bluuebunny/crossref_metadata_2025 huggingface dataset:
Real and pseudo oxygen gradients in Ca‐alginate beads monitored during polarographic Po2‐measurements using Pt‐needle microelectrodes
Authors: Müller W. , Winnefeld A. , Kohls O. , Scheper T. , Zimelka W. , Baumgärtl H.
Published: December 2004

Polarographic microcoaxial needle electrodes were used to measure internal profiles of dissolved oxygen tension (Po2) within single Ca‐alginate beads of different diameter containing entrapped cells of Saccharomyces cerevisiae. For the investigations, single beads coming from variable growing conditions and distinct cultivation stages were fixed in a special holding device. In dependence on microbial growth steep oxygen gradients were observed. The Oxygen penetration depth at steady state lay between 50 and 100 μm. After 8 h of cultivation time, the anaerobic space within the beads (ϕ 2 mm; cultivation in a packed bed reactor) is beginning at ∼ 130 μm, whereas the anaerobic space within the beads (ϕ 2 mm) coming from the shaker flask culture is located ∼440 μm below the bead surface. Surprisingly, steep gradients were also observed, when recording profiles from cell‐free Ca‐alginate beads of different diameter and alginate concentrations. The steep oxygen gradients apparently had to be interpreted as pseudo‐Po2‐gradients. These results were borne by several effects, such as formation of artifacts and diffusion barriers in front of the electrode tip or oxygen “availability” at the tip and consumption of oxygen by the electrode itself. These phenomena could be documented by microscopic observation and photography. Thus, to obtain real Po2‐profiles it is important to be exactly informed about the physical, chemical, and biological properties of the material to be investigated. Furthermore, it is necessary to apply a special stepwise puncture technique with distinct step‐in/step‐out movements of the electrode: e.g., unidirectional or contradirectional puncture techniques. © 1994 John Wiley & Sons, Inc.
On the use of mild hydrophobic interaction chromatography for “method scouting” protein purification strategies in aqueous two‐phase systems: A study using model proteins
Authors: Huddleston J. G. , Wang R. , Lyddiatt A.
Published: December 2004

The behavior of a series of pure proteins partitioned in aqueous two‐phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid‐liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two‐phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. © 1994 John Wiley & Sons, Inc.
A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors
Authors: Zhong Jian‐Jiang , Fujiyama Kazuhito , Seki Tatsuji , Yoshida Toshiomi
Published: December 2004

The short‐time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well‐defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5‐L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s−1 or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. © 1994 John Wiley & Sons, Inc.
Separation of amino‐acid enantiomers using micellar‐enhanced ultrafiltration
Authors: Creagh A. L. , Hasenack B. B. E. , Van der Padt A. , Sudhölter E. J. R. , Van't Riet K.
Published: December 2004

Micellar‐enhanced ultrafiltration (MEUF) is investigated as a large‐scale technique for separating amino acid enantiomers. Specifically, L‐5‐cholesterol glutamate, a chiral ligand‐exchange cosurfactant, is used together with a nonionic surfactant to form mixed micelles that preferentially bind D‐phenylalanine over L‐phenylalanine in the presence of copper(II). Operational selectivities as high as 4.2 are obtained. Potentiometric titrations using a water‐soluble model compound similar to the chiral cosurfactant indicate that the ternary copper complex with phenylalanine has a stereoselectivity for the D enantiomer which is significantly smaller than that observed in the MEUF system. Thus, the selectivity of the chiral legend's local solvent and structural environment. © 1994 John Wiley & Sons, Inc.
Behavior of spores of Penicillium roquefortii during fed‐batch bioconversion of octanoic acid into 2‐heptanone
Authors: Larroche C. , Besson I. , Gros J. B.
Published: December 2004

The bioconversion of octanoic acid into 2‐heptanone by spores of Penicillium roquefortii is performed using a fed‐batch technique with pH control by addition of the liquid substrate itself. The early stage of this process takes place with a high bioconversion rate and high yield. These values then decrease as a result of germination and growth the biocatalyst. An optimization strategy for the process would thus be to improve the characteristics of this first period, i.e., increase its duration and the reaction rate. An increase in duration is evidenced in two cases: (I) under oxygen limitation: and (ii) when the spore content in the medium is less than 107 spores/mL. These conditions give insufficient overall bioconversion rates: better optimization should be achieved without oxygen limitation and with high spore content. Characterization of the first period by material and bioenergetic balances suggests that an increase in the ethanol content of the medium, which acts as an energy source and a permeabilizer, and the use of specific inhibitor of the Krebs cycle, may be a way to further improve the biocatalyst performance and stability. © 1994 John Wiley & Sons, Inc.
Cell death in bioreactors: A role for apoptosis
Authors: Singh R. P. , Al.‐Rubeai M. , Gregory C. D. , Emery A. N.
Published: December 2004

The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease‐mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above‐mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity. © 1994 John Wiley & Sons, Inc.
Characterization of a recombinant antibody produced in the course of a high yield fed‐batch process
Authors: Robinson D. K. , Chan C. P. , Yu Lp C. , Tsai P. K. , Tung J. , Seamans T. C. , Lenny A. B. , Lee D. K. , Irwin J. , Silberklang M.
Published: December 2004

Many mammalian cell fed‐batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti‐HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed‐batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed‐batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N‐acetylglucosamine and highmannose N‐glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. © 1994 John Wiley & Sons, Inc.
Improving protein extraction yield in reversed micellar systems through surface charge engineering
Authors: Pires M. J. , Martel P. , Baptista A. , Petersen S. B. , Willson R. C. , Cabral J. M. S.
Published: December 2004

The mechanism of extraction of rat cytochrome b5 from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b5 and modified proteins with substitutions of the type glutamic acid → lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. © 1994 John Wiley & Sons, Inc.
High‐density continuous cultures of hybridoma cells in a depth filter perfusion system
Authors: Oh Duk Jae , Choi Sang Kyo , Chang Ho Nam
Published: December 2004

A depth filter perfusion system (DFPS) for animal cell culture was developed and its use in continuous highdensity cultures of hybridoma cells was investigated. In the DFPS, based on cell immobilization in a cylindrical depth filter matrix, cells were easily immobilized and cultivated by simple medium recirculation. The cell density in the 20‐μm pore size filter matrix reached up to 3 × 107 cells/mLin less than 10 days. This resulted in a high monoclonal antibody productivity of 744 mg/L/day, which was 25‐35 times higher than that of continuous‐suspension cultures using the same cell line. The 20‐μm pore filter retained more cells than the 30‐μm filterin a shorter period. The DFPS provides advantages of low‐cost set‐up, easy operation, and scale‐up in the cultures of anchorage‐independent cells. It also has a high potential for anchorage‐dependent cell cultures because of its unusually high surface‐to‐volume ratio of 450‐600 cm2/cm3. © 1994 John Wiley & Sons, Inc.
Effects of intermolecular thiol–disulfide interchange reactions on bsa fouling during microfiltration
Authors: Kelly Sean T. , Zydney Andrew L.
Published: December 2004

Several studies have shown that one of the critical factors governing protein fouling of microfiltration membranes is the presence of denaturedand/or aggregated protein in the bulk solutions. Experiments were performed to evaluate the role of intermolecular disulfide interchange reactionson protein aggregation and membrane fouling during stirred cell microfiltration of bovine serum albumin (BSA). The flux decline during BSA filtration was quite dramatic due to the formation of a protein deposit thatfully covered the membrane pores. This flux decline could be completely eliminated by capping the free sulfhydryl group present on the BSA with eithera carboxymethyl or cysteinyl group, demonstrating the critical importance of this free thiol in the intermolecular aggregation reactions and, in turn, protein fouling. BSA aggregation during storage could be reduced by the addition of metal chelators (EDTA and citrate) or dithiothreitol, orby storage at lower pH (7.0) these solutions all had a significantly lower rate of fouling upon subsequent filtration. This behavior is completely consistent with the known chemistry of the thiol–disulfide interchange reaction, demonstrating that an understanding of these intermolecular (aggregation) reactions can provide a rational framework for the analysis and control of protein fouling in these membrane systems. © 1994 John Wiley & Sons, Inc.
Improvement of cloned α‐amylase gene expression in fed‐batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller
Authors: Shiba Sumihisa , Nishida Yoshio , Park Yong Soo , Lijima Shinji , Kobayashi Takeshi
Published: December 2004

The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B‐12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the α‐amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. The increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy ontroller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed‐batch culture. The synthesis of α‐amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory α‐amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory α‐amylase activities of cells harboring plasmids pNA3 and pNA7 in fed‐batch culture were 175 and 395 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled. © 1994 John Wiley & Sons, Inc.
Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose
Authors: Medve József , Ståhlberg Jerry , Tjerneld Folke
Published: December 2004

Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohy‐drolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30–90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased with increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I. © 1994 John Wiley & Sons, Inc.
Flow parameters associated with hydrodynamic cell injury
Authors: Garcia‐Briones Migual A. , Chalmers Jeffrey J.
Published: December 2004

Two flow parameters are proposed for the analysis of flows that have potential to damage animal cells. They are the state of stress (characterized by the second invariant of the stress tensor) and the flow classification parameter RD (which is related to the possibility of stress relaxation). We consider the flow that occurs when a 1.7‐mm bubble collapses at a liquid interface. Using these two parameters, we show the regions in which the flow is strong in terms of high hydrodynamic stresses and elongation characteristics. © 1994 John Wiley & Sons, Inc.
A fluid dynamic study of the retrofitting of large agitated bioreactors: Turbulent flow
Authors: Nienow A. W. , Hunt G. , Buckland B. C.
Published: December 2004

Studies were conducted in three 19‐m3 fermentors (14 m3 working volume, aspect ratio = 3:1), one fitted with four Rushton turbines (D/T = 0.35), one with three Lightnin' A315 hydrofoil impellers (D/T = 0.46). The power drawn under the same aerated conditions relative to the unaerated ones was always greater with the hydrofoils, which gives them the potential for enhanced mass transfer rates under practical operating conditions. However, the power draw was also sensitive to the magnitude of the unaerated power. Indeed, at low unaerated specific power (∼0.6 W‐kg) and high air flow rates (∼1vvm), the relative power draw with the hydrofoils could be even greater than 1. The hold‐up with each of the impellers was broadly similar at the same aeration rate and power input, though the later had a much smaller impact in these large vessels than has been reported in the literature based on smaller scale work. As usual, repressed coalescence caused increased hold‐up, and, with the hydrofoils, this increase was associated with a lower power draw. Because of the greater mechanical vibration of the reactors with the hydrofoils, vibration characteristics of the vessels were measured and they were very similar. The results showed that provided care is taken in the mechanical design of the system, such impellers can operate reliably in large‐scale fermentations with the potential for enhanced biological performance. © 1994 John Wiley & Sons, Inc.
Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks
Authors: Goodacre Royston , Trew Sally , Wrigley‐Jones Carys , Neal Mark J. , Maddock John , Ottley Thomas W. , Porter Neil , Kell Douglas B.
Published: December 2004

Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant‐producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. © 1994 John Wiley & Sons, Inc.
Diffusion of acetophenone and phenethyl alcohol in the calcium‐alginate–bakers' yeast–hexane system
Authors: Wu W. , Sidhoum M. , DeLancey G. B.
Published: December 2004

The intrabead diffusion coefficients of acetophenone and phenethyl alcohol were measured at 30°C in the triphasic immobilized yeast‐water‐hexane system. Saccharomyces cerevisiae cells were deactivated with hydrochloric acid and entrapped in calcium‐alginate beads. Measurements of dry cell loss during deactivation, shrinkage of the beads during deactivation and the final porosity of the beads were made for various cell loadings. Final concentrations of wet cells in the beads ranged from approximately 0.25 to 0.30 g/mL. Mass transfer in the hexane phase, external to the beads, was eliminated experimentally. The estimated error of 5% to 10% in the diffusion coefficients is within the experimental error associated with the bead method. The effect of significant sampling volumes on the diffusivities was estimated theoretically and accounted for experimentally. The intrabead concentration of acetophenone and phenethyl alcohol was 150 to 800 ppm. The deactivated cells were shown to be impervious to acetophenone so that the measured diffusivities are extracellular parameters. The cell volume fraction in the beads ranged from 0.70 to 0.90, significantly higher than previously reported data. The effective diffusion coefficients conform to the random pore model. No diffusional interaction between acetophenone and phenethyl alcohol was observed. The addition of 2 vol% ethanol or methanol slightly increased the diffusivities. The thermodynamic partition coefficients were measured in the bead‐free water‐organic system and found to be an order of magnitude lower than the values calculated from the analysis of the diffusion data for the organic‐bead system, suggesting that bead‐free equilibrium data cannot be used in triphasic systems. © 1994 John Wiley & Sons, Inc.
Protein separation by differential drainage from foam
Authors: Mohan S. B. , Lyddiatt A.
Published: December 2004

Commercially available beer, which is a dilute solution containing components of yeast, malt, and hop used in the manufacture of the beer, was used as a model system to demonstrate the potential of foam fractionation beyond the primary foaming stage. Most of the components present in the beer concentrated in the initial foam, but they drained differentially in the subsequent collapsed foam collected over a period of 30 min. This resulted in further enrichment, in particular, of components which were present in low concentration in the original beer, Preferential drainage from foam, hence, might provide a novel way of fractionating further the proteins concentrated initially in the liquid films of foam. © 1994 John Wiley & Sons, Inc.
Effect of alcohol feeding mode on the biosynthesis of poly (3‐hydroxybutyrate‐co‐3‐hyroxyvalerate)
Authors: Park Chang‐Ho , Damodaran Vijay
Published: December 2004

An alcohol utilizing Alcaligenes eutrophus produced poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) copolymer under phosphate limitation. Fermentation was performed for 42−46 h in a computer‐controlled 5‐L working volume fed‐batch fermentor using ethanol and propanol as carbon sources. The culture experienced phosphate limitation in approximately 19 h. When propanol was used as a sole carbon source, 24 g/L of copolymer with 36.5 mol % of hydroxyvalerate (HV) was produced at a polymer yield of 0.41 g polymer/g alcohol (g/g) and an average polymer production rate of 0.08 g polymer/g residual biomass‐h (g/g‐h). Two experiments switching alcohol after phosphate exhaustion resulted in better polymer production (g/L), polymer yield (g/g) on alcohol, HV yield (g/g) on propanol, and average polymer production rate (g/g‐h) as compared to propanol run without alcohol switching. One switching experiment was from a mixture of 50% ethanol and 50% propanol to 100% propanol and the other experiment was from 100% ethanol to a mixture of 65% ethanol and 35% propanol. Polymer yield for these two experiments was 0.51 g/g and 0.46 g/g, respectively. However, HV mol % in the copolymer for these two runs (30.8 mol % 12.6 mol % respectively) was lower compared to propanol run without alcohol switching (3605 mol %). Direct switch from ethanol to propanol did not support cell growth and polymer production. Polymer production rate and polymer yield changed with time, and the pattern was dependent upon the alcohol feeding mode. © 1994 John Wiley & Sons, Inc.
Cultivation of attenuated hepatitis A virus antigen in a titanium static mixer reactor
Authors: Junker B. H. , Seamans T. C. , Ramasubramanyan K. , Aunins J. , Paul E. , Buckland B. C.
Published: December 2004

The titanium static mixer reactor, demonstrated for a variety of vaccine processes during the late 197s, was investigated for the production of attenuated hepatitis A virus antigen from anchorage‐dependent MRC‐5 cells. This reactor system used Charles River Biotechnological Services cabinets for monitoring and process control. Cell inoculation protocols, using 6000−10,000 cells/cm2, resulted on over 95% attachment at both the laboratory and pilot scales. Indirect monitoring techniques using oxygen, glucose, L‐serine, and L‐glutamine uptake rates were indicative of cell growth prior to virus inoculation as well as environmental and/or nutrient limitations. Seven laboratory‐scale (3900 cm2) runs and one pilotscale (265,000 cm2) run were conducted to investigate refeeding regiments, parallel versus perpendicular element orientation, increased element surface area per unit volume, and scale‐up performance. In general, lysate antigen yields achieved were similar to those of parallel T‐flasks cultivated under similar conditions. © 1994 John Wiley & Sons, Inc.
Antibiotic production by Streptomyces aureofaciens using self‐cycling fermentation
Authors: Zenaitis Michael G. , Cooper David G.
Published: December 2004

The self‐cycling frementation (;rSCF) technique was applied to culture of Streptomyces aureofaciens. SCF is a method of continuous fermentation in which the metabolism of a microorganism is monitored by a measurement such as dissolved oxygen. These data are sent to a computer to allow it to control the system. Tetracycline production was observed only at exceedingly low iron concentrations in the growth medium. Repeatability of cycles was found to be dependent upon the presence of tetracycline in the fermentation broth as well as the strain of microorganism grown in the fermentor. Tetracycline was produced by an improved specific rate when compared to results in the literature for this organism grown using the batch method. © 1994 John Wiley & Sons, Inc.
Correlation between steady‐state cell concentration and cell death of hybridoma cultures in chemostat
Authors: Lee Yuan‐Kun , Yap Peng‐Kang , Teoh Ai‐Peng
Published: December 2004

In the present study, the steady‐state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (kd) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of kd vs. X/D yielded linear relation, which suggests that cell death was due to a non‐growth‐linked inhibitory product of the cells. The kd value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h−1. The kd for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low‐molecular‐weight component of possibly less than 3000 MW was detected to be cell‐death‐inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell‐line specific. © 1995 John Wiley & Sons, Inc.
Enzymatic interesterification of triglyceride with surfactant‐coated lipase in organic media
Authors: Goto Masahiro , Goto Muneharu , Kamiya Noriho , Nakashio Fumiyuki
Published: December 2004

Several surfactant‐coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C18Δ9GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant‐coated lipase was carried out in organic media. The surfactant‐coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant‐coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di‐substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. © 1995 John Wiley & Sons, Inc.
An in situ dissolved‐hydrogen probe for monitoring anaerobic digesters under overload conditions
Authors: Strong G. E. , Cord‐Ruwisch R.
Published: December 2004

The concentration of diatomic hydrogen in the liquid phase of an anaerobic digester was used to determine the onset of digester failure induced by substrate overloading. The construction of an inexpensive probe to measure dissolved hydrogen, having a partial pressure detection limit of 30 Pa, is described. An increase in the partial pressure of dissolved hydrogen, from less than 30 Pa to 400 Pa, was observed when the D‐glucose concentration in a laboratory‐scaled digester was increased rapidly to 10 mM. However, when the digester was gradually overloaded, an increase in the dissolved‐hydrogen partial pressure was not observed until after the digester failed. The accumulation of volatile fatty acids and digester failure were observed at dissolved‐hydrogen partial pressures below 30 Pa. © 1995 John Wiley & Sons, Inc.
Buffer exchange using size exclusion chromatography, countercurrent dialysis, and tangential flow filtration: Models, development, and industrial application
Authors: Kurnik Ronald T. , Yu Audrey W. , Blank Gregory S. , Burton Arlene R. , Smith David , Athalye Atul M. , van Reis Robert
Published: December 2004

There are three major methods for buffer exchange of proteins at industrial scale: size exclusion chromatography (SEC), tangential flow filtration (TFF), and countercurrent dialysis (CCD). In order to determine the optimal technology for a given process, a study was done to compare these technologies on a technological and economic basis. This comparison required that new mathematical models be developed which enable the common features of each unit operation to be directly compared. The new concept of a diavolume equivalent for SEC, defined as the inverse of the fractional loading, was also introduced to aid in this comparison. Variables that were examined for each unit operation included range of buffer exchange, dilution of protein solution, yield, buffer requirements, total operating time, throughput, plant space, capital, raw materials, and labor costs. It was found that TFF and CCD have a greater range of buffer exchange than SEC. TFF also provides the advantage that concentration of the protein can readily be accomplished in the same step. For processes of equal batch size and yield, TFF and CCD also provide a two‐ to five‐ fold improvement in each of the remaining variables. The major economic advantage in using TFF and CCD over SEC is the decreased plant size required for manufacturing and thus the longer term use of existing facilities. Situations where SEC (or CCD) would be favored over TFF are when protein denaturation occurs in TFF but does not occur in SEC. © 1995 John Wiley & Sons, Inc.
Fluid shear effects on suspension cultures of Morinda citrifolia
Authors: Kieran P. M. , O'Donnell H. J. , Malone D. M. , MacLoughlin P. F.
Published: December 2004

The shear susceptibility of cell suspension cultures of the plant cell Morinda citrifolia was investigated by subjecting the cells to the well‐defined shear field generated in turbulent flow through a capillary. Suspensions were circulated using a peristaltic pump and average shear stresses between 25 and 350 N m−2 were generated in the capillary test section. Control experiments were performed to assess the possible contribution of the peristaltic pump to the observed cell damage. There was clear evidence of pump‐induced damage at the more severe test conditions and all viability measurements were corrected accordingly. Both shake flask suspension cultures (aged between 9 and 15 days) and repeated batch fermentation cultures, grown in a stirred tank reactor (STR) under a variety of controlled agitation conditions, were tested in the capillary shear loop. The cell damage incurred was evaluated in terms of suspension viability, as determined by a dye exclusion technique. Viability loss was found to conform closely to a first‐order model in which the rate constant was observed to increase with the imposed shear stress. Furthermore, a linear relationship was identified between the specific death constant and the cumulative energy dissipated. Post‐shear morphological measurements showed that the chain length distribution is shifted toward markedly lower values. In comparison with shake flask cultures, repeated batch fermentation cultures exhibited a marked increase in sensitivity to capillary shear. Based upon the determined morphological characteristics, this result is primarily attributable to the increased chain lengths characteristic of the repeated batch cultures. © 1995 John Wiley & Sons, Inc.
Relation between dissolved oxygen concentration and ajmalicine production rate in high‐density cultures of Catharanthus roseus
Authors: Schlatmann J. E. , Vinke J. L. , ten Hoopen H. J. G. , Heijnen J. J.
Published: December 2004

The relation between dissolved oxygen (DO) and the ajmalicine production rate of Catharanthus roseus was investigated in 15‐L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the ajmalicine production rate was less than 0.06 μmol/g/d. Above a DO of 43% the ajmalicine production rate was constant at 0.21 μmol/g/d. Between a DO of 29% and 43% there was a strong relation between the ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO ⩽29% the culture lacked the ability to adapt to a DO ⩾57%. A kinetic equation is proposed for the relation between DO and the specific ajmalicine production rate. © 1995 John Wiley & Sons, Inc.
Enhancing effect of albumin hydrolysate on ethanol production employing Saccharomyces sake
Authors: Shin Chul Soo , Song Ji Yeon , Ryu Ok Hee , Wang Shaw S.
Published: December 2004

The enhancing effect of albumin hydrolysate on ethanol production was investigated in ethanol fermentations using Saccharomyces sake. In batchwise ethanol production, addition of supplemental albumin hydrolysate and phosphatidylcholine, or albumin hydrolysate alone, brought about a more than 60% increase in final ethanol concentration (148 or 144 g/L compared with 88 g/L with no supplementation [control] after 72 h). The effect of the supplements is believed to be due to an enhanced alcohol tolerance of cells grown in media containing the supplements. Cells grown in media containing albumin hydrolysate were enriched in phenyalanine, tyrosine, and methionine in their plasma membranes. All three amino acids were also present in considerable amounts in the albumin hydrolysate. This fact suggests that the three amino acids, which are present in albumin hydrolysate, are incorporated into the plasma membranes of cells. Under ethanol production conditions in which only one amino acid among the components of albumin hydrolysate was excluded, namely phenlalanine, tyrosine, or methionine, significant reductions in ethanol production resulted. © 1995 John Wiley & Sons, Inc.
Dynamics of biofilm detachment in biofilm airlift suspension reactors
Authors: Tijhuis L. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

The dynamic change in the overall detachment rate of spherical biofilms in a biofilm airlift suspension reactor was measured after a downshift of the substrate loading rate to zero while all other conditions remained constant. In contrast to the expectations, the overall detachment rate decreased rapidly to a nearly stable level. Correlations available from literature were not able to describe this phenomenon. Concepts were formulated which can describe the observations from this study. Research under dynamic conditions and careful monitoring of the biofilm surface area and biofilm morphology are necessary to elucidate and discriminate biofilm detachment mechanisms. © 1995 John Wiley & Sons, Inc.
Effect of cDNA copy number on secretion rate of activated protein C
Authors: Guarna M. M. , Fann C. H. , Busby S. J. , Walker K. M. , Kilburn D. G. , Piret J. M.
Published: December 2004

The secretion rate of activated protein C (APC) by BHK cells was increased 35‐fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the relation between APC secretion and cDNA copy number was not linear and the rate of APC secretion per cDNA copy increased sevenfold. This apparent cooperative effect of multiple cDNA copies could be related to their integration in tandem. For cDNA copy numbers higher than 240, the APC secreation rate per cDNA and per cell decreased dramatically. The γ‐carboxylation of glutamic acid residues, a posttranslational modification required for APC biological activity, was also investigated. The proportion of APC that was fully γ‐carboxylated decreased as the secretion rate of APC increased. © 1995 John Wiley & Sons, Inc.
Metabolic flux distributions in Penicillium chrysogenum during fed‐batch cultivations
Authors: Jørgensen Henrik , Nielsen Jens , Villadsen John , Møllgaard Henrik
Published: December 2004

Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo–steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if α‐aminoadipate is converted to 6‐oxo‐piperidine‐2‐carboxylic acid (OPC). If only 40% of the α‐aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, γ‐aminobutyrate, and 21 amino acids were measured during fed‐batch cultivations. The rates of formation of penicillin V, δ‐(L‐α)‐aminoadipyl‐L‐cysteinyl‐D‐valine (ACV), OPC, and the pool of isopenicillin N, 6‐APA, and 8‐HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide‐adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model YxATP is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations mATP is found to 3 mmol ATP/g DW/h. © 1995 John Wiley & Sons, Inc.
Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery
Authors: Chang Qing‐long , Chen Jia‐yong
Published: December 2004

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid–liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT‐reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. © 1995 John Wiley & Sons, Inc.
Adhesion of mammalian cells to a recombinant attachment factor, CBD/RGD, analyzed by image analysis
Authors: Wierzba Andrew , Reichl Udo , Turner Robin F. B. , Warren R. Antony J. , Kilburn Douglas G.
Published: December 2004

A luminance thresholding procedure was developed to quantify cell attachment of a variety of cell lines to CBD/RGD, a hybrid attachment factor comprising a cellulose binding domain and the fibronectin‐like RGD attachment peptide. The technique used local thresholding, median filtering, and opening to separate and count cells on each image. Cell lines exhibited three different patterns of attachment to CBD/RGD, depending on whether it was immobilized on polystyrene or cellulose acetate. Vero, COS, HFF, 3T3, 293, and U373 cells attached well to CBD/RGD immobilized on polystyrene or cellulose acetate. CHO, MRC‐5, and HEp‐2 cells attached to CBD/RGD immobilized on polystyrene, but not to CBD/RGD immobilized on cellulose acetate. BHK and L cells failed to attach to CBD/RGD immobilized on either polystyrene or cellulose acetate. The attachment of many cell lines to CBD/RGD was comparable with attachment of these cells to fibronectin. © 1995 John Wiley & Sons, Inc.
Cartilage production by rabbit articular chondrocytes on polyglycolic acid scaffolds in a closed bioreactor system
Authors: Dunkelman Noushin S. , Zimber Michael P. , LeBaron Richard G. , Pavelec Rebecca , Kwan Michael , Purchio A. F.
Published: December 2004

Rabbit articular chondrocytes were seeded onto three‐dimensional polyglycolic acid (PGA) scaffolds and placed into a closed bioreactor system. After 4 weeks of growth, meshes were examined for cartilage formation. Gross examination revealed solid, glistening material that had the appearance of cartilaginous tissue. Histologic examination revealed cell growth and deposition of extracellular matrix throughout the mesh with a less dense central core. Alcian blue and Safranin 0 staining showed deposition of glycosaminoglycans (GAGs). Immunostaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen. Biochemical analysis showed collagen and GAG values to be 15% and 25% dry weight, respectively. Our results indicate that this type of system compares well with those previously described and should be useful for producing cartilage for evaluation in a clinical setting. © 1995 John Wiley & Sons, Inc.
Hydrodynamic effects on BHK cells grown as suspended natural aggregates
Authors: Moreira José L. , Alves Paula M. , Aunins John G. , Carrondo Manuel J. T.
Published: December 2004

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25–100 rpm) led to aggregates with maximum sizes of 150 μm. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to −1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. © 1995 John Wiley & Sons, Inc.
Kinetics and mass transfer for lactic acid recovered with anion exchange method in fermentation solution
Authors: Zihao Wang , Kefeng Zhao
Published: December 2004

An anion exchange method for lactic acid recovered from lactic acid–glucose solution in an ion‐exchange membrane‐based extractive fermentation system was examined. The exchange isotherms of anion exchange resins for lactic acid recovered were measured batchwise, and the exchange–desorption kinetics of lactic acid passing through the exchange column was investigated. The determined typical breakthrough and elution curves were measured and simulated by conventional mode. The mass transfer coefficients were identified by numberical method. The effects of the velocity of the fluid on the dynamics were studied. Aqueous NaOH solution was found to be the best solvent for elution. An experiment on anioun exchange from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the ion exchange system from a borth. The ion‐exchange mass‐transfer coefficient and efficiency from the fermentation broth is found to be lower when compared with aqueous solutions of pure lactic acid. The results show that the separation method with anion exchange resins may be used in the production of lactic acid by fermentation.© 1995 John Wiley & Sons, Inc.