bluuebunny/crossref_metadata_2025 huggingface dataset:
Diffusion and reaction within porous packing media: A phenomenological model
Authors: Jones Warren L. , Dockery Jack D. , Vogel Curtis R. , Sturman Paul J.
Published: December 2004

A phenomenological model has been developed to describe biomass distribution and substrate depletion in porous diatomaceous earth (DE) pellets colonized by Pseudomonas aeruginosa. The essential features of the model are diffusion, attachment and detachment to/from pore walls of the biomass, diffusion of substrate within the pellet, and external mass transfer of both substrate and biomass in the bulk fluid of a packed bed containing the pellets. A bench‐scale reactor filled with DE pellets was inoculated with P. aeruginosa and operated in plug flow without recycle using a feed containing glucose as the limiting nutrient. Steady‐state effluent glucose concentrations were measured at various residence times, and biomass distribution within the pellet was measured at the lowest residence time. In the model, microorganism/substrate kinetics and mass transfer characteristics were predicted from the literature. Only the attachment and detachment parameters were treated as unknowns, and were determined by fitting biomass distribution data within the pellets to the mathematical model. The rate‐limiting step in substrate conversion was determined to be internal mass transfer resistance; external mass transfer resistance and microbial kinetic limitations were found to be nearly negligible. Only the outer 5% of the pellets contributed to substrate conversion. © 1993 Wiley & Sons, Inc.
Determining metabolic sensitivity coefficients directly from experimental data
Authors: Schlosser Paul M. , Holcomb Tyler , Bailey James E.
Published: December 2004

The application of metabolic control theory (MCT), or other methods of determining metabolic sensitivity to the rates of specific cellular processes, such as enzymatic reactions, requires knowledge of the elasticity coefficients (system partial derivatives) for the processes under study. Although rate equations are available in the literature for some enzymatic reactions, there are many reactions and processes for which this is not the case. Although one could perform the experiments necessary to determine the rate equations for a given system, these equations are, in fact, not required for the calculation of sensitivities–only the elasticities (the derivatives) are needed. A more direct and efficient approach would be to compute elasticities directly from experimental data. Errors can analysis and alternative regression techniques are presented which not only allow one to eliminate data components with excessive noise, but also provide guidance as to what additional data may be require for accurate sensitivity analysis. This information indicates which measurements require more accuracy and what additional experiments should be conducted to reduce errors in calculated metabolic sensitivity coefficients. © 1993 Wiley & Sons, Inc.
Separation albumin–PEG: Transmission of PEG through ultrafiltration membranes
Authors: Lentsch Sandrine , Aimar Pierre , Orozco Jose Luis
Published: December 2004

Transmission of polyethylene glycol (PEG) through ultrafiltration membranes has been studied under various operating conditions of pressure, crossflow, and concentration, using different membranes cut‐offs and two module designs with the aim of understanding the separation of PEG from BSA. The influence of protein adsorption and fouling of the choice of a membrane has also been considered. Retention depends in general on the molecule to average pore size ratio, as expected, but also on concentration polarization. Accordingly, all operating and design parameters favoring concentration polarization lead to higher transmission. At high fluxes, flexible macromolecules can pass through the membrane, even if the random coil is larger than the apparent average pore. From a process selectivity point of view, the best way to separate PEG from BSA would be to use a membrane totally retaining BSA and to enhance concentration polarization of PEG. Unfortunately, such conditions also increase fouling and concentration polarization by BSA, which limits flux and thus PEG concentration polarization and transmission. Consequences of such conditions on separation efficiency are discussed. © 1993 Wiley & Sons, Inc.
A novel BOD sensor based on bacterial luminescence
Authors: Hyun Chang‐Kee , Tamiya Eiichi , Takeuchi Toshifumi , Karube Isao , Inoue Noriuki
Published: December 2004

A reagent‐type BOD sensor with a new principle employing a luminous bacterium, Photobacterium phosphoreum, was developed. The increased intensity of luminescence resulting from the cellular assimilation of organic compounds in wastewater was detected by a photodiode. The BOD response of the bacterial reagent could be obtained within 15 min with ±7% error. The temperature condition for optimal BOD response was 18° to 25°C at pH 7 to 8, indicating that it is possible to measure BOD at room temperature without having to stabilize the temperature of the measuring system. For practical use, two procedures for long‐term preservation of the bacterial reagent, vacuum drying method and freezing method, are suggested. The metabolic characteristics of employed luminous bacteria were investigated by comparing the BOD values for several pure organic substrates estimated by the BOD sensor with conventional 5‐day BOD values. In comparison with the 5‐day measurement for some wastewater samples, BOD values estimated by the sensor showed comparatively good agreement with those measured by the 5‐day method. © 1993 Wiley & Sons, Inc.
Symmetric branching model for the kinetics of mycelial growth
Authors: Viniegra‐González G. , Saucedo‐Castañeda G. , López‐Isunza F. , Favela‐Torres E.
Published: December 2004

A mathematical model, linking microscopic to macroscopic parameters of the kinetics of mycelial growth is presented. The model consists of two parts: (a) a microscopic description, based on the assumption that growth of a mycelium can be represented approximately by the growth of a symmetric binary tree, where the branching level (microscopic state variable) is logarithmically related to the number of tips and segments; and (b) a macroscopic description which makes use of the microscopic description in order to define the parameters related to the evolution of biomass (macroscopic state variable) as a function of time. The latter uses a distribution of arrested tips in a population of mycelia, in order to estimate the fraction of non‐growing biomass in terms of a power law function with coefficient, n, of the biomass concentration. The microscopic description explains the fact that the germ tube specific growth rate of Aspergillus nidulans measured in a growth chamber, is about the double the specific growth rate of this organism, when measured in shake flasks. It predicts that the length of the hyphal growth unit of the mycelium of Geotrichum candidum would be approximately the double the germ tube length measured at the time just before the first branching event. It also allows the derivation of useful expressions for predicting macroscopic parameters, such as the maximal specific growth rate, the initial amount of biomass, and the amount of biomass before the branching process starts. Those estimates are done in terms of microscopic quantities, i.e., the amount of germinated spores, the diameters of the spores and hyphae, the average rate of tip extension, and the average internodal segment length. Estimation of coefficient n by fitting the macroscopic description to a growth curve of A. niger gives an indication on the degree of skewness of the distribution of arrested mycelia. Estimated macroscopic parameters are in relative good agreement with measured average segment length. © 1993 John Wiley & Sons, Inc.
Biochemical production capabilities of escherichia coli
Authors: Varma Amit , Boesch Brian W. , Palsson Bernhard O.
Published: December 2004

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. © 1993 John Wiley & Sons, Inc.
Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain
Authors: Wang Zhengjun , Da Silva Nancy A.
Published: December 2004

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid‐k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid‐k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFα1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80–90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long‐term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid‐containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid‐k locus was observed. © 1993 John Wiley & Sons, Inc.
Loss of antibody productivity is highly reproducible in multiple hybridoma subclones
Authors: Merritt Steven E. , Palsson Bernhard O.
Published: December 2004

An immunoglobulin G (IgG2b) producing hybridoma cell line (S3H5/γ2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum‐free medium. Following adaptation to serum‐free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. © 1993 John Wiley & Sons, Inc.
Activated sludge process performance using a multistage tower aeration tank
Authors: Shimizu Tatsuo , Kudo Kenzo , Nasu Yoshikazu
Published: December 2004

This study's objective was to clarify both experimentally and theoretically whether a vertical multistage tower aeration tank system is advantageous as compared with a completely mixed system, particularly with respect to purification efficiency, sludge settleability, and excess sludge production. In comparing the two systems: (1) purification efficiency in the multistage tower aeration system with partial fluid mixing with a large Peclet number was higher than in a corresponding completely mixed system for all applied organic loadings; (2) the multistage tower aeration system had some definite advantages with respect to sludge settleability and excess sludge production; (3) the activated sludge system's higher performance with partial fluid mixing was shown quantitatively with the axial dispersion model in conjunction with growth kinetics which involved rapid uptake such as biosorption and subsequent oxidative biodegradation processes of organic substances. © 1993 John Wiley & Sons, Inc.
Production of tPA in recombinant CHO cells under oxygen‐limited conditions
Authors: Lin Andy A. , Kimura Roy , Miller William M.
Published: December 2004

Animal cell bioreactors are often limited by the oxygen supply. The reduction in oxygen consumption per cell that occurs under hypoxic conditions may be exploited as a method for increasing reactor capacity if additional glucose is provided to offset increased glycolytic activity. The effects of oxygen deprivation on recombinant tPA (tissue‐type plasminogen activator) production were investigated using midexponential and slowly growing CHO cells. The specific oxygen consumption rate can be reduced by at least 50% (mild hypoxic conditions) without affecting the cell growth rate, maximum cell concentration, tPA production rate, or tPA quality (as characterized by the tPA‐specific activity and SDS‐PAGE analysis). This suggests that mild‐hypoxic conditions (with sufficient glucose) can be used to double the cell concentration and volumetric tPA production rate (at a constant volumetric oxygen supply rate) without sacrificing product quality. However, anoxic conditions should be avoided. When slowly growing cultures were exposed to anoxia, the tPA production rate decreased by 80% without affecting tPA quality. However, when midexponential cultures were exposed to anoxia, the drop in tPA production was accompanied by a decrease in tPA quality that ranged from a 40% decrease in tPA specific activity to extensive tPA degradation. © 1993 John Wiley & Sons, Inc.
Microencapsulation selection for isolation of yeast mutants with increased secretion of Aspergillus awamori glucoamylase
Authors: Wittrup K. D. , Weber A. L. , Tsai P.
Published: December 2004

We have developed a microencapsulation selection method which allows the rapid and quantitative screening of >106 yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400‐fold single‐pass enrichment for high‐secreting mutants, and can be straightforwardly adapted for application to growth‐based selection schemes with other microorganisms and enzymes. © 1993 John Wiley & Sons, Inc.
Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions
Authors: Shi Yuan , Ryu Dewey D. Y. , Park Sun Ho
Published: December 2004

The metabolic pattern and cell culture kinetics of high‐cell‐density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non‐oxygen‐limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen‐limited conditions was almost 4.0 times higher than that under non‐oxygen‐limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen‐limited cultures than those in non‐oxygen‐limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high‐cell‐density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. © 1993 John Wiley & Sons, Inc.
Competition between two microbial populations in a sequencing fed‐batch reactor: Theory, experimental verification, and implications for waste treatment applications
Authors: Dikshitulu S. , Baltzis B. C. , Lewandowski G. A. , Pavlou S.
Published: December 2004

Competition between two microbial populations for a single pollutant (phenol) was studied in a sequencing fed‐batch reactor (SFBR). A mathematical model describing this system was developed and tested experimentally. It is based on specific growth rate expressions revealed from pure culture batch experiments. The species employed were Pseudomonas putida (ATCC 17514) and Pseudomonas resinovorans (ATCC 14235). It was found that both species biodegrade phenol following inhibitory kinetics which can be described by Andrews' expression. The model predicts that the dynamics of a SFBR, and the kinetics of biodegradation, result in a complex set of operating regimes in which neither species, only one species, or both species can survive at steady cycle. The model also predicts the existence of multiple outcomes, achievable from different start‐up conditions, in some domains of the operating parameter space. Experimental results confirmed the model predictions. There was excellent agreement between predicted and measured concentrations of phenol, total biomass, and the biomass of each individual species. This study shows how serious discrepancies can arise in scale‐up of biodegradation data if population dynamics are not taken into account. It also further confirms experimentally the theory of microbial competition in periodically forced bioreactors. © 1993 John Wiley & Sons, Inc.
Enhanced production of α‐amylase in fed‐batch cultures of Bacillus subtilis TN106[pAT5]
Authors: Lee Jeewon , Parulekar Satish J.
Published: December 2004

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid‐encoded protein (α‐amylase) are investigated in batch, continuous, and fed‐batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of α‐amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of α‐amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed‐batch experiments with constant volumetric feed rate demonstrate that α‐amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable α‐amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed‐batch experiments, production of α‐amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two‐stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and α‐amylase production in the second stage, in attaining increased bulk α‐amylase activity is demonstrated. © 1993 John Wiley & Sons, Inc.
Repeated fed‐batch culture of hybridoma cells in nutrient‐fortified high‐density medium
Authors: Jo Eui‐Cheol , Park Hae‐Joon , Kim Dong‐II , Moon Hong Mo
Published: December 2004

Long‐term high‐density cultivation of the hybridoma 2c3.1 was successfully carried out in a repeated fed‐batch mode using high‐density media that were constructed to meet in vitro cell growth limitations. The high‐density culture was possible in a range of 0.5 ∼ 1.0 × 107 cells/mL in MBRI 40‐02 medium for over 2500 h by the repeated supplementation of the most fortified medium, MBRI 40‐03, and consequently, distinct enhancement of MAb production was achieved. MAb concentrations were maintained around 1 g/L for about 1000 h of the process and the maximum MAb concentration was around 1.56 g/L. The result supported strongly the fact that the nutritional fortification was the most critical factor for high‐density cell culture in vitro. The mean chromosome number of the hybridoma 2c3.1 was maintained stably for about 1500 h, whereas gradual loss of the MAb activity was apparent during the long‐term cultivation. © 1993 John Wiley & Sons, Inc.
Adsorption–desorption of recombinant hepatitis B surface antigen (r‐HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification
Authors: Agraz Alberto , Quiñones Yair , Expósito Néstor , Breña Fidel , Madruga Joel , Pentón Eduardo , Herrera Luis
Published: December 2004

Recombinant hepatitis B surface antigen (r‐HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption–elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r‐HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r‐HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r‐HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three‐ and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled‐up for production purposes. © 1993 John Wiley & Sons, Inc.
Scale‐up on basis of structured mixing models: A new concept
Authors: Mayr B. , Moser A. , Nagy E. , Horvat P.
Published: December 2004

A new scale‐up concept based upon mixing models for bioreactors equipped with Rushton turbines using the tanks‐in‐series concept is presented. The physical mixing model includes four adjustable parameters, i.e., radial and axial circulation time, number of ideally mixed elements in one cascade, and the volume of the ideally mixed turbine region. The values of the model parameters were adjusted with the application of a modified Monte‐Carlo optimization method, which fitted the simulated response function to the experimental curve. The number of cascade elements turned out to be constant (N = 4). The model parameter radial circulation time is in good agreement with the one obtained by the pumping capacity. In case of remaining parameters a first or second order formal equation was developed, including four operational parameters (stirring and aeration intensity, scale, viscosity). This concept can be extended to several other types of bioreactors as well, and it seems to be a suitable tool to compare the bioprocess performance of different types of bioreactors. © 1994 John Wiley & Sons, Inc.
Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen
Authors: Tsao Jia‐Huey , Kaneshiro Stacey M. , Yu Shu‐San , Clark Douglas S.
Published: December 2004

Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep‐sea hydrothermal vent, was grown at 80°C in continuous culture on a mineral salts medium gassed with H2 and CO2 at three different flow rates. The maximum specific growth rate was 0.56 h−1, and the maximum specific methane productivity was 0.32 (mol g−1 h−1). Uncoupling of growth and methane production was evidenced by an increase in teh non‐growth‐associated rate of methane formation, β, with increasing gaseous input. The specific hydrogenase activity exhibited growth‐assiciated behaviour at low growth rates, but showed no dependence on growth at higher growth rates. The growth dependence of hydrogenase activity is consistent with the pressure dependence of hydrogenase activity measured in previous experiments. In contrast, the specific protease activity was independent of the growth rate over the entire range of dilution rates studied. © 1994 John Wiley & Sons, Inc.
Predictions for oxygen supply control to enhance population stability of engineered production strains
Authors: Varma Amit , Palsson Bernhard O.
Published: December 2004

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. © 1994 John Wiley & Sons, Inc.
Multifunctional poly(ethylene glycol) semi‐interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting
Authors: Drumheller Paul D. , Elbert Donald L. , Hubbell Jeffrey A.
Published: December 2004

Novel artificial extracellular matrices were synthesized in the form of semi‐interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG–co‐AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG–co‐AA were grafted with peptides at densities of 475 ± 40 pmol/cm2. Polymer networks containing immobilized PEG‐co‐AA N‐terminally grafted with GRGDS supported cell adhesion efficiencies of 42 ± 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 ± 0%, even when challenged with high seeding densities (to 100,000 cell/cm2) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell‐surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.
Cerebral neurons, skeletal myoblasts, and cardiac muscle cells cultured on macroporous beads
Authors: Shahar A. , Reuveny S. , David Y. , Budu C. , Shainberg A.
Published: December 2004

Biodegradable macroporous spherical microcarriers (MCs) offer a suitable substrate for adhesion, growth, and differentiation of cerebral neurons, skeletal myoblasts, and cardiac cells. The cavernous structure of these gelatin beads supplies a large surface and a tridimensional habitat for cell propagation. Within hours from their seeding, all three cell types became firmly attached to the MCs, forming cell–MC aggregates, which remained floating in the medium. Neuronal aggregates were composed mainly of single or groups of perikarya, and their sprouted nerve processes formed a ramified network. In skeletal muscle aggregates, fusion of myoblasts into myotubes occured within 5 days. The myotubes, arranged in bundles having the same orientation, became striated and the whole aggregate, or parts of it, contracted spontaneously. Cardiac cells divided in the aggregate to form one or more layers of flat cells, which exhibited single microvilli. The cells were interconnected at uneven intervals and the whole aggregate contracted actively. Morhological and biochemical analysis of sampled aggregates ensured that cells reached the proper stage of maturation and could be used for either physiological and pharmacological studies or implantation into injured or dystrophic tissue.
Baculovirus expression system scaleup by perfusion of high‐density Sf‐9 cell cultures
Authors: Caron Antoine W. , Tom Rosanne L. , Kamen Amine A. , Massie Bernard
Published: December 2004

A perfusion system based on a 4‐L stirred tank bioreactor and a custom‐designed tangential (cross‐flow) filter was assembled to realize a scaleup of the Baculovirus Expression Vector System (BEVS). When perfused with 1 to 1.5 vol/day, Spodoptera frugiperda (Sf‐9) insect cell cultures grew from 4 × 106 to 15 × 106 cells/mL over 3 to 4 days. The possibility of maintaining high specific production of recombinant VP6 protein (from bovine rotavirus) after baculovirus infection of the high‐density cultures was then assessed. The process consisted of a growth phase in TNMFH + 10% FBS, followed by infection with Bac‐BRV6L recombinant baculovirus and a shift to a low‐serum (0 to 1%) medium for perfusion during the production phase. Multiple runs were executed, each including a battery of shaker flask controls at various cell densities and serum concentrations. On average, specific rVP6 production in the bioreactor amounted to 76% of that found in 20‐mL shaker cultures simulatingthe bioreactor's high cell density, low serum concentration, and medium renewal rate. Mechanical stress generated by cell/medium separation in theperfusion process reduced cell growth rate but had minimal effect on rVP6production. Our results also indicated that serum concentration during the infection phase affected the rVP6 specific production in a cell density–dependent fashion. Although the feasibility of the cell density scale up was demonstrated, optimization is still needed to achieve a truly cost‐effective process.
Synthesis of aspartame precursor with an immobilized thermolysin in tert‐amyl alcohol
Authors: Nagayasu Takeshi , Miyanaga Masamitsu , Tanaka Takaaki , Sakiyama Takaharu , Nakanishi Kazuhiro
Published: December 2004

N‐(Benzyloxycarbonyl)‐L‐aspartyl‐L‐phenylalanine methyl ester (Z‐AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N‐(benzyloxycarbolyl)‐L‐aspartic acid (Z‐Asp) and L‐phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert‐amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70°C in tert‐amyl alcohol in the absence of the subatrate, and up to 50°C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.

The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z‐AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z‐Asp as the substrate for over 300 h at 45°C and a space velocity of 1 h−1 without any loss of acivity. © 1994 John Wiley & Sons, Inc.
Stoichiometric analysis of animal cell growth and its application in medium design
Authors: Xie Liangzhi , Wang Daniel I. C.
Published: December 2004

Animal cell cultivation in vitro has been studied for more than 40 years. However, the culture medium composition has not been designed on the basis of the stoichiometric nutritional demands for animal cell growth. In this article, a model was developed to study the stoichiometric demands for nutrients (including glucose, 20 amino acids, and 10 vitamins)for the synthesis of cell mass and product. The coefficients for these nutrients in the stoichiometric equation governing animal cell growth were determined based on cell composition. In addition, a detailed analysis of the nutrients′ roles in the synthesis of cell mass and product was also performed. Applications of the stoichiometric analysis in animal cell cultivation, such as culture medium design, supplemental medium formulation, and feeding strategy will also be discussed. The stoichiometric analysis can be potentially employed to analyze results from animal cell cultures, to improve the performance of culture processes, and to design new process rationally. It can also help to provide a better understanding of animal cell metabolism. Simplifications on the cellular energy metabolism were made in order to simplify the model and to provide the preliminary bases to test the process performance. However, this could introduce inaccuracies for the model and results in errors in the calculations of glucose and glutamine concentrations when employed in medium design. © 1994 John Wiley & Sons, Inc.
Cultivation of an L‐lactate dehydrogenase mutant of Bacillus stearothermophilus in continuous culture with cell recycle
Authors: Martin R. San , Bushell D. , Leak D. J. , Hartley B. S.
Published: December 2004

Continuous fermentation with cell recycle proved very effective in increasing the ethanol volumetric productivity of the thermophilic facultative anaerobe, Bacillus stearothermophilus strain LLD‐15, on sucrose at 70°C. When complete cell recycle was used, cell viability decreased after a few residence times and sucrose consumption was reduced. Operation using a constant bleed rate resulted in greater stability and higher ethanol volumetric productivities. A mathematical model based on maintenance energy requirements provided an adequate description of the system. © 1994 John Wiley & Sons, Inc.
Desulfurization of coal by microbial column flotation
Authors: Ohmura Naoya , Saiki Hiroshi
Published: December 2004

Twenty‐three strains capable of oxidizing iron were isolated from coal and ore storage sites as well as coal and ore mines, volcanic areas, and hot spring. Four strains were found to have high iron‐oxidizing activity. One strain (T‐4) was selected for this experiment since the strain showed the fastest leaching rate of iron and sulfate from pyrite among the four strains. The T‐4 strain was assigned for Thiobacillus ferrooxidans from its cultural and morphological characteristics.

Bacterial treatment was applied to column flotation. An increase of cell density in the microbial column flotation resulted in the increase of pyrite removal from a coal‐pyrite mixture (high sulfur imitated coal) with corresponding decrease of coal recovery. The addition of kerosene into the microbial column flotation increased the recovery of the imitated coal from 55% (without kerosene) to 81% (with 50 μL/L kerosene) with the reduction of pyrite sulfur content from 11% (feed coal) to 3.9% (product coal). The kerosene addition could reduce the pyritic sulfur content by collecting the coal in the recovery. However, the addition could not enhance separation of pyrite from the coal‐pyrite mixture, since pyrite rejection was not affected by the increase of the kerosene addition. An excellent separation was obtained by the microbial flotation using a long column which had a length‐diameter (L/D) ratio of 12.7. The long column flotation reduced the pyritic sulfur content from 11% (feed coal) to 1.8% (product coal) when 80% of the feed coal was recovered without the kerosene addition. The long column flotation not only attained an excellent separation but also reduced the amount of cells for desulfurization to as little as one‐tenth of the reported amount. © 1994 John Wiley & Sons, Inc.
On protein solubility in organic solvent
Authors: Chin Jennifer T. , Wheeler Sarah L. , Klibanov Alexander M.
Published: December 2004

Solubility of a model protein, hen egg‐white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non‐aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co‐solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5‐pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. © 1994 John Wiley & Sons, Inc.
On‐line characterization of a hybridoma cell culture process
Authors: Zhou Weichang , Hu Wei‐Shou
Published: December 2004

The on‐line determination of the physiological state of a cell culture process requires reliable on‐line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on‐line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on‐line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on‐line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on‐line. Using these on‐line measurements and calculations, the hybridoma culture process was characterized on‐line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed‐batch and perfusion cultures. © 1994 John Wiley & Sons, Inc.
A fast bioassay for phytotoxicity measurements using immobilized photosynthetic membranes
Authors: Loranger Chantal , Carpentier Robert
Published: December 2004

The potential of thylakoid membranes immobilized in an albumin‐glutaraldehyde crosslinked matrix in a fast bioassay for phytotoxicity measurements in aqueous samples is studied. Free and immobilized preparations are compared for their electron transport activity measured as the initial rate of oxygen evolution with 2,5‐cichlorobenzoquinone as the artificial electron acceptor. Immobilized thylakoids were much stable under storage conditions; in the dark, at 4°C, they were fully stable in terms of photosynthetic activity for a period of 200 h. The immobilized membranes were as sensitive as the free thylakoids for the detection of most of the compounds tested (metal cations, sulfite, nitrite, and herbicides), all known as inhibitors of photosynthetic electron transport. In some instances, the immobilized preparations were even more sensitive than the free counterparts. The sensitivity could be further increased by lowering chlorophyll concentration in the assay. The short incubation period required (∼10 to 15 min) and the small volume of the assay (3 mL) suggest that this type of material should be useful in the detection of locations or effluents with phytotoxic character. © 1994 John Wiley & Sons, Inc.
Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata
Authors: Fett‐Neto Arthur G. , Zhang Wen Yi , Dicosmo Frank
Published: December 2004

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non‐growth related (higher in the stationary phase) and at relatively low levels (up to 4 μg/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4μg/mL) and another during the late (0.1‐μg/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 μg/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. © 1994 John Wiley & Sons, Inc.
Biological destruction of CCI4: II. Kinetic modeling
Authors: Hooker Brain S. , Skeen Rodney S. , Petersen James N.
Published: December 2004

A denitrifying consortium capable of transforming carbon tetrachloride (CCI4) was cultured from an aquifer soil sample from the U.S. Department of Energy's Hanford Site in southeastern Washington State. A mathematical description of the kinetics of CCI4 destruction by this microbial consortium is presented, and its prediction are compared to experimental data. The model successfully predicted the concentrations of acetate, nitrate, nitrite, biomass, and CCI4 for all 12 experiments (a total of 60 concentration‐vs.‐time data sets). In addition, no statistically significant interactions exist between parameter values and individual test conditions. The ability of the model to predict the results of a treatability test for CCI4 degradation in Hanford groundwater, without adjusting any model parameters, is discussed. © 1994 John Wiley & Sons, Inc.
Kinetic investigation of microbial souring in porous media using microbial consortia from oil reservoirs
Authors: Chen Ching‐I , Reinsel Mark A. , Mueller Robert F.
Published: December 2004

Microbial souring (H2S production) in porous media was investigated in an anaerobic upflow porous media reactor at 60°C using microbial consortia obtained from oil reservoirs. Multiple carbon sources (formate, acetate, propionate, iso‐ and n‐butyrates) found in reservoir waters as well as sulfate as the electron acceptor was used. Kinetics and rates of souring in the reactor system were analyzed. Higher volumetric substrate consumption rates (organic acids and sulfate) and a higher volumetric H2S production rate were found at the from part of the reactor column after H2S production had stabilized. Concentration gradients for the substrates (organic acids and sulfate) and H2S were generated along the column. Biomass accumulation throughout the entire column was observed. The average specific sulfate reduction rate (H2S production rate) in the present reactor after H2S production had stabilized was calculated to be 11062 ±2.22 mg sulfate‐S/day g biomass. © 1994 John Wiley & Sons, Inc.
Monitoring of ethanol during fermentation of a lignocellulose hydrolysate by on‐line microdialysis sampling, column liquid chromatography, and an alcohol biosensor
Authors: Buttler Torbjörn , Gorton Lo , Jarskog Helena , Marko‐Varga György , Hahn‐HäGerdal Bärbel , Meinander Nina , Olsson Lisbeth
Published: December 2004

During a 70‐h fermentation of a lignocellulose hydrolysate, the ethanol produced was monitored on‐line using a microdialysis probe as an in situ sampling device. The dialysate components were then separated in a column liquid chromatographic system and the ethanol was selectively detected by an amperometric alcohol biosensor. The result was compared with two off‐line analysis methods: one chromatographic method with refractive index (RI) detection and one enzymatic method based on spectrophotometric detection. The two methods base on enzymes were shown to give lower values than the chromatographic method based on RI detection, which is discussed n terms of selectivity. The investigated on‐line setup was found to be a flexible system for monitoring of fermentations, allowing a sampling frequency of at least 12 h−1 and with a delay between sampling and detection of less than 5 min. © 1994 John Wiley & Sons, Inc.