bluuebunny/crossref_metadata_2025 huggingface dataset:
Microencapsulated human bone marrow cultures: A potential culture system for the clonal outgrowth of hematopoietic progenitor cells
Authors: Levee Minette G. , Lee Gyun‐Min , Paek Se‐Hwan , Palsson Bernhard O.
Published: December 2004

Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate–poly‐L‐lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24‐fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst‐forming units erythroid (BFU‐E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.
Biosorption of lead and nickel by biomass of marine algae
Authors: Holan Z. R. , Volesky B.
Published: December 2004

Screening tests of different marine algae biomas types revealed a high passive biosorptive uptake of lead up to 270 mg Pb/g of biomass in some brown marine algae. Members of the order Fucales perfomed particularly well in this descending sequence: Fucus > Ascophyllum > Sargassum. Although decreasing the swelling of wetted biomass particles, their reinforcement by crosslinking may significantly affect the biosorption performance. Lead uptakes up to 370 mg Pb/g were observed in crosslinked Fucus vesiculosus and Ascophyllum nodosum. At low equilibrium residual concentrations of lead in solution, however, ion exchange resin Amberlite IR‐120 had a higher lead uptake than the biosorbent materials. An order‐of‐magnitude lower uptake of nickel was observed in all of the sorbent materials examined. © 1994 John Wiley & Sons, Inc.
Fed‐batch cultivation of animal cells using different medium design concepts and feeding strategies
Authors: Xie Liangzhi , Wang Daniel I. C.
Published: December 2004

In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end‐products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL‐1606) was cultivated in T‐flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed‐batch culture and by 26‐fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed‐batch culture and by 50‐fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five‐ and tenfold respectively, compared with conventional batch culture. © 1994 John Wiley & Sons, Inc.
Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin
Authors: Huth Jeffrey R. , Weijun Feng , Ruddon Raymond W.
Published: December 2004

The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the β subunit of human chorionic gonadotropin (hCG‐ β), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG‐β were investigated: the rate‐limiting events in the folding of this protein, and the assembly of hCG‐β with, hCG‐α. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate‐limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG‐β folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10μM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half‐time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the β subunit under these conditions yields a functional protein, based on its ability to assemble with the α subunit. The rates of assembly of hCG‐β with hCG‐α in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t1/2/sb> = 9 to 12 min). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine. © 1994 John Wiley & Sons, Inc.
Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease
Authors: Nohara Daisuke , Yamada Toshiyasu , Watanabe Akiko , Sakai Tomoya
Published: December 2004

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10°C and 85°C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol−1 at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol−1 and 18.8kJ mol−1. The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. © 1994 John Wiley & Sons, Inc.
Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture
Authors: Park Sun Ho , Ryu Dewey D. Y.
Published: December 2004

The flow‐cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. © 1994 John Wiley & Sons, Inc.
Morphological kinetics and distribution in somatic embryo cultures
Authors: Chi Chung‐Ming , Vits Hugo , Staba E. John , Cooke Todd J. , Hu Wei‐Shou
Published: December 2004

The environmental effects on developing somatic embryos should be characterized not only by the growth based on biomass, but also by the morphological properties and size. We have previously developed a discrete classifier to separate developing embryos into distinct morphological classes. In this study, a continuous descriptor using the distributions of magnitude of features representing morphological characteristics and size information was used to describe the developing embryo populations. The identity of the population was examined by comparing either the distributions of all features or key features. The method was applied to characterize the kinetics of carrot embryo populations cultivated in the presence and absence of triiodobenzoic acid(TIBA), an inhibitor of auxin polar transport. Optimal sample size for morphological characterization was determined by the invariance of feature distributions with further increase in sample size. The overall growth and substrate consumption kinetics were only slightly affected by the presence of TIBA. However, the distribution of morphological features was significantly affected. The features showing the highest statistical significance were related to those corresponding to the roughness. The continuous descriptor for characterizing developing embryo population is potentially useful for quality control in large‐scale operations. © 1994 John Wiley & Sons, Inc.
Ethene removal from a synthetic waste gas using a dry biobed
Authors: De Heyder Bart , Overmeire Ann , Van Langenhove Herman , Verstraete Willy
Published: December 2004

A packed granular activated carbon (GAC) biobed, inoculated with the ethane‐degrading strain Mycobacterium E3, was used to study ethene removal from a synthetic waste gas. Ethene, for which the dimensionless partition coefficient for an air‐water system at 20°C is about 7.6, was used as a model compound for poorly water soluble gaseous pollutants. In a first mode or operation, the GAC biobed was sprinkled intermittently and the waste gas influent was continuously pre‐humidified, establishing relatively moist conditions (water content >40% to 45%). A volumetric ethene removal rate of 0.382 kg COD · m−3 · d−1 (0.112 kg ethene · m−3 · d−1) was obtained for an influent concentration of 125 ppm, a superficial waste gas velocity of 3.6E−3 m · s−1 and a pseudo residence time of 45 s. However, in the second mode of operation, omitting the pre‐humidification of the waste gas influent and establishing a “dry” biobed (water content <40% to 45%), and thus obtaining better mass transfer to the biofilm, the ethene removal could be doubled for otherwise comparable operating parameters. Furthermore, under decreased wetting and for the given experimental conditions (influent concentration 125 to 816 ppm, waste gas superficial velocity 3.0E−3 m ·s−1, pseudo waste gas residence time 43 s), the ethene removal was not limited by mass transfer of ethene through the water layer covering the biofilm. © 1994 John Wiley & Sons, Inc.
Protein complexation with acrylic polyampholytes
Authors: Patrickios Costas S. , Hertler Walter R. , Hatton T. Alan
Published: December 2004

The interaction of dilute mixtures of proteins and ABC triblock methacrylic polyampholytes at different values of pH was investigated turbidimetrically. The onset of interaction was manifested by large changes in turbidity at certain critical pHs which lie close to the isoelectric points of the two interacting components. Protein precipitation yields in protein‐polyampholyte binary mixtures followed the corresponding turbidity profiles and varied from 10% to 90%. The synthetic polyampholytes self‐aggregate around their isoelectric point. The kinetics of precipitation of one of the same polymer with soybean trypsin inhibitor were studied, with turbidity‐based characteristic times (exponential fit) of 2–3 min. The kinetics of precipitation of the protein‐polymer mixture are slower than that of pure polymer because a small, but steady, long‐term increase in turbidity is observed in the former case. The pH‐dependence of the turbidity of binary mixtures of one protein and one synthetic polyampholyte, as well as a tertiary mixture of two proteins and one polyampholyte, were measured 30 min after the pH adjustment. The observations in these experiments along with the measured protein precipitation yields in the binary mixtures and the polyampholyte self‐aggregation can be used for polymer removal and recycling. The latter constitutes a significant advantage over the use of homopolyelectrolytes which cannot easily be recycled. © 1994 John Wiley & Sons, Inc.
Immobilization of “Disguised” yeast in chemically crosslinked chitosan beads
Authors: Freeman Amihay , Dror Yael
Published: December 2004

A new simple method for the preparation of chemically crosslinked chitosan beads is presented. It consists of the dropwise addition of 2–3% (w/v) low molecular weight chitosan solution containing 2% (w/v) glyoxal in 1% (w/v) tetrasodiumdiphosphate, pH 8.0. Immobilized viable baker's yeast (Saccharomyces cerevisiae) could be obtained via gel entrapment within the new beads when means preventing their direct contact with soluble chitosan were provided, “disguising” the cells until gelation and crosslinking were completed. Such means included cell suspension in castor oil or mixing with carboxymethyl‐cellulose powder. Application of these means was shown to be necessary, as cells exposed to soluble chitosan immediately lost their viability and glycolytic activity. Yeast disguised in castor oil was also protected from bead reinforcement by glutaraldehyde treatment, significantly strengthening bead stability while operating under acidic conditions. This capability was demonstrated by continuous ethanol production by chitosan entrapped yeast. © 1994 John Wiley & Sons, Inc.
A dense cell retention culture system using stirred ceramic membrane reactor
Authors: Suzuki Takahiro , Sato Takeshi , Kominami Minoru
Published: December 2004

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the kLa to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed‐batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. © 1994 John Wiley & Sons, Inc.
Hydrogen production from organic substrates in an aerobic nitrogen‐fixing marine unicellular cyanobacterium Synechococcus sp. strain Miami BG 043511
Authors: Luo Yao‐Hua , Mitsui Akira
Published: December 2004

Synechococus sp. strain Miami BG 043511 exhibits very high H2 photoproduction from water, but the H2 photoproduction capability is lost rapidly with the age of the batch culture. The decreases of the capability coincides with the decrease of cellular glucose (glycogen) content. However, H2 photoproduction capability can be restored by the addition of organic substrates. Among 40 organic compounds tested, carbohydrates such as glucose, fructose, maltose, and sucrose were effective electron donors. Among organic acids tested, only pyruvate was an effective electron donor. Among alcohols tested, glycerol was a good electron donor. These results demonstrate that this unicellular cyanobacterium exhibits a wide substrate specificity for H2 photoproduction but has a different substrate specificity compared to photosynthetic bacteria. The maximum rates of H2 photoproduction from a 6‐day‐old batch culture with 25 mmol of pyruvate, glucose, maltose, sucrose, fructose, and glycerol were 1.11, 0.62, 0.50, 0.47, 0.30, and 0.39 µmoles per mg cell dry weight per hour respectively. Therefore, this cyanobacterium strain may have a potential significance in removing organic materials from the wastewater and simultaneously transforming them to H2 gas, a pollution free energy. The activity of nitrogenase, which catalyzes hydrogen production, completely disappeared when intracellular glucose (glycogen) was used up, but it could be restored by the addition of organic substrates such as glucose and pyruvate. © 1994 John Wiley & Sons, Inc.
Catalytic behavior of Pseudomonas cepacia lipase in w/o microemulsions
Authors: Stamatis H. , Xenakis A. , Dimitriadis E. , Kolisis F. N.
Published: December 2004

The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis‐(2‐ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping‐pong bi–bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be Km octanol = 310 mM, Km lauric acid = 78 mM, and Vmax = 250 μmol min−1 mg−1. The same system was used for the synthesis of mono‐ and diglycerides from glycerol and lauric acid, which was successful at very low wo values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C12E4). The optimum activity was found at about wo = 8. The apparent values of Vmax app and Km app for octanol were calculated and found to be 100 μmol min−1 mg−1 and 76 mM, respectively. © 1995 John Wiley & Sons, Inc.
Influence of cell‐ and media‐derived factors on the integrity of a human monoclonal antibody after secretion into serum‐free cell culture supernatants
Authors: Ackermann Marcus , Jäger Volker , Marx Uwe
Published: December 2004

To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB‐SF‐21 AE) and four mammalian cell lines [CHO K1, BHK‐21 (C13), C1271, P3‐X63‐Ag8.653], all of them commonly used for the production of recombinant proteins, and the human–human–mouse heterohybridoma cell line itself (H‐CB‐hahE), were adapted to serum‐free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum‐free media supplemented with 30 μg mL−1 of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS‐PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB‐SF‐21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well‐known advantages in posttranslational modifications (e.g., formation of complex type N‐glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus‐infected insect cells. © 1995 John Wiley & Sons, Inc.
Cultivation of hybridoma cells in an inclined bioreactor
Authors: Lu George Z. , Gray Murray R. , Thompson B. G. , Suresh M. R.
Published: December 2004

Murine hybridoma cells were grown in a bubble column that was inclined up to 45° from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30° gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. © 1995 John Wiley & Sons, Inc.
Diffusion of Cu2+ in calcium alginate gel beads: Further analyses
Authors: Chaiken Robert F.
Published: December 2004

The diffusivity of Cu2+, as determined by previous authors from analysis of experimental data in terms of the shrinking core (SCM) and linear absorption (LAM) models, is examined in light of the ability of the models to curve fit all the data. It is concluded from this further analysis that previous conclusions depicting the LAM to have an advantage over the SCM for predictive value are not justified. It is also shown that equally good curve fits can be obtained with a recent absorption/desorption model of diffusion which considers directly, through distribution theory, the effect of heterogeneity of material properties on the rate of diffusion. © 1995 John Wiley & Sons, Inc.
A modeling study of anaerobic biofilm systems: II. Reactor modeling
Authors: Flora Joseph R. V. , Suidan Makram T. , Biswas Pratim , Sayles Gregory D.
Published: December 2004

A rigorous steady‐state model of anaerobic biofilm reactors taking into account acid–base and gas–phase equilibria in the reactor in conjunction with detailed chemical equilibria and mass transfer in acetate‐utilizing methanogenic biofilms is presented. The performances of ideal completely stirred tank reactors (CSTRs) and plug‐flow reactors, as well as reactors with nonideal hydraulic conditions, are simulated. Decreasing the surface loading rate increases the acetate removal efficiency, while decreasing the influent pH and increasing the buffering capacity improves the removal efficiency only if the bulk pH of the reactor shifts toward more optimal values between 6.8 to 7.0. The reactor can have negative or positive removal efficiencies depending on the start‐up conditions. The respiration coefficient plays a critical role in determining the minimum influent pH required for reactor recovery after failure. Having multiple CSTRs‐in‐series generally increases the overall removal efficiency for the influent conditions investigated. Monitoring of the influent feed quality is critical for plug‐flow reactors, becasue failure of the initial sections of the reactor may cause a cascading effect that may lead to a rapid reactor failure. © 1995 John Wiley & Sons, Inc.
Development of a predictive description of an immobilized‐cell, three‐phase, fluidized‐bed bioreactor
Authors: Petersen James N. , Davison Brian H.
Published: December 2004

A fully predictive mathematical description of a three‐phase, tapered, fluidized‐bed bioreactor is developed. This mathematical model includes the effects of the tapered bed, variable dispersion coefficient, and variable solid holdup upon the concentration profiles developed in the bed. In addition, the effect of the concentration profile which is developed inside the biocatalyst bead is included by means of an effectiveness factor calculation. Using accepted correlations for the dispersion coefficient and for the liquid, gas, and solid holdup in the bed, the model is fully predictive. The model was found to adequately predict experimental obtained concentration profiles. Then, the model was used to examine the various phase holdups through the bed and the degree to which the dispersion coefficient varied through the bed. The effect of changes in these calculated variables upon the reaction rate is discussed. © 1995 John Wiley & Sons, Inc.
Nitrite inhibition of denitrification by Pseudomonas fluorescens
Authors: Almeida J. S. , Júlio S. M. , Reis M. A. M. , Carrondo M. J. T.
Published: December 2004

Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred‐tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 μg N/L. Denitrification activity was partially associated with growth, as described by the Luedeking–Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. © 1995 John Wiley & Sons, Inc.
Cultivation of cell–polymer tissue constructs in simulated microgravity
Authors: Freed L. E. , Vunjak‐Novakovic G.
Published: December 2004

Tissue‐engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three‐dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free‐fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid‐construct velocity was comparable to the experimentally measured construct settling velocity (2–3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm2). Rotating bioreactors thus provide a powerful research tool for cultivating tissue‐engineered cartilage and studying 3D tissue morphogenesis under well‐defined fluid dynamic conditions. © 1995 John Wiley & Sons, Inc.
Mass transfer and temperature effects on substrate utilization in brewery granules
Authors: Wu May M. , Criddle Craig S. , Hickey Robert F.
Published: December 2004

Liquid film and diffusional resistances of brewery granules during acetate, propionate, and ethanol utilization were investigated. Substrate utilization rate increased with decreased granule size. Effectiveness factors for acetate, propionate, and ethanol were calculated by comparing the maximum rates of substrate utilization of whole granules (1.8 to 3.0 mm) and fine flocs (20 to 75 μm) derived by disrupting whole granules. For acetate, propionate, and ethanol, maximum specific substrate utilization rates (km′ g/g VS · d) for the flocs, were 5.11, 6.25, and 5.49, respectively, and half‐velocity coefficients (Kg′ mM) were 0.45, 0.40, and 3.37, respectively. Calculated effectiveness factors were 0.32, 0.41, and 0.75 for acetate, propionate, and ethanol, respectively. The effect of temperature on substrate utilization was examined at 26°C, 31°C, and 37°C using acetate as sole carbon source. Utilization rates increased with temperature. Flocs were most sensitive to temperature, and whole granules were least affected. The behavior of flocs was well described by the Van't Hoff–Arrhenius equation. Effectiveness factors for acetate utilization by the granules were 0.36, 0.35, and 0.32 at 26°C, 31°C, and 37°C, respectively, indicating little effect of temperature. Based on these results, we conclude that both liquid film and diffusional resistances influenced the rate of substrate utilization in a UASB reactor with granular sludge. Temperature effects were much less important than diffusional limitations within the granules. © 1995 John Wiley & Sons, Inc.
Polysulfone affinity membranes for the treatment of amino acid mixtures
Authors: Rodemann Klaus , Staude Eberhard
Published: December 2004

Affinity membranes for the treatment of solutions containing amino acids were obtained via lithiating polysulfone that was subsequently converted with glycidylether. From this polymer asymmetric ultrafiltration membranes were cast. The membranes were reacted with iminodiacetic acid yielding membranes fitted out with bidentate chelates. The same reaction path was applied to commercially available symmetric microfiltration membranes. The chelate‐bearing membranes were complexed with Cu, Ni, and Zn ions. For the experiments with amino acids only the Cu‐complexed membranes were used. The complexation constants for histidine and tryptophan for six different membranes were determined. Because of the affinity of these two amino acids for the complexed Cu ions, they could easily be separated from solutions containing amino acids such as alanine, glycine, and valine. Also, concentrating very dilute amino acid solutions was carried out successfully. © 1995 John Wiley & Sons, Inc.
Process optimization and modeling of trichlorophenol degradation by Phanerochaete chrysosporium
Authors: Pal Nirupam , Lewandowski Gordon , Armenante Piero M.
Published: December 2004

The biodegradation of 2,4,6‐trichlorophenol and 2,4,5‐trichlorophenol by the white rot fungus Phanerochaete chrysosporium was studied in batch and continuous reactor systems. Experiments were conducted in shake flasks as well as in packed‐bed reactors in which the fungus was immobilized. The degradation rates in the packed‐bed reactors were found to be two orders of magnitude greater than those obtained in the shake flasks in which the fungus was just suspended. The degradation rate was found to be influenced by the concentrations of the carbon and nitrogen sources, pH, and fluid shear stress. Optimal ranges of these parameters to maximize biodegradation were determined. A mathematical model was developed in which the degradation process was assumed to consist of two sequential reaction steps, the first catalyzed by an extracellular enzyme system and the second requiring the presence of the mycelium. The deactivation of the extracellular enzyme system was also accounted for in the model. The Michaelis–Menten and the enzyme deactivation parameters were determined independently. Good agreement between the experimental data and the results produced by the regression was found. © 1995 John Wiley & Sons, Inc.
Effects of temperature and cycloheximide on secretion of cloned invertase from recombinant Saccharomyces cerevisiae
Authors: Marten Mark R. , Chung Bong‐Hwan , Seo Jin‐Ho
Published: December 2004

The effects of temperature on the kinetics and efficiency of secretion of cloned invertase were investigated in a recombinant yeast system. This system consisted of the baker's yeast Saccharomyces cerevisiae (SEY2102) transformed with the 2μ‐based plasmid pBR58 which contains the entire SUC2 gene including the promoter, signal sequence, and structural gene. The recombinant yeast produces the naturally secreted yeast enzyme invertase. In transition experiments done at temperatures ranging from 25° to 45°C, the maximum invertase level and secretion rate exhibited maxima of 5.5 U/mL · OD and 4.6 U/mL · OD per hour, respectively, at 35°C. Experiments involving the use of cycloheximide showed that it took approximately 15 min for secreted invertase to move through the secretion pathway, which held 0.4 U/mL · OD of specific activity. © 1995 John Wiley & Sons, Inc.
Protein recovery from cell debris using rotary and tangential crossflow microfiltration
Authors: Parnham Charles S. , Davis Robert H.
Published: December 2004

Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long‐term permeate flux values of 10−4–10−3 cm/s (where I cm/s = 3.6 × 104 L/m2 – h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one‐third power or greater for the flat filter and the one‐half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. © 1995 John Wiley & Sons, Inc.
Characterization of a pH‐inducible promoter system for high‐level expression of recombinant proteins in Escherichia coli
Authors: Chou Chih‐Hsiung , Aristidou Aristos A. , Meng Shi‐Yuan , Bennett George N. , San Ka‐Yiu
Published: December 2004

A pH‐inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552‐545C−, in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ′ reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200‐fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h−1was observed. © 1995 John Wiley & Sons, Inc.
Affinity purification of proteins using ligands derived from peptide libraries
Authors: Huang Ping Y. , Carbonell Ruben G.
Published: December 2004

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try‐Asn‐Phe‐Glu‐Val‐Leu, as a ligand for the purification of S‐protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphaze™ gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S‐protein decrease. S‐protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S‐protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.© 1995 John Wiley & Sons, Inc
High‐rate continuous biodegradation of concentrated chlorinated aliphatics by a durable enrichment of methanogenic origin under carrier‐dependent conditions
Authors: Boucquey J.‐B. , Renard P. , Amerlynck P. , Filho P. Modesto , Agathos S. N. , Naveau H. , Nyns E.‐J.
Published: December 2004

The simultaneous biodegradation of toxic compounds in mixtures is a major current concern. To bioremediate a toxic mixture, we designed a strategy combining an ad‐sorbent carrier with an ecological and nutritional system which allowed work close to heavily polluted conditions in nature. Starting from a methanogenic community, we developed a microbial consortium acclimated to a mixture of about 30 chlorinated aliphatics in a fixed‐film stationary‐bed bioreactor. Prior to the establishment of a durable period of dechlorination, an interval of progressive dechlorination of the toxic mixture was observed during which the excess of the toxic compounds was stored on the carrier. The latter, consisting of activated carbon in a polyurethane foam, allowed us to work at concentrations far above the solubility of the toxic compounds (apparent concentrations of about 10 g/L). The complete disappearance of hexachloroethane as well as its lower homologues, penta‐, tetra‐, and trichloroethane, present in the toxic mixture, was observed. Additionally, octachlorocyclopentene, carbon tetrachloride, trichloro‐ethylene, tetrachloroethylene, and hexachloro‐1,3‐butadiene also completely disappeared. For the four latter compounds, from mass balances in the bioreactor, degradation rates around 10 μmol/L per day were determined with total dechlorination. The enrichment culture thus developed exhibited high degradation performances similar to those reported in the literature for pure or enriched anaerobic microbial cultures in contact with a single toxic compound. The results demonstrate the possibility of concurrent high‐rate degradation of several highly chlorinated toxic compounds, under conditions approximating field situations.© 1995 John Wiley & Sons, Inc
Mathematical modeling and analysis of glucose and glutamine utilization and regulation in cultures of continuous mammalian cells
Authors: Zeng A.‐P. , Deckwer W.‐D.
Published: December 2004

A number of factors have been shown to affect the metabolism of glucose and glutamine in mammalian cells and their mechanisms have been partially elucidated. Despite these efforts, a quantitative knowledge of the significance of these factors, the regulation of glucose and glutamine utilization, and particularly the interactions of these two nutrients is still lacking. Controversies exist in the literature. To clarify some of these controversies, mathematical models are proposed in this work which enable to separate and identify the effects of individual factors. Experimental data from five cell lines obtained in batch, fed‐batch, and continuous cultures, both under steady‐state and transient conditions, were used to verify the model formulations. The resulting kinetic models successfully describe all these cultures. According to the models, the specific consumption rate of glucose (QGlc) of continuous animal cells under normal culture conditions can be expressed as a sum of three parts: a part owing to cell growth; a part owing to glucose excess; and a part owing to glutamine regulation. The specific consumption rate of glutamine (qGlc7) can be expressed as a sum of only two parts: a part owing to cell growth; and a part owing to glutamine excess. Using the kinetic models the interaction and regulation of glucose and glutamine utilizations are quantitatively analyzed. The results indicate that, whereas qGlc is affected by glutamine, qGln appears to be not or less significantly affected by glucose. It is also shown that the relative utilizations of glucose and glutamine by anabolism and catabolism are mainly affected by the residual concentrations of the respective compounds and are less sensitive to growth rate and the nature of growth limitation.© 1995 John Wiley & Sons, Inc
Optimization of step gradient separations: Consideration of nonlinear adsorption
Authors: Gallant Stuart R. , Kundu Amitava , Cramer Steven M.
Published: December 2004

Nonlinear adsorption plays an important role in determining the chromatographic behavior of proteins in preparative ion‐exchange chromatography. In this article, the steric mass action (SMA) isotherm is used in conjunction with a mass transport model to describe nonlinear cation‐exchange chromatography. Excellent agreement is observed between simulated and experimental step gradient separations of the proteins α‐chymotryp‐sinogen A, cytochrome C, and lysozyme. A systematic method of selecting the optimum step gradient program for a given separation problem is presented and employed to study optimization of step gradient chromatography under conditions of high mass loading. This article includes consideration of the effects of the adsorption properties of the feed stream, the feed stream concentration, protein solubility, and otherconstraints on the optimum separation conditions.© John Wiley & Sons, Inc.
Description of two‐metal biosorption equilibria by Langmuir‐type models
Authors: Chong K. H. , Volesky B.
Published: December 2004

A biosorbent prepared from Ascophyllum nodosum seaweed biomass, FCAN2, was examined for its sorption capacity. Equilibrium batch sorption studies were performed using two‐matal systems containing either (Cu + Zn), (Cu + Cd), or (Zn + Cd). In the evaluation of the two‐metal sorption system performance, simple isotherm curves had to be replaced by three‐dimensional sorption isotherm surfaces. In order to describe the isotherm surfaces mathematically, three Langmuir‐type models were evaluated. The apparent one‐parameter Langmuir constant (b) was used to quantify FCAN2 “affinity” for one metal in the presence of another one. The uptake of Zn decreased drastically when Cu or Cd were present. The uptake of Cd wasmuch more sensitive to the presence of Cu than to that of Zn. The presence of Cd and Zn alter the “affinity” of FCAN2 for Cu the least at high Cu equilibrium concentrations. The mathematical model of the two‐metal sorption system enabled quantitative estimation of one‐metal (bio)sorption inhibition due to the influence of a second metal. © 1995 John Wiley & Sons Inc.
Purification of an L‐asparaginase—atrial natriuretic peptide fusion protein expressed in Escherichia coli
Authors: Ma Nien‐Tung , Wang Yun‐Fang , Roe Bruce A. , Harrison Roger G.
Published: December 2004

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L‐Asparaginase from Erwinia chrysanthemi is fused to the N‐terminus of a model peptide, α‐human atrial natriuretic peptide (α‐hANP). L‐Asparaginase was chosen because of its selective affinity for L‐asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L‐asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L‐asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of α‐hANP was observed by coupled HPLC/mass spectrometry. © 1995 John Wiley & Sons Inc.