bluuebunny/crossref_metadata_2025 huggingface dataset:
Induced flocculation of animal cells in suspension culture
Authors: Aunins John G. , Wang Daniel I. C.
Published: May 2005

Recycle unit operations for suspension cell cultures may be improved by flocculation of the cells. A flocculant search was conducted, and it was found that where strongly cationic polymers were severly toxic to the cells, neutral and anionic polymers were nontoxic and ineffective at flocculating the cells. A weak and poorly soluble polycation, poly‐L‐histidine, was capable of flocculating cultures of CHO, HeLa, U‐937, and CRL 1606 hybridoma cells with no toxicity to the majority of cells. In addition to the lowered acute toxicity, the polymer treatment left the cells in a growing state for recycle. Flocculation was found to be mediated by precipitates of the polymer. The low toxicity of poly‐L‐histidine is probably due to its low solubility and charge at physiological pH. Nonelectrostatic interactions may also play a role.
Interaction of infectious viral particles with a quaternary ammonium chlorid (QAC) surface
Authors: Tsao I‐Fu , Wang Henry Y. , Shipman Charles
Published: May 2005

The antiviral activity of a surface‐bonded quaternary ammonium chloride (QAC) was examined in this study. The mechanism of inactivation was elucidated by a combination of infectivity assay, radioactive labeling assay, and sedimentation analysis. Although the virions are still infectious when attached onto the chemically modified surface, we found these viruses are inactivated if they are eluted from the surface. The inactivation is caused by the disruption of the viral envelope with subsequent release of the nucleocapsid. No evidence indicates the released nucleocapsid is further disrupted. An enveloped virus shows a much higher affinity for the QAC‐treated surface than a nonenveloped one due to hydrophobic interaction. The QAC‐treated beads can effectively remove the enveloped viruses at low protein concentrations. The titer of herpes simplex virus was reduced by a factor of nearly 5 logarithm units in a 0.5 wt % bovine serum albumin solution with less that 10% protein loss. However, the presence of proteins in the solution reduced both the rate and capacity of this nonspecific adsorption–inactivation process. As a consequence, the removal efficiency is relatively poor in solutions with high protein content.
Subsite mapping of Aspergillus niger glucoamylases I and II with malto‐ and isomaltooligosaccharides
Authors: Meagher Michael M. , Nikolov Zivko L. , Reilly Peter J.
Published: May 2005

Glucoamylase, industrially derived from Aspergillus niger, was chromatographically separated into forms I and II and purified to near homogeneity. Preparations were proved to be free of D‐glucosyltransferase by electrophoretic and differential inhibition tests. Maximum rates and Michaelis constants were obtained for both glucoamylases I and II with maltooligosaccharides from maltose to maltoheptaose and with isomaltooligosac‐charides from isomaltose to isomaltohexaose. Subsite maps were calculated from these kinetic data and were not significantly different for the two forms. Subsites in both forms had lower affinities for D‐glucosyl residues contained in isomaltooligosaccharides than for D‐glucosyl residues in maltooligosaccharides.
Kinetics, equilibria, and modeling of the formation of oligosaccharides from D‐glucose with Aspergillus niger glucoamylases I and II
Authors: Nikolov Zivko L. , Meagher Michael M. , Reilly Peter J.
Published: May 2005

Near‐homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were incubated with D‐glucose at a number of temperatures and pH values. Kinetics and equilibria of the formation of α,β‐trehalose, kojibiose, nigerose, maltose, isomaltose, panose, and isomaltotriose, which with isomaltotetraose were the only products formed, were determined. There was no difference in the abilities of GA I and GA II to form these products. Activation energies for the formation of maltose and panose were lower than those of the other Oligosaccharides. Relative rates of oligosaccharide production based on glucoamylase hydrolytic activity did not vary significantly between pH 3.5 and 4.5 but were lower at pH 5.5. Maltose was formed much faster than any other product. Equilibrium concentrations at higher dissolved solids concentrations decreased in the order isomaltose, isomaltotriose, kojibiose, nigerose, maltose, α, β‐Mrehalose, panose, and isomaltotetraose. They were not appreciably affected by changes in temperature or pH. A kinetic model based on adsorption of D‐glucose and the seven di‐ and trisaccharides by the first three glucoamylase subsites was formulated. Oligosaccharide formation was simulated with the model, using equilibrium data gathered for this article and subsite binding energies and kinetic parameters for oligosaccharide hydrolysis measured earlier. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent except at very high solid concentrations.
On the coexistence of competing microbial species in a chemostat under cycling
Authors: Pavlou S. , Kevrekidis I. G. , Lyberatos G.
Published: May 2005

It is known that two microbial species competing for a single rate‐limiting nutrient cannot grow together in a chemostat under steady‐state operation, but eventually the species with the lower specific growth rate at the particular operating conditions will become extinct. Coexistence of the two populations has been shown obtainable in chemostats under periodic operation. This is possible in cases where the specific growth rate functions of the two species are such that for certain values of the nutrient concentration the first species grows faster than the second, and for other values of the nutrient concentration the second species is the one growing faster. In a previous article it was demonstrated that, even in cases where the specific growth rate functions of the two species are such that one of the species grows faster than the other for all values of the nutrient concentration, extinction of either species is possible provided that time delay in the response of the species to changes in their fermentation environment is accounted for, and that the faster growing species is also faster in its response. Here, we show that coexistence of the two species is also possible in a significant range of the operating parameters. We develop a numerical algorithm with which we trace the boundary of the coexistence region in the entire operating parameter space and construct the operating diagram of the system.
A modeling study of carbon and light limitation in algal biofilms
Authors: Liehr Sarah K. , Suidan Makram T. , Eheart J. Wayland
Published: May 2005

In an effort to develop an understanding of the interaction of inorganic carbon and light limitation in algal biofilms, a mathematical model was developed, based on well‐established mathematical descriptions of the biological, chemical, and physical processes involved. The results showed that the growth of algal biofilms is a very complex function of the inorganic carbon and light conditions, due to the interaction of inorganic carbon speciation with pH. These results are discussed in terms of the distribution of reaction rate through the biofilm and in terms of maximum potential growth rate for given bulk conditions.
Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus
Authors: Biswas S. R. , Jana S. C. , Mishra A. K. , Nanda G.
Published: May 2005

Enhancement of the productivity of xylanase and β‐xy‐losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N‐methyl‐N‐nitro‐N‐nitro‐soguanidine (NTG). The hyperxylanolytic mutant (NG‐13), which showed good clearing on the surface of the xylan‐agar plate, secretes xylanase and β‐xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG‐13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45°C though its activity was maximal at 50°C. The molecular weight of the enzyme was estimated to be 4.3 × 104 by SDS‐polyacrylamide gel electrophoresis and 5 × 104 by gel filtration on Sephadex G‐75. The kinetic data showed that the Km and Vmax values for xylan were 1 × 10−3M and 19.6 μmol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K+and was inactivated by thiol reagents such as Hg2+, p‐hydroxymercuribenzoate (PHMB), 3′, 5′‐dithiobis (2′‐nitrobenzoic acid) (DTNB), and N‐ethylmaleimide (NEM).
The use of a single‐fiber reactor for the enzymatic removal of amino acids from solutions
Authors: Reiken Steven R. , Briedis Daina M.
Published: May 2005

In this article we describe the use of bench‐scale single‐fiber dialyzers for the development and testing of an immobilized enzyme reactor for the treatment of leukemia. The treatment is based on the enzymatic removal of specific amino acids from the blood of leukemia patients. L‐Lysine α‐oxidase and catalase were coimmobilized within the void space of the porous region of asymmetric hollow‐fiber membranes for the removal of L‐lysine from simulated human plasma solutions. Hollow‐fiber reactor performance was evaluated using a small single‐fiber dialyzer (SFD) consisting of a single fiber encased in a protective glass shell. This small reactor affords ease of use, requires small amounts of chemicals and biochemicals, and gives useful reactor performance data. Single‐fiber dialyzers were constructed using polyamide fibers with a molecular weight cutoff of 10,000 (PA10 fibers); these fibers demonstrated the best compatibility with and retention of the enzymes. The SFD performance in removing L‐lysine from solution was evaluated under both steady and pulsatile flow operation. Pulsatile flow was tested for two reasons: (1) to enhance the radial mass transfer of lysine within the SFD and (2) to simulate the pulsatile flow of blood in dialysis treatment. The use of pulsatile flow increased lysine conversion by 15% over the steady‐flow case. Approximately 40% of the lysine was removed from simulated plasma by the SFD in a 4‐h experiment using pulsatile flow in the recycle mode.
Diffusion of phenol through a biofilm grown on activated carbon particles in a draft‐tube three‐phase fluidized‐bed bioreactor
Authors: Fan L.‐S. , Leyva‐Ramos R. , Wisecarver K. D. , Zehner B. J.
Published: May 2005

Diffusion of phenol through a biofilm attached to activated carbon particles was investigated. The biofilm was grown on activated carbon particles in a draft‐tube three‐phase fluidized‐bed bioreactor operating in a fed‐batch mode. It was found that phenol did not adsorb on the biofilm and that the diffusion coefficient of phenol within the biofilm varied from 13 to 39% of its corresponding value in water. The diffusion coefficient of phenol within the biofilm was reduced by increasing the biofilm density. An extensive literature review of diffusion of substrates through biofilms indicated that this conclusion could be extended to biofilms grown on flat surfaces, rotating cylinders, and even bioflocs.
Prediction of diffusion coefficients of proteins
Authors: Tyn Myo T. , Gusek Todd W.
Published: May 2005

A correlation for predicting the diffusion coefficients of proteins at standard conditions is proposed by adapting the Stokes‐Einstein equation to a model for the equivalent hydrodynamic sphere. The radius of gyration, which accounts for the size and shape of protein molecules in solution, was used in deriving the new correlation. The correlation successfully predicted the diffusion coefficients of proteins, for which experimental data were available, with a higher degree of accuracy than achieved by previous correlation methods.
A process for the production of human proinsulin in Saccharomyces cerevisiae
Authors: Tøttrup Hanne V. , Carlsen Søren
Published: May 2005

In order to develop a large‐scale fermentation process for the production of human proinsulin in yeast, the intra‐cellular expression of a human superoxide dismutase‐human proinsulin fusion product (SOD‐PI) has been studied. The expression of SOD‐PI in Saccharomyces cerevisiae is regulated by a hybrid alcohol dehydrogenase 2/glyceraldehyde‐3‐phosphate dehydrogenase promoter. The promoter is repressed by glucose and derepressed by depletion of glucose. Although the genetic stability of the construction is shown to be poor under product‐inducing conditions, it is demonstrated in shake flask experiments that a stable expression potential can be maintained in a complex medium for more than 60 generations by maintaining excess glucose throughout the cultivations. These results have been confirmed in continuous cultures in chemostat and turbidostat experiments. Addition of the glucose analogs glucosamine, 2‐desoxyglucose, methylglucose, and thioglucose also leads to repression of SOD‐PI formation. The analogs, however, are not suitable for improving genetic stability during propagation because of growth inhibition. In batch fermentation experiments in a complex medium at 30°C, it has been demonstrated that initial glucose concentrations up to 50 g/L result in high specific SOD‐PI yields giving an overall yield of up to 700 mg SOD‐PI/L whereas higher glucose concentrations lead to both lower specific and overall yields due to depletion of critical medium components in the production period. In fed‐batch experiments at 30°C it has been possible to obtain high specific SOD‐PI yields even at high biomass concentrations by feeding glucose at a constant rate of 1.5 g/L/h for 40 h followed by a feeding of ethanol at 1.0 g/L/h for 24 h, thus giving an overall yield of 1200 mg/L. Decreasing the temperature from 30 to 26°C leads to improved yields in batch as well as fed‐batch experiments. The optimized fed‐batch fermentation process which is suitable to be scaled up to the cubic meter level has been tested in 200‐L fermentations resulting in yields of more than 1500 mg/L of the fusion protein which conveniently can be used as a precursor in the production of recombinant human proinsulin.
Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen‐vectors
Authors: Rols J. L. , Condoret J. S. , Fonade C. , Goma G.
Published: May 2005

Limitations of oxygen transfer in fermentation can be solved using auxiliary liquids immiscible in the aqueous phase. The liquids (called oxygen‐vectors) used in this study were hydrocarbon (n‐dodecane) and perfluorocarbon (forane F66E) in which oxygen is highly soluble (54.9 mg/L in n‐dodecane and 118 mg/L in forane F66E at 35°C in contact with air at atmospheric pressure). It has been demonstrated that the use of n‐dodecane emulsion in a culture of Aerobacter aerogenes enabled a 3. 5‐fold increase of the volumetric oxygen transfer coefficient(kLa) calculated on a per‐liter aqueous phase basis. The droplet size of the vector played a crucial role in the phenomena. When a static contact between gas bubble and vector droplet was established in water, the vector covered the bubble, in agreement with positive values of the spreading coefficient for these fluids. The determination of the oxygen transfer coefficients (kL) in a reactor with a definite interfacial area enabled the main resistance to be located in the boundary layer of the waterside either for a gas‐water or a vector‐water interface. Because oxygen consumption by weakly hydrophobic cells can only occur in the aqueous phase, the oxygen transfer is achieved according to the following pathway: gas‐vector‐water‐cell. Finally, a mechanism for oxygen transfer within this four‐phased system is proposed.
Mass transfer phenomena in an airlift reactor: Effects of solids loading and temperature
Authors: Smith Bryan C. , Skidmore Duane R.
Published: May 2005

Gas holdups and volumetric mass transfer coefficients were measured in a concentric tube airlift reactor designed for the microbial desulfurization of coal. The solutions studied were comprised of an acidified basal salts solution containing thirteen different weight percentages (0 to 40) of coal (74 μm Ohio #1) at three different temperatures (30, 50, and 72°C). Gas holdup ϵG decreased with solids loading for the entire range studied. An enhancement in the volumetric mass transfer coefficient KLa with respect to that in pure solution was observed from zero to approximately 5 wt % (solids volume fraction ϵs = 0.035), the maximum enhancement occurring at approximately 2 wt % (ϵs = 0.014). At higher solids fractions, the mass transfer coefficient decreased with further solids additions. Gas holdups and the mass transfer coefficients increased with temperature over the studied range. The KLa and ϵG were correlated to three process variables separately and the separate correlations combined to yield generalized correlations for the mass transfer coefficient and gas holdup for this system. The correlations may be used for design, operation, and ost estimation of such systems.
Effect of biofilm growth on steady‐state biofilm models
Authors: Skowlund Christopher T.
Published: May 2005

The results of numerical simulations for a growing biological film are presented to justify the use of steady‐state biofilm models for approximating the behavior of both unlimited and shear‐limited biofilms. For an unlimited biofilm we show that although the total biofilm thickness may continue to increase over time, the active biofilm volume will reach a constant value. We also show that the profile of active microorganisms within the biofilm will become constant with respect to the biofilm/fluid interface and simply move outward as the biofilm thickness increases. For a shear‐limited biofilm we similarly show that once a “limiting” thickness has been reached the active biofilm volume, substrate consumption, and profile of active microorganisms within the biofilm will also be independent of the biofilm thickness.
Kinetic model for microbial uptake of insoluble solid‐state substrate
Authors: Huang Shih‐Yow , Chou Ming‐Shean
Published: May 2005

A kinetic model for anaerobic digestion of insoluble solid‐state substrates was developed. Rate equations for cell growth and substrate consumption were derived based on the assumption that the microorganisms assimilate the substrate mainly at the point of contact where they grow. The model emphasizes effects of substrate particle size, organic loading, and cell concentration on the rates of cell growth and substrate utilization. Batch digestion of a stearic acid emulsion with a mean particle size of 2.0 μm and a biological sludge was conducted at 30 and 37°C to verify the proposed model. Agreement between the experimental and calculated results indicated the validity of the model for describing the microbial degradation of insoluble solid‐state substrates. Further examinationof the model revealed that with low cell substrate affinity or at low cell concentration, it coincided with a Michaelis–Menten type kinetics in which the effect of particle size was taken into consideration.
Immobilized cell biocatalyst activation and pseudo‐steady‐state behavior: Model and experiment
Authors: Monbouquette H. G. , Sayles G. D. , Ollis D. F.
Published: May 2005

An intrinsic, unstructured model has been utilized to describethe startup dynamics of a continuous Caalginate‐immobilized Zymomonas mobilis (ATCC 10988) fermentation. This model predicts, at least qualitatively, transients in the fermenter effluent glucose, ethanol, and biomass concentrations as well as radial gradients in immobilized‐cell concentration and activity within the gelbiocatalysts. Predicted intrabiocatalyst gradients in immobilized‐cell specific growth rate were used to calculate the corresponding gradients in intracellular RNA level based on a reported linear relationship between the two. Mathematical simulations of immobilized biomass concentration profiles and RNA content were verified using a novel, scanning microfluorimetry technique.
Comparison of simultaneous wet milling and enzymatic hydrolysis of cellulose in ball mill and attrition mill reactors
Authors: Furcht P. W. , Silla H.
Published: May 2005

Simultaneous wet milling and enzymatic hydrolysis of newsprint in a ball mill reactor (BMR) and in an attrition mill reactor (AMR) were studied. The effect of milling on the enzyme deactivation was also investigated. Variables studied included milling media size, material, and the presence or absence of an air–liquid interface in the reactor. At a reaction temperature of 45°C and a pH of 4.5 and in the presence of an air–liquid interface, the amount of reducing sugars produced by hydrolysis using a BMR was typically 5 to 15%less than the amount produced using an AMR. Without an air–liquid interface there was no significant difference. The effect of an air–;liquid interface on enzyme deactivation without any substrate was also studied. Enzyme activity after six hours of milling ranged from 1 to 85% ofthe starting activity. In both the AMR and the BMR, the extent of enzyme deactivation was 20 to 50% greater with an air–liquid interface present than with the air–liquid interface absent.
Production of indole alkaloids by surface immobilized C. roseus cells
Authors: Archambault J. , Volesky B. , Kurz W. G. W.
Published: May 2005

A technique was developed to surface immobilize plant cells and was scaled up in laboratory size bioreactors. This technique was shown not to hinder the biosynthetic potential of Catharanthus roseus immobilized cells and to induce a partial release (300 μg/L) of serpentine into the culture medium contrary to suspension cultured cells. The release pattern seemed to follow the biosynthesis trends of the product. This release mechanism could be stimulated by a factor of 10 within 2 h by increasing the pH of the culture from 5.0 to 5.5.
Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria
Authors: Bentley William E. , Mirjalili Noushin , Andersen Dana C. , Davis Robert H. , Kompala Dhinakar S.
Published: May 2005

Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (ampr, camr, and letr). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1‐like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol‐acetyl‐transferase (CAT) and β‐lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.

Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically‐structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.
Enzyme production by genetically engineered Streptomyces strains: Influence of culture conditions
Authors: Erpicum Thomas , Granier Benoît , Delcour Marianne , Lenzini V. Mauro , Nguyen–Distèche Marline , Dusart Jean , Frère Jean–Marie
Published: May 2005

Production of various extracellular enzymes (the β‐lactamases from Streptomyces albus G, Streptomyces cacaoi, Actinomadura R39, and the DD‐carboxypeptidase from Streptomyces R61) by genetically engineered Streptomyces lividans TK24 in Lennox broth medium reached a maximum after 36 to 48 h. Subsequently, the enzyme activity drastically decreased probably due to an increased pH value and the production of an inactivator by Streptomyces lividans. Protease activity did not seem to play a major role. The increased pH and inactivator synthesis are related to amino acid catabolism and generally result in cellularlysis. The use of a medium where the catabolism of amino acids was made less likely by the presence of glucose and NH4Cl and by buffering at pH 7.4 considerably inproved the yield. Furthermore, the water activity of the medium seemed to be an important parameter for the production of extracellular proteins by genetically engineered Streptomyces. Better production was observed when the water activity was decreased to 0.96–0.98 by addition of sucrose.

Under those conditions, the concentration of extracellular enzyme reached about 0.3 g (1 g in the best case)/L of culture supernantant.
Optimum ph and temperature conditions for xylose fermentation by Pichia stipitis
Authors: Slininger P. J. , Bothast R. J. , Ladisch M. R. , Okos M. R.
Published: May 2005

Pichia stipitis NRRL Y‐7124 is a xylose‐fermenting yeast able to accumulate ca. 57 g/L ethanol. Because optimum process conditions are important, data were collected to determine the effects of temperature and pH on growth and fermentation rates and product accumulations. Temperatures (26–35°C) providing optimum biomass and ethanol productivities did not necessarily provide maximum ethanol accumulation. Xylitol and residual xylose concentrations increased with temperature. Maximum ethanol selectivity was achieved at 25–26°C with minimal sacrifice to production rates. The temperature optimum for xylose could not be generalized to glucose fermentations, in which ethanol productivity and accumulation were optimum at 34°C. The optimum pH range for growth and fermentation on xylose was 4–7 at 25°C.
Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues
Authors: Mulchandani A. , Male K. B. , Luong J. H. T.
Published: May 2005

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine‐5′‐monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.
Use of regulated secretion in protein production from animal cells: An evaluation with the AtT‐20 model cell line
Authors: Sambanis A. , Stephanopoulos Gregory , Sinskey A. J. , Lodish H. F.
Published: May 2005

Regulated secretion, i.e., the ability of certain specialized animal cells to store secretory proteins intracellularly and release them upon stimulation, may be used to realize production schemes that facilitate downstream processing of protein products. Mouse AtT‐20 cells expressing recombinant human insulin and human growth hormone (hGH) were found to secrete the proteins at relatively low and constant rates when exposed to media with no secretion agonists: basal rates were 1.0–1.6 μU insulin‐reiated peptides and 0.38 ng hGH/105 cells‐h. When induced with 8 brorno‐cyclic AMP (BrcAMP), the cells secreted recombinant proteins at initial rates 3.5–9‐fold higher. A cycling secretion experiment was conducted with the insulin‐producing cells in which the cells were exposed alternately to complete growth medium and to secretion medium with BrcAMP. During the first three cycles, the cells secreted immunoreactive insulin at the foregoing high induced rates when they were exposed to BrcAMP. The cells then started to detach from the culture surface, leading to a reduction of BrcAMP‐induced secretion. Operational modifications that may result in improved system performance are discussed.
Kinetics of carrot somatic embryo development in suspension culture
Authors: Cazzulino Dana L. , Pedersen Henrik , Chin Chee‐Kok , Styer Donald
Published: May 2005

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.
Detection of microbial contamination in fermentation processes: Mass spectrometric determination of gram‐negative bacteria in Leuconostoc mesenteroides cultures
Authors: Elmroth Ingrid , Valeur Anders , Odham Göran , Larsson Lennart
Published: May 2005

Gas chromatography/mass spectrometry was used to detect the presence of Enterobacter cloacae in cultures of Leuconostoc mesenteroides, an organism used in an industrial process for production of dextrane. The penta‐fluorobenzoyl‐methyl ester derivative of 3‐hydroxy‐myristic acid, a characteristic compound of gram‐negative bacteria, was used as the analyte. By using gas chromatography/mass spectrometry with selected ion monitoring, E. cloacae was determined over the range of 1 ppm to 1% in cultures of L. mesenteroides. The proposed analytical approach represents a useful alternative to conventional methods for determining contaminating organisms in industrial fermentation processes.
The pH‐auxostat as a tool for studying microbial dynamics in continuous fermentation
Authors: Larsson G. , Enfors S.‐O. , Pham H.
Published: May 2005

If a microorganism has a growth coupled production or consumption of acid or alkali, it is possible to use the pH‐auxostat as a means of control in continuous fermentation. In using the pH‐auxostat, it is possible to separate the inlet substrate flow in two different streams. These will both be pH controlled, with one main flow, consisting of nutrients and a second minor but concentrated flow, of acid or alkali. Hereby, it is possible to vary the difference in pH between the fermentor and the inlet medium. This pH difference is proportional to the steady‐state cell mass concentration.1,2 It is shown that by separating the inlet flow in two different streams and cultivating without any substrate limitation, the maximum growth rate may be obtained while the cell mass concentration will be controlled. This will also give the possibility to reach high cell mass concentrations at μmax without the risk of wash‐out. A modified expression, based on hydrogen, of the steady‐state bio‐mass concentration, X, is developed as

where YX/H is the yield coefficient of cell mass per acid produced. The indexes Hin and Min refer to the inflows of alkali and medium, respectively; CHin is the inlet concentration of hydrogen ions. The boundary condition for the cell mass shows that Sin > X/YX/S, where Sin is the medium substrate concentration and YX/S is the yield of biomass per consumed substrate. It is shown that when the cell mass concentration exceeds this value, the flow stops. The applicability of the pH‐auxostat method is then verified from different experiments. It is hereby used to detect a deviation from the maximal growth rate showing effects on the microbial physiology. With Escherichia coli used as the model organism, the effect on the growth rate of temperature and high concentration of ammonia were investigated.
Effect of β‐cyclodextrin on production of L‐phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae
Authors: Mahmoud Wafaa M. , El‐Sayed Abdel‐Halim M. M. , Coughlin Robert W.
Published: May 2005

The rate and extent of microbial transformation of higher concentrations of benzaldehyde substrate to L‐phenylacetyl carbinol (L‐PAC) by immobilized cells of Saccharomyces cerevisiae ATCC 834 was markedly stimulated by addition of different concentrations of β‐cyclodextrin (BCD) to the fermentation medium. With 0.5, 1.0, and 1.5% BCD in the fermentation medium and cumulative doses of benzaldehyde of 12 and 14 g/L, significantly higher yields of L‐PAC were obtained, about one‐ to twofold that of the yields of the control experiments. The favorable effects of BCD were evident in spite of its presence in stoichiometric concentrations significantly lower than those of benzaldehyde. The presence of BCD also appeared to stimulate microbial growth slightly. Enhanced cellular activity was reflected by faster D‐glucose consumption and faster benzaldehyde utilization in the presence of BCD.
Use of novel immobilized β‐galactosidase reactor to hydrolyze the lactose constituent of skim milk
Authors: Bakken Andrew P. , Hill Charles G. , Amundson Clyde H.
Published: May 2005

β‐galactosidase from Aspergillus oryzae immobilized in an axial‐annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30°C, 40°C, and 50°C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three‐parameter rate expression which includes the separate competitive inhibition effects of α‐ and β‐galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the β anomer of galactose (EA = 10.3 kcal/mol). At 40°C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial‐annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.
Recovery of a charged‐fusion protein from cell extracts by polyelectrolyte precipitation
Authors: Parker Diane E. , Glatz Charles E. , Ford Clark F. , Gendel Steven M. , Suominen Ilari , Rougvie Malcolm A.
Published: May 2005

β‐Galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low‐cost, easily scaled separation method—precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartaies. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivitiy is attributed to the binding strength of the polyanionic tails to the polycationic PEI.
Damage mechanisms of suspended animal cells in agitated bioreactors with and without bubble entrainment
Authors: Kunas Kurt T. , Papoutsakis Eleftherios T.
Published: May 2005

We show that when freely suspended hybridoma cells are cultured in an agitated bioreactor, two fluid‐mechanical mechanisms can cause cell damage and growth retardation. The first is present only when there is a gas phase, and is associated with vortex formation accompanied by bubble entrainment and breakup. In the absence of a vortex and bubble entrainment, cells can be damaged only at very high agitation rates, above approximately 700 rpm, by stresses in the bulk turbulent liquid. Cell damage then correlates with Kolmogorov eddy sizes similar to or smaller than the cell size. In the absence of a vortex, the entrainment and motion of very fine bubbles cause no growth retardation even at agitation rates as high as 600 rpm.
Some comments on respiratory quotient (RQ) determination from the analysis of exit gas from a fermentor
Authors: Aiba Shuichi , Furuse Hisamoto
Published: May 2005

Recently, the respiratory quotient (RQ) of microbes measured in situ in a fermentor by exit‐gas analysis has been used successfully, for instance, in a fed‐batch culture of baker's yeast as a criterion to control the feeding rate.1–3 It is significant here to keep RQ values close to unity throughout; any deviations of RQ from unity give rise to deterioration of the cell growth yield.

However easy it might be to keep RQ values around unity by controlling the feeding rate, the question of whether or not RQ values determined by gas analysis at the fermentor exit could generally represent those in vivo deserves attention. Indeed, for a fermentation carried out at an alkaline side, gas analysis would give RQ values that differ remarkably from true values because of the medium's “storage” of CO2 released from microbes. The purpose of this communication is to make clear those factors that would affect true RQ values in the analysis of exit gas from a fermentor.
A structured, segregated model for genetically modified Escherichia coli cells and its use for prediction of plasmid stability
Authors: Kim Byung Gee , Shuler M. L.
Published: May 2005

A structured, segregated model is presented for an asynchronously growing population of genetically modified Escherichia coli cells. A finite representation method was modified so that 272 cells could be used to represent a microbial population. The concept of a “limbo” compartment was introduced to allow random plasmid distribution to daughter cells upon cell division while restricting the number of computer cells included in the calculation. This scheme enabled us to predict plasmid instability and distribution of plasmid‐originated properties in a population without a priori determination of growth rates or probability of forming plasmid‐free cells from plasmid‐containing cells. Predictions of population behavior using a single‐cell model requires no adjustable parameters. The results comparing different induction strategies suggest that in continuous culture, there exists an optimum efficiency of partial induction that maximizes the long‐term productivity of the gene product due to plasmid stability. With the optimum efficiency of partial induction, constant induction appears to prove more stable than cycling induction.
Lipoprotein lipase immobilization onto polyacrolein microspheres
Authors: Hayashi Toshio , Ikada Yoshito
Published: May 2005

A lipoprotein lipase (LPL) was made water insoluble by immobilizing onto the surface of polyacrolein (PAA) microspheres with and without oligoglycines as spacer. The activity of the immobilized LPL was found to remain high toward a small ester substrate, p‐nitrophenyl laurate (pNPL). The relative activity of the immobilized LPL without spacer decreased gradually with the decreasing surface concentration of the immobilized LPL on the PAA microsphere. On the contrary, the immobilized LPL with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than that without any spacer. The Michaelis constant Km and the maximum reaction velocity Vm were estimated for the free and the immobilized LPL. The apparent Km was larger for the immobilized LPL than for the free one, while Vm was smaller for the immobilized LPL. The pH, thermal, and storage stabilities of the immobilized LPL were higher than those of the free one. The initial enzymatic activity of the immobilized LPL maintained almost unchanged without any leakage and inactivation of LPL when the batch enzyme reaction was performed repeatedly, indicating the excellent durability of the immobilized LPL.
Protein–protein interaction in low water organic media
Authors: Gaertner Hubert F. , Puigserver Antoine J.
Published: May 2005

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein–protein interactions in water‐immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe–NH2 was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol‐modified trypsin in organic media containing 1–2% water (v/v). The organic solvent‐soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate‐organic solvent interface. Peptide mapping of bovine insulin digest by reversed‐phase high‐performance liquid chromatography definitely showed that enzyme‐catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.
The large‐scale immobilization of Penicillium chrysogenum: Batch and continuous operation in an air‐lift reactor
Authors: Keshavarz T. , Eglin R. , Walker E. , Bucke C. , Holt G. , Bull A. T. , Lilly M. D.
Published: May 2005

A temperature‐sensitive cell division cycle mutant of Penicillium chrysogenum P2 has been immobilized on Celite and grown in a 250–320‐L working volume air‐lift fermenter. The ability to uncouple growth and penicillin synthesis by raising the temperature to 30 °C also overcame the problem of the free cell mass which appeared after 300 h operation with the parent organism. After 500 h operation, penicillin and ACV dimer were still being synthesized.
A viscous pump bioreactor
Authors: Kleis S. J. , Schreck S. , Nerem R. M.
Published: May 2005

The fluid dynamic design and characterization of a low mechanical stress agitator and bioreactor vessel for large‐scale bioprocessing using anchorage dependent mammalian cells is considered. The complex and fragile nature of mammalian cells puts stringent constraints on the design of agitators for stirred tank bioreactors. Traditional agitators have difficulty meeting the competing requirements of fluidization and mixing while maintaining low enough mechanical stress levels to avoid cell damage. A rotating disc agitator design is proposed. Flow visualization and laser Doppler velocimeter measurements reveal fluidization of microcarriers with a gentle (low turbulence level), highly three‐dimensional flow characterized by good mixing at low hydrodynamic shear stress levels.
Kinetic studies and unstructured models of lymphocyte metabolism in fed‐batch culture
Authors: Truskey George A. , Nicolakis Dimitri P. , DiMasi Don , Haberman Allan , Swartz Randall W.
Published: May 2005

The growth of two lymphocyte cell lines, a hybridoma cell line and a human cutaneous T cell lymphoma (HuT78), was studied in fed‐batch culture, and unstructured models of growth developed. A criteria was established to insure that the growth rate varied by less than a specified tolerance throughout the culture period. Glutamine and serum were growth‐limiting nutrients for both cell lines with half‐maximal growth rates at 0. 53 mM glutamine and 0. 55%(v/v) serum for the hybridoma cells and 0. 21 mM glutamine and 1. 5% serum for the HuT‐78 cells. Over the range of glucose concentrations from 5. 5 mM to 28 mM, the specific growth rate of hybridoma cells was independent of glucose concentration, whereas glucose concentrations above 5. 5 mM inhibited HuT‐78 growth. For both cell lines, the growth rate was significantly inhibited by the addition of ammonium, although the hybridoma cell line was more affected by ammonia than was the HuT‐78 cell line. Growth of HuT‐78 cells increased in the presence of interleukin‐2. Unstructured models for the hybridoma cells were similar to other models presented in the literature. Applications of these models to adoptive immunotherapy are discussed.
Changes in the activity of the general amino acid permease from Saccharomyces cerevisiae var. ellipsoideus during fermentation
Authors: Lglesias R. , Ferreras J. M. , Arias F. J. , Muñoz R. , Girbès T.
Published: May 2005

The evolution of the activity of the general amino acid permease and ethanol and glucose concentrations in the medium were studied in a mild fermentation process carried out by a wine strain of Saccharomyces cerevisiae var. ellipsoideus isolated from grape musts in spontaneous fermentation. The cells displayed a reduction in the activity of the general amino acid permease parallel to the increase of ethanol in the medium. This ethanol increase was not enough to promote a substantial inhibition on the total polypeptide synthesis measured as polyuridylic‐acid‐directed polyphenylalanine synthesis.
Glucose oxidase immobilized polyethylene‐g‐acrylic acid membrane for glucose oxidase sensor
Authors: Hsiue Ging‐Ho , Wang Chee‐Chang
Published: May 2005

A polyethylene‐g‐acrylic acid (PE‐g‐AA) graft copolymer was prepared via gamma‐ray‐irradiation‐induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water‐soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE‐g‐AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 °C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme‐immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0–1000 ppm. The obtained glucose oxidase‐immobilized PE‐g‐AA membranes were kept in pH 5. 6 acetate buffer solution at 4 °C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.