rate differential may be due to a “building block” limitation. © 1992 John Wiley & Sons, Inc.
Cell growth on immobilized cell growth factor. 8. Protein‐free cell culture on insulin‐immobilized microcarriers
Authors: Ito Yoshihiro , Uno Tsuyoshi , Liu Shu Qin , Imanishi Yukio
Published: December 2004

In order to develop a new protein‐free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage‐dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM1C1, and the anchorage‐independent cells, mouse hybridoma cells SJK132‐20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin‐immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage‐dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin‐immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use. © 1992 John Wiley & Sons, Inc.
Studies of the cell‐wall properties of Saccharomyces cerevisiae during fermentation
Authors: Bowen W. Richard , Sabuni Hoze A. M. , Ventham Timothy J.
Published: December 2004

The cell‐wall properties of three strains of the yeast Sacharomyces cerevisiae have been experimentally studied at various times during fermentation. The cell walls have been characterized by electrophoretic mobility measurements, from which zeta potentials may be calculated. They have also been characterized by computerized pH titration, which gives direct information on the number and nature of groups in the yeast cell wall. The data have been quantitatively analyzed in three ways. First, a simplified analysis of the electrokinetic data of a type used by previous workers has been applied. Second, such a simplified analysis of the electrokinetic data has been developed more rigorously by means of a two‐dimensional site‐dissociation model of the outer cell wall–solution interface. Third, a description of the yeast cell‐wall electrochemical properties in terms of a three‐dimensional gel model incorporating site dissociation has been developed. The advantages and disadvantages of the three analyses are discussed. Only the three‐dimensional gel model can account simultaneously for both the electrokinetic and pH surface titration data. It provides new insights into the changes that occur to the yeast cell wall during fermentation. © 1992 John Wiley & Sons, Inc.
Effects of nonionic surfactants on the solubilization and mineralization of phenanthrene in soil–water systems
Authors: Laha Shonali , Luthy Richard G.
Published: December 2004

The solubilization and mineralization of 14C‐phenanthrene in soil–water systems was examined with several commercially available surface‐active agents, viz., an alkyl ethoxylate C12E4; two alkylphenol ethoxylate surfactants: C8PE9.5 and C9PE10.5; two sorbitan ethoxylate surfactants: the sorbitan monolaurate (Tween 20) and the sorbitan monooleate (Tween 80); two pairs of nonionic ethoxylate surfactant mixtures: C12E4/C12E23 at a 1:1 ratio, and C12–15E3/C12–15E9 at a 1:3 ratio; and two surfactants possessing relatively high critical micelle concentration (CMC) values and low aggregation numbers: CHAPS and octyglucoside. Surface tension experiments were performed to evaluate surfactant sorption onto soil and the surfactant doses required to attain the CMC in the soil–water systems. Surfactant solubilization of 14C‐phenanthrene commenced with the onset of micellization. The addition of surface‐active agents was observed not to be beneficial to the microbial mineralization of phenanthrene in the soil–water systems and, for supra‐CMC surfactant doses, phenanthrene mineralization was completely inhibited for all the surfactants tested. A comparison of solubilization, surface tension, and mineralization data confirms that the inhibitory effect on microbial degradation of phenanthrene is related to the CMC of the surfactant in the presence of soil. Additional tests demonstrated the recovery of mineralization upon dilution of surfactant concentration to sub‐CMC levels, and a relatively high exit rate for phenanthrene from micelles. These tests suggest that the inhibitory effect is probably related to a reversible physiological surfactant micelle–bacteria interaction, possibly through partial complexing or release of membrane material with disrupting membrane lamellar structure. This study indicates that nonionic surfactant solubilization of sorbed hydrophobic organic compounds from soil may not be beneficial for the concomitant enhancement of soil bioremediation. Additional work is needed to address physicochemical processes for bioavailability enhancement, and effects of solubilizing agents on microorganisms for remediation and treatment of hydrophobic organic compounds and nonaqueous phase liquids. © 1992 John Wiley & Sons Inc.