bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy
Authors: Hamilton Dylan , Goodwin Joseph , Clarke Mary Beth , du Moulin Gary C. , Liu Victor , Caplan Barry , Babbitt Bruce
Published: December 2004

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in‐process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of 3H‐thymidine incorporated, was demonstrated to have intra‐assay coefficients of variation (cv′s) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter‐assay cv′s that were typically less than 15% were obtained by individual analysts, and overall cv′s of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.
Collagen fabrics as biomaterials
Authors: Cavallaro John F. , Kemp Paul D. , Kraus Karl H.
Published: December 2004

Tissue‐engineered implants require appropriate biomaterials to serve the required physical function of the tissue being repaired or replaced while facilitating remodeling of the implant. We report on the development of implantable fabrics manufactured from continuous collagen threads. The collagen threads are formed by extrusion of native, acid‐extracted bovine colagen into a buffered solution of polyethylene glycol, followed by rinsing and air drying. The high manufacturing rate of such threads permits the production of colagen fabrics of various configurations. The fiber diameter can be controlled, and threads with dry diameters as low as 25 μm have been produced. Braids and bundles of collagen threads implanted as a replacement of the anterior cruciate ligament in a dog model were completely remodeled into host tissue by 12 weeks. Knitted collagen fabrics implanted in a rat abdominal repair model prevented herniation, and connective tissue ingrowth was observed within the fabric by 12 weeks.
Spatial distribution of mammalian cells dictated by material surface chemistry
Authors: Healy Kevin E. , Lom Barbara , Hockberger Philip E.
Published: December 2004

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n‐octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N‐(2‐aminoethyl)‐3‐aminopropyl‐trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time‐dependent process. Angle dependent x‐ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was imobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.
Crossflow filtration of yeast broth cultivated in molasses
Authors: Tanaka Takaaki , Kamimura Ryoji , Fujiwara Ryo , Nakanishi Kazuhiro
Published: December 2004

A broth of yeast cells cultivated in molasses was crossfiltered with a thin‐channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 μm). The steady‐state flux was one‐twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead‐end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 μm in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead‐end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 μm, but using larger pores of 3 to 5 μm it returned almost to the bases line. © 1994 John Wiley & Sons, Inc.
Continuous propionate production from whey permeate using a novel fibrous bed bioreactor
Authors: Yang Shang‐Tian , Zhu Hui , Li Ying , Hong Gene
Published: December 2004

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de‐lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was ∼46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30°C, but the temperature effect was not dramatic in the range of 25 to 35°C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long‐term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, ∼50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10‐fold increase in productivity, acceptance of low‐nutrient feedstocks such as whey permeate, and resistance to contamination. © 1994 John Wiley & Sons, Inc.
Growth characteristics in fed‐batch culture of hybridoma cells with control of glucose and glutamine concentrations
Authors: Kurokawa Hideo , Park Yong Soo , Iijima Shinji , Kobayashi Takeshi
Published: December 2004

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive‐control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one‐half and the lactate concentration by one‐third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 × 106 cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. © 1994 John Wiley & Sons, Inc.
Degradation of pencillin‐V in fermentation media
Authors: Christensen Lars H. , Nielsen Jens , Villadsen John
Published: December 2004

In Industrial production of penicillin there is a noticeable loss of the product through degradation reactions. It is shown that the degradation of penicillin‐V, both in a complex and in a chemically defined medium, can be separated into a phosphate‐catalyzed conversion of penicillin‐V to penicilloic‐V acid, overlaid by at least one other reaction in which penicillin V is degraded to as yet unknown products. Parameter values for the phosphatecatalyzed degradation are found to be independent of the type of fermentation medium. The rate of formation of other degradation products of penicillin‐V is found to be significantly higher in a complex fermentation medium with corn‐steep liquor in a chemically defined medium. © 1994 John Wiley & Sons, Inc.
Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences
Authors: Gjaltema A. , Arts P. A. M. , van Loosdrecht M. C. M. , Kuenen J. G. , Heijnen J. J.
Published: December 2004

A rotating annular reactor (Roto Torque) was used for qualitative and quantitative studied on biofilm heterogeneity. In contrast to the classic image of biofilms as smooth, homogeneous layers of biomass on a substratum, studies using various pure and mixed cultures consistently revealed more‐dimensional structures that resembled dunes and ridges, among others. These heterogeneities were categorized and their underlying causes analyzed. Contrary to expectations, motility of the microorganisms not a decisive factor in determining biofilm homogeneity. Small Variations in substratum geometry homogeneity. Small variations in substratum geometry and flow patterns were clearly reflected in the biofilm pattern. Nonhomogeneous flow and shear patterns in the reactor, together with inadequate mixing resulted in significant, position‐dependent differences in surface growth. It was therefore not possible to take representative samples of the attached biomass. Like many other types of reactors, the Roto Torque reactor is valuable for qualitative and morphological biofilm experiments but less suitable for quantitative physiological and kinetics studies using attached microorganisms. © 1994 John Wiley & Sons, Inc.
Flotation of cadmium‐loaded biomass
Authors: Matis K. A. , Zouboulis A. I.
Published: December 2004

Biosorption of heavy metal ions such as Cd2+ by dead biomass has been recognized as a potential alternative to existing removal technologies applied to wastewater treatment. Two bacterial strains were studied in the laboratory, streptomyces griseus and S. clavuligerus, an industrial by‐product. Both washed and unwashed samples were examined. Foam flotation proposed in this work as the separation state following biosorption. Effective biomass separation was conducted in the presence of a frother, ethanol. The pH of the solution was a crucial parameter for flotation and also for metal binding. Other basic parameters of flotation examined were the initial cadmium concentration in the dilute aqueous solution and the quantity of biomass used. A study of ζ‐potential measurements of the actinomycetes was carried out under the conditions used in the separation; surface tension was also measured. These provided useful information on the process. © 1994 John Wiley & Sons, Inc.
A new method for studying microaerobic fermentations. II. An experimental investigation of xylose fermentation
Authors: Franzén Carl Johan , Lidén Gunnar , Niklasson Claes
Published: December 2004

A new experimental technique, called oxygen programmed fermentation (OPF), was used to study microbial cultures of the years Pichia stipitis and Candida utilis growing on xylose as carbon and energy source. In the oxygen programmed fermentation, the inlet oxygen mole fraction was continuously changed to scan through a wide range of oxygen uptake rates in a continuous culture. The largest ethanol yields and productivities of P. stipitis were found at oxygen transfer rates below 1.5 mmol L−1 h−1. It was found that the ratio between the culture fluorescence and near‐IR absorbance increased at oxygen transfer rates lower than 1.5 mmol L−1 h−1. Small amounts of ethanol were produced also by C. utilis when the oxygen transfer rate was between 0 and 3 mmol L−1 h−1. It is suggested that OPF will form a nice complement to ordinary, microaerobic chemostat experiments, by making the identification of interesting regions of oxygen transfer rates possible in an efficient and time‐saving initial experiment. © 1994 John Wiley & Sons, Inc.
Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low‐strength soluble wastewaters
Authors: Kato Mario T. , Field Jim A. , Versteeg Paul , Lettinga Gatze
Published: December 2004

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low‐strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory‐scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L · d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low‐strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate‐biomass contact and adequate expansion of the substrate‐biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (Vup) was greater than 2.5 m/h. Under such conditions, an extremely low apparent Ks value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at Vup lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory‐scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at Vup higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L· d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold‐up, the operation should be limited to an organic loading rate of 7 g COD/L d· and to a liquid up‐flow velocity between 2.5 and 5.5 m/h © 1994 John Wiley & Sons, Inc.
Enzymatic hydrolysis of whey proteins. II. Molecular‐weight range
Authors: Tello P. González‐ , Camacho F. , Jurado E. , Páez M. P. , Guadix E. M.
Published: December 2004

Using high‐pressure liquid chromatography we studied the distribution of molecular weights in whey‐protein hydrolysates using the following commercially obtained proteases: Alcalasa 0.6 L and Protease 660 L, both bacterial in origin, and PEM 2500 S, of animal origin. In each of the systems, the range of molecular weights in the hydrolysate depended solely on the degree of hydrolysis (DH) achieved. For DH ≥ 20, between 65% and 95% of the hydrolysate is made up of peptides with a molecular weight of less than 1,000 Da. © 1994 John Wiley & Sons, Inc.
Interactions between benzene, toluene, and p‐xylene (BTX) during their biodegradation
Authors: Oh Young‐Sook , Shareefdeen Zarook , Baltzis Basil C. , Bartha Richard
Published: December 2004

A microbial consortium and Pseudomonas strain (PPO1) were used in studying biodegradation of benzene, toluene, and p‐xylene under aeorbic conditions. Studies involved removal of each compound individually as well as in mixture with the others. Both cultures exhibited a qualitatively similar behavior toward each compound. Both the pure culture and the consortium grew on benzene following Monod kinetics, on toluene following inhibitory (Andrews) kinetics, whereas neither could grow on P‐xylene. Benzene and toluene mixtures were removed under cross‐inhibitory (competitive inhibition) kinetics. In the presence of benzene and/or toluene, p‐xylene was cometabolically utilized by both cultures, but was not completely mineralized. Metabolic intermediates of p‐xylene accumulated in the medium and were identified. Benzene and toluene were completely mineralized. Cometabolic removal of p‐xylene reduced the yields on both benzene and toluene. Except for cometabolism, kinetic constants were determined from data analysis and are compared with values published recently by other researchers. © 1994 John Wiley & Sons, Inc.
Liquid flow in heterogeneous biofilms
Authors: de Beer Dirk , Stoodley Paul , Lewandowski Zbigniew
Published: December 2004

Liquid flow was studied in aerobic biofilms, consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. Fluorescein microinjection was used as a qualitative technique to determine the presence of flow in cell clusters and voids. Flow velocity profiles were determined by tracking fluorescent latex spheres using confocal microscopy. Liquid was flowing through the voids and was stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, whereas in cell clusters diffusion is the dominant factor. The flow velocity in the biofilm depended on the average flow velocity of the bulk liquid. The velocity profiles in biofilms were linear and the velocity was zero at the substratum surface. The velocity gradients within biofilms were 50% of that near walls without biofilm coverage. The influence of the biofilm roughness on the flow velocity profiles was similar to that caused by rigid roughness elements. © 1994 John Wiley & Sons, Inc.
A practical approach to biosurfactant production using nonaseptic fermentation of mixed cultures
Authors: Ghurye G. L. , Vipulanandan C. , Willson R. C.
Published: December 2004

Non‐aseptic production of biosurfactant from molasses by a mixed culture was investigated in stirred batch reactors. Biosurfactant production was quantified by surface tension reduction, critical micelle dilution (CMD), and emulsification capacity (EC). Biosurfactant production was directly correlated with biomass production, and was improved by pH control and addition of yeast extract. Centrifugation of the whole broth increased emulsifying capacity and reduced surface tension. Acidification of the whole broth increased the emulsification capacity but reduced the apparent biosurfactant concentration (CMD), without affecting the surface tension. The emulsification capacity of the cell‐free broth was equivalent to that of a 100 mg/L solution of sodium dodecyl sulfate. The emulsification capacity of the whole broth and cell‐free broth were reduced by about 50% at and above NaCl concentrations of 100mM. Preliminary characterization suggests that the biosurfactant activity is primarily associated with one or more protease‐sensitive species, released from cells in larger quantities after more vigorous centrifugation. © 1994 John Wiley & Sons, Inc.
Kinetics of growth and elemental sulfur oxidation in batch culture of thiobacillus ferrooxidans
Authors: Konishi Yasuhiro , Takasaka Yuichiro , Asai Satoru
Published: December 2004

The kinetics of oxidation of elemental sulfur by Thiobacillus ferrooxidans in a batch reactor was followed by measuring the concentration of adsorbed cells on the sulfur surface, the concentration of free cells in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate, the liquid‐phase concentration of free cells continued to increase with time, whereas the surface concentration of adsorbed cells per unit weight of sulfur approached a limiting value, i.e., the maximum adsorption capacity. During sulfur oxidation, there was a close correlation between the concentrations of adsorbed and free cells, and these data were well correlated with the Langmuir isotherm. The observed rates of batch growth and sulfur oxidation were consistent with a kinetic model, assuming that the growth rate of batch growth and sulfur oxidation were consistent with a kinetic model. Assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration of adsorbed cells and the fraction of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were evaluated using the experimental data and were compared with parameters determined previously for a few metal sulfides. © 1994 John Wiley & Sons, Inc.
Modeling and optimization of the baculovirus expression vector system in batch suspension culture
Authors: Power John F. , Reid Steven , Radford Kathryn M. , Greenfield Paul F. , Nielsen Lars K.
Published: December 2004

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum‐supplemented IPL‐41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2‐L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque‐forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large‐scale systems for the production of recombinant products using the baculovirus expression vector system. © 1994 John Wiley & Sons, Inc.
Effects of substrate branching characteristics on kinetics of enzymatic depolymerizaion of mixed linear and branched polysaccharides: I. Amylose/amylopectin α‐amylolysis
Authors: Park Jun T. , Rollings James E.
Published: December 2004

Hydrolysis reactions of homopolysaccharides, which differ in their degree of branching, and mixtures of linear and branched polymers were carried out with α‐amylase. The branching structures of both the original amylopectin substrate and the cluster domains of amylopectin, obtained by ethanol precipitation of the products of the action of α‐amylase, were characterized via enzymatic digestion with debranching enzyme (i.e., isoamylase), followed by the fractions of the resulting products using gel filtration chromatography. The structural properties (i.e., molecular weight, molecular weight distribution, and branching characteristics) of the resulting products during depolymerization of amylose, amylopectin and their mixtures via α‐amylase were characterized by size exclusion chromatography coupled with a low angle laser right scattering (SEC/LALLS) technique. It was determined that substrate branching characteristics strongly influence both the observed enzymatic activity as well as the enzyme's action pattern. A simplified kinetic model that represents the hydrolysis reactions of amylose and amylopectin mixtures via endo‐acting α‐amylase is proposed. We found that that reaction kinetics (i.e., enzyme affinity) was also governed by the substrate's conformation in solution. The relationships between the mass fraction of branched polymers and the kinetic parameters during α‐amylolysis were compared with those predicted by the kinetic model. Excellent agreement was found between the model predictions and the experimental observations. The results reported here imply and interrelationship between enzyme action and polymeric substrate structural properties. © 1994 John Wiley & Sons, Inc.
An investigation of the physico‐chemical basis of foaming in fungal fermentations
Authors: Noble Ian , Collins Mark , Porter Neil , Varley Julie
Published: December 2004

A detailed physico‐chemical analysis of two foaming fungal fermentations was carried out to identify that key groups of compounds responsible for foam formation. Fermentations were carried out on a 20‐L scale in a stirred aerated tank, over 7 days, using a commercial, defined medium. The organisms investigated were Penicillium herqueii, a hyphomycete, and an unidentified Ingoldian fungus. Samples of broth and, where possible, foam were analyzed to determine which groups of compounds were concentrated into generated foams. Surface tension, bulk viscosity, and antifoam A concentration were additionally determined in broth samples. To date the cause of foaming in fermentations has been attributed to the surfactant properties of extracellular proteins. This assumption was tested and found to be incomplete as many additional groups of biochemicals were found to be enriched into the foam. The results of the investigation revealed the presence of proteins, carbohydrates, α‐keto acids, and lipophilic biosurfactants, particularly extracellular pigments, enriched within stable foams. © 1994 John Wiley & Sons, Inc.
Solids retention time in spherical biofilms in a biofilm airlift suspension reactor
Authors: Tijhuis L. , van Benthum W. A. J. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

Fluorescent microparticles were used as tracer beads to measure the dynamics of solids in spherical biofilms in a biofilm airlift suspension reactor. Attachment to, release from, and penetration into the biofilms of the tracer beads were measured. The coverage of the biofilm surface was low and the steady state particle concentration on the surface was dependent on the biofilm surface characteristics. The measured attachment rate constant was identical in both experiments and appeared to be determined by the hydrodynamic conditions in the turbulent reactor. The attachment rate was much faster than the release rate of the tracer beads and, therefore, the solidsretention time in the biofilm particle is not due to a simple reversible adsorption–desorption process. The heterogeneity of the distribution oftracer beads on different sectors on the biofilm surface decreased duringthe attachment period. Due to random detachment processes the heterogeneity of the tracer bead distribution increased during the release period

The tracer beads quickly penetrated into the biofilm and became distributed throughout the active layer of the biofilm. The observed penetration into biofilms, the nonuniform distribution on the biofilm surface, and the fast uptake and slow release of tracer beads cannot be described by a simple model based on a reversible adsorption–desorption mechanism, nor withexisting biofilm models. These biofilm models, which balance growth and advection assuming a uniform biofilm with a homogeneous surface, are inadequate for the description of the observed solids retention time in biofilms. Therefore, a new concept of biofilm dynamics is proposed, in which formation of cracks and fissures, which are rapidly filled with growing biomass, combined with nonuniform local detachment, explains the observed fast penetration into the biofilm of tracer beads, the long residence time, and the nonuniform distibution of fluorescent microparticles. © 1994 John Wiley & Sons, Inc.
Evaluation of the hok/sok killer locus for enhanced plasmid stability
Authors: Wu Kuowei , Wood Thomas K.
Published: December 2004

The effectiveness of the hok/sok plasmid stability locus and mechanism of cloned‐gene loss was evaluated in shake‐flask cultures. Addition of the hok/sok locus dramitically increasedapparent plasmid segregational stability to the hok/sok− control. In terms of the number of generations before 10%of the population became plasmid‐free, segregational stability was increased by 11‐ to 20‐fold in different media in the absence of induction of the cloned‐gene (hok/sok+ plasmid stable for over 200 generations in all media tested). With constant expression of β‐galactosidase in the absence of an tibiotic, the segregational stability of the plasmid containing hok/sok was incresed more than 17‐ to 30‐fold when β‐galactosidase was expressed at 7–15 wt % of total cell protein. Although the hok/sok system stabilized the plasmid well infour different media (Luria‐Bertani (LB), LB glucose, M9C Trp, and a representative fedbatch medium), the ability of hok/sok to maintain the plasmid with induction of the cloned gene decreased as the complexity of the media increased. This result is better interpreted in terms of the influence of cloned‐gene expression on plasmidmaintenance; plasmid segregational stability decreased linearly as specificβ‐galactosidase activity increased. © 1994 John Wiley & Sons, Inc.
Pilot scale recovery of recombinant annexin V from unclarified escherichia coli homogenate using expanded bed adsorption
Authors: Barnfield Frej Ann‐Kristin , Hjorth Rolf , Hammarström Åke
Published: December 2004

Expanded bed adsorption is a new downstream processing technique forcapture of proteins directly from unclarified feedstocks. Expanded bed adsorption reduces the number of operations in purification processes by combining clarification, concentration, and capture into one operation. It is based on stable fluidization and uses adsorbent particles with well‐defined size and density distributions, together with columns designed to giveeven liquid flow distribution. The bed expands as the adsorbent particles are lifted by an upward liquid flow through the column. The behavior of the expanded bed is similar to a packed chromatography bed due to very little back‐mixing of the adsorbent particles. The major benefit of using anexpanded bed is that adsorption can be carried out with unclarified feedstocks; there is no need for centrifugation or filtration to remove cells and debris. When the feedstock is applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed with the bed in an expanded mode, followed by elution of bound protein in a sedimented mode with downward flow

Described in this article is the use of expanded bed adsorption for pilot scale recovery of recombinant human placental annexin V from an Escherichia coli ho mogenate. The description includes the whole procedure, from small‐scale method optimization to pilot scale. The recovery of annexin V was approximately 95% at both lab scale and pilot scale. During the trials, it was discovered that the expanded bed was affected by the biomass content and viscosity of the homogenate. The upper limits for these parameters were therefore investigated further. For the E. coli used in the application described here, homogenates with biomass dry weightup to 5% and viscosities up to 10 mPa s (at a shear rate of 1 s−1) worked best. It was, however, feasible to use homogenates with dry weight up to 7–8% and viscosities up to 50 mPa s (1 s−1). © 1994 John Wiley & Sons, Inc.
Soluble microbial products (SMP) in anaerobic chemostats
Authors: Noguera Daniel R. , Araki Nobuo , Rittmann Bruce E.
Published: December 2004

The production of soluble microbial products (SMP) in anaerobic systems was evaluated using chemostat reactors. Results from steady‐state and tracer experiments with 14C‐glucose and 14C‐acetate showed that significant amounts of SMP were produced during the acidogenesis of glucose, but that SMP did not accumulate during methanogenesis from acetate. In addition, at a retention time of 40 days, SMP comprised almost all of the effluent COD from the glucose‐fed chemostat. For shorter retention times, as low as 10 days, the SMP concentration remained almost constant, but its significance in the effluent COD was reduced due to the accumulation of intermediate volatile fatty acids. The results from a 14C‐tracer experiment in the glucose‐fed chemostat were used to evaluate the importance of including SMP formation and degradation in kinetic modeling of the methanogenic chemostats. Three models were evaluated: a model without SMP production, a model with SMP production but no degradation, and a model with SMP production and degradation, The results of this kinetic analysis indicate that the model that includes SMP production and degradation was the only one able to adequately represent the fate of 14C in the tracer experiment. The kinetic parameters were successfully used to predict steady‐state concentrations of SMP and to characterize the formation and degradation characteristics of the SMP. © 1994 John Wiley & Sons, Inc.
Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma
Authors: Barnabé N. , Butler M.
Published: December 2004

The specific monoclonal antibody productivity (qMab) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33°C to 39°C. qMab was constant at each temperature and was independent of the phase of culture. The qMab increased 97% at 39°C and decreased by 21% at 33°C compared with controls at 37°C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, YLac/Llc' was constant for 33°C to 39°C and YAmm/Gin was constant for 37°C to 39°C. YAmm/Gin at 33°C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U‐ratio (UTP/UDP–GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP‐/N acetyl galactosamine, UDP‐N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP‐sugars) ratio, purine/pyrimidine, ATP/GTP, and U‐ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of qMab observed at the higher temperatures. © 1994 John Wiley & Sons, Inc.
Evaluation of D‐amino acid oxidase from Rhodotorula gracilis for the production of α‐keto acids: A reactor system
Authors: Butò Simona , Pollegioni Loredano , D'Angiuro Luigi , Pilone Mirella S.
Published: December 2004

The study reports on the development of a bioreactor for the production of α‐keto acids from D,L‐ or D‐amino acids using Rhodotorula gracilis D‐amino acid oxidase. D‐Amino acid oxidase was co‐immobilized with catalase on Affi‐Gel 10 matrix, and the reactor was operated as a continuous‐stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half‐life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D‐amino acid oxidase. © 1994 John Wiley & Sons, Inc.
Purification and processing of cellulose‐binding domain‐alkaline phosphatase fusion proteins
Authors: Greenwood Jeffrey M. , Gilkes Neil R. , Miller Robert C. , Kilburn Douglas G. , Warren R. Antony J.
Published: December 2004

Fusion of the leader peptide and the cellulose‐binding domain (CBD) of endoglucanase A (CenA) from Cellulomonas fimi, with of without linker sequences, to the N‐terminus of alkaline phosphatase (PhoA) from Escherichia coli leads to the accumulation of significant amounts of the CBD–PhoA fusion proteins in the supernatants of E. coli cultures. The fusion proteins can be purified from the supernatants by affinity chromatography on cellulose. The fusion protein can be desorbed from the cellulose with water or guanidine‐HCl. If the sequence IEGR in present between the CBD and PhoA, the CBD can be cleaved from the PhoA with factor Xa. The efficiency of hydrolysis by factor Xa is strongly in fluenced by the amino acids on either side of the IEGR sequence. The CBD released by factor Xa is removed by adsorption to cellulose. A nonspecific proteases from C. fimi, which hydrolyzes native CenA between the CBD and the catalytic domain, may be useful for removing the CBD from some fusion proteins. © 1994 John Wiley & Sons, Inc.
Effect of vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant chinese hamster ovary cells
Authors: Pendse Girish J. , Bailey James E.
Published: December 2004

Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen‐limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone‐inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb‐expressing clone and the parental CHO‐tPA expressing cells. The VHb‐expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO‐tPA culture. © 1994 John Wiley & Sons, Inc.
On‐line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH
Authors: Oeggerli A. , Eyer K. , Heinzle E.
Published: December 2004

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k1, respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. © 1995 John Wiley & Sons, Inc.
Starvation‐induced programmed death of hybridoma cells: Prevention by amino acid mixtures
Authors: Franěk F.
Published: December 2004

Association of the availability of nutrients with the phenomenon of programmed cell death—apoptosis—was investigated using hybridoma cells cultured in protein‐free medium under conditions of starvation, i.e., in RPMl‐1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth‐supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. © 1995 John Wiley & Sons, Inc.
The Monod equation and mass transfer
Authors: Merchuk J. C. , Asenjo J. A.
Published: December 2004

An alternative interpretation of the growth rate–substrate concentration dependence is presented. This is based on the assumption that the main factors affecting growth rate are transfer of substrate from the medium and the maximum growth velocity, which is that observed when no substrate limitations occur. This approach allows the approximate prediction of one of the two kinetic constants required, and may be of great use, especially for continuous cultures. It is the first attempt to provide a phenomenological explanation for the large variations observed in the values of the Monod constant, Ks, reported in the literature. © 1995 John Wiley & Sons, Inc.
Molecular assembly of proteins and conjugated polymers: Toward development of biosensors
Authors: Ayyagari Madhu S. , Pande Rajiv , Kamtekar Snjay , Marx Kenneth A. , Tripathy Sukant K. , Gao Harry , Kumar Jayant , Akkara Joseph A. , Kaplan David L.
Published: December 2004

A molecular assembly in which a conjugated polymer is interfaced with a photodynamic protein is described. The conjugated polymer, functionalized with biotion, is designed such that it can be physisorbed on or chemically grown off a glass surface. The streptavidin‐derivatized protein is immobilized on the biotinylated polymer matrix through the strong biotin–streptavidin interactions. The assembly, built on the surface of an optical fiber or on the inside walls of a glass capillary, forms an integral part of a biosensor for the detection of environmental pollutants such as organophosphorus‐based insecticides. The Protein in the system can be replaced by any biological macromolecule of interest. We study one specific case, the enzyme alkaline phosphatase. The enzyme catalyzes a reaction producing an intermediate compound that chemiluminesces, and the chemiluminescence singnal is monitored to detect and quantify insecticides such as paraoxon and methyl parathion. Preliminary results indicate ppb level detection with response time less than 1 minute. © 1995 John Wiley & Sons, Inc.
A study of mass transfer in yeast in a pulsed baffled bioreactor
Authors: Ni Xiongwei , Gao Siwen , Pritchard Dave W.
Published: December 2004

We report experimental data of mass transfer of oxygen into yeast resuspension in a pulsed baffled bioreactor. The bioreactor consists of a 50‐mm‐diameter column with the presence of a series of either wall (orifice) or central (disc) baffles or a mixture of both where fluid oscillation can also be supermposed during the experiments. Air bubbles are sparged into the bottom of the pulsed baffled bioreactor, and the kinetics of liquid oxygen concentration in the yeast solution is followed using a dissolved oxygen probe with a fast response time of 3 s together with the dynamic gassing‐out technique. Among the three different baffle geometries investigated, the orifice baffles gave the highest and sharpest increase in the oxygen transfer rate, and the trends in the kLa measurements are consistent with the fluid mechanics observed within both the systems and previous work. In addition, we have also compared the kLa values with those obtained in a stirred tank; an 11% increase in the KLa is reported. © 1995 John Wiley & Sons, Inc.
A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose
Authors: Yang Shang‐Tian , Huang Yan , Hong Gene
Published: December 2004

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de‐lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5‐L column reactor, which was connected to a 5‐L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took ∼55 h to ferment whey permeate containing ∼45 g/L lactose to ∼20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L · h to 0.47 g/L · h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L · g cell. The productivity increased to 0.68 g/L · h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. © 1995 John Wiley & Sons, Inc.