bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli
Authors: Betenbaugh Michael J. , Beaty Christine , Dhurjati Prasad 
Published: December 2004

AbstractAn experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature‐sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.
Characterization of a cellodextrin glucohydrolase with soluble oligomeric substrates: Experimental results and modeling of concentration‐time‐course data
Authors: Schmid G. , Wandrey C. 
Published: December 2004

AbstractA previously isolated cellodextrin glucohydrolase (β‐glucosidase) from Trichoderma reesei QM 9414 is characterized using β‐1,4‐glucose oligomers with defined degrees of polymerization as soluble substrates. The enzyme splits off glucose units from the nonreducing chain ends of cellooligomers. Besides this hydrolytic activity there is also evidence for transfer activity depending on the concentration and degree of polymerization of substrates. Concentration‐time‐course data have been gathered for the degradation of cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose covering a wide range of enzyme and substrate concentrations. A Michaelis‐Menten type kinetic model has been developed, which is able to satisfactorily describe the complex system of parallel and series reactions during the conversion of oligomers to glucose. The only kind of inhibition considered is competitive inhibition by the final product glucose. The model takes into account the formation of multiple enzyme‐substrate complexes and is limited to those conditions, in which no transglucosylation products are observed. Cellodextrins with higher degrees of polymerization are found to be better substrates for this enzyme than is the dimer cellobiose.
Periodic operation of immobilized cell systems: Analysis
Authors: Sayles Gregory D. , Ollis David F. 
Published: December 2004

AbstractThe authors' mathematical model of transient immobilized cell growth and product formation is applied here to examine the performance of an immobilized cell system subject to periodic cycling of the rate‐limiting substrate supply. The model system consists of a single hydrogel‐like (porous) particle entrapping viable microorganisms. Proper nutrient cycling is shown to yield a relaxed periodic system and to virtually eliminate the leakage of biomass from the support that is commonly observed experimentally in steady (continuous nutrient supply) operation of these systems. The use of cyclic operation is evaluated by calculating the average product yield (the ratio of product formed to substrate consumed) and the average product flux from the particle (a measure of the total productivity of the system), for various cycling rates. Cycling increased the average product yield by at least a factor of three in nongrowth‐related fermentations, relative to steady operation, without any significant sacrifice in average total productivity. Growth‐related fermentations lost significant total productivity under most cycling conditions, while the average product yield was approximately unchanged at all cycling rates. Thus, immobilization in conjunction with periodic operation should be considered as an alternative process design for the production of nongrowth‐related products such as penicillin and monoclonal antibodies.
Cybernetic modeling of bacteriol cultures at low growth rates: Single‐substrate systems
Authors: Turner Brian G. , Ramkrishna D. , Jansen Norman B. 
Published: December 2004

AbstractThe cybernetic framework developed by Ramkrishna and co‐workers has been expanded to include the effects of cellular maintenance energy requirements on biomass levels in slow‐growing, carbon‐substrate‐limited cultures. A simple structured model, based on the existence of distinct key enzymes for growth and maintenance functions, is presented. Comparisons of the model with experimental data for the growth of Klebsiella oxytoca in constant fed‐batch culture on glucose, fructose, arabinose, and xylose show good agreement. In addition, perturbed fed‐batch culture experiments indicate that slow‐growing cultures respond less rapidly to a removal of the growth limitation than do faster‐growing ones. The possibility of a growth‐rate dependent “critical resource” is discussed.
Expression of β‐lactamase by recombinant Escherichia coli strains containing plasmids of different sizes—effects of pH, phosphate, and dissolved oxygen
Authors: Ryan Wen , Parulekar Satish J. , Stark Benjamin C. 
Published: December 2004

AbstractThe characteristics of growth and synthesis of plasmid‐encoded protein were studied for strains of recombinant E. coli JM103 which carried the β‐lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying‐sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0–8.0, the bulk β‐lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7–8.7 kb), at which β‐lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for β‐lactamase synthesis. The bulk β‐lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and β‐lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid‐encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.
Autoradiographic determination of mass‐transfer limitations in immobilized cell reactors
Authors: Karel Steven F. , Robertson Channing R. 
Published: December 2004

AbstractPseudomonas putida cells were grown in confined volumes in dual‐membrane immobilized cell reactors constructed from microporous polyethylene hollow fibers and silicone rubber tubules as a model system for the study of mass transport in microbial aggregates. Local cell concentrations in the reactors reached 300 g dry mass/L. Pulse‐chase radioisotope labeling with 35SO42− was used to estimate the rates of cell mass synthesis and degradation. Sulfur incorporation consistently exceeded sulfur release, implying that the cell mass concentration continually increases. The location and size of the cell growth region was determined using liquid emulsion autoradiography of thin sections prepared from labeled reactors. Cell growth occurs in a region less than 25 μm in depth adjacent to the oxygen supply, and the expansion of the cells caused by cell growth promotes convection of the cell mass into regions of the reactor where starving cells accumulate. The combination of mass‐balance and spatial distribution measurements that can be made using radioisotope tracers provides a versatile method for determining metabolic rates and limitations caused by mass transfer in immobilized cell reactors.
Semicontinuous cultivation of Digitalis lanata cells: Production of β‐methyldigoxin in a 300‐L airlift bioreactor
Authors: Reinhard E. , Kreis W. , Barthlen U. , Helmbold U. 
Published: December 2004

AbstractSelected Digitalis lanata cell lines cultivated in 1‐L shake flasks or 20‐L airlift bioreactors converted β‐methyldigitoxin into β‐methyldigoxin with almost no side reactions. This biotransformation process was optimized with regard to substrate supply and culture medium composition, and was then scaled up to a volume of 210 L using a 300‐L airlift bioreactor. A semicontinuous process was developed in which 513.3 g β‐methyldigoxin were produced after 89 days of cultivation.
Xylose fermentation by yeasts. 5. Use of ATP balances for modeling oxygen‐limited growth and fermentation of yeast Pichia stipitis with xylose as carbon source
Authors: Rizzi Manfred , Klein Christian , Schulze Constanze , Bui‐Thanh Ngoc‐Anh , Dellweg Hanswerner 
Published: December 2004

AbstractKinetic studies are presented for the growth and fermentation of the yeast Pichia stipitis with xylose as the carbon source. Ethanol is produced from xylose under anaerobic as well as under oxygen‐limiting conditions but only at dissolved oxygen concentrations up to 3 μmol/L Maximum yields and production rates were obtained under oxygen‐limiting conditions, where the xylose metabolism may be considered to be consisted of three different components (assimilation, respiration, fermentation). The contribution of each pathway is determined by the availability of oxygen and the energy yield of each pathway. In order to describe the course of oxygen‐limited fermentations, a mathematical model has been developed with the assumption that growth is coupled to the energy production. The resulting model requires only four independent parameters (Y, Y, mATP, and P/O). These parameters were estimated on the basis of eight separate batch fermentations.
Reduced product formation following perturbation of ethanol‐ and propionate‐fed methanogenic CSTRs
Authors: Smith Daniel P. , McCarty Perry L. 
Published: December 2004

AbstractEnergetic analysis was applied to reduced product formation following perturbation of ethanol‐ and propionate‐fed methanogenic continuous stirred tank reactors (CSTRs). Formation and dissipation of longer‐chained n‐carboxylic acids corresponded with the variation in Gibbs free energy change associated with beta‐oxidation reactions. Formation appeared to occur from acetate and propionate by reductive back‐reactions, made energetically favorable by elevated hydrogen partial pressure (P), and possibly mediated by biosynthetic enzymes. The formed longer‐chained acids dissipated when the P fell and equilibrium shifted to favor beta‐oxidations. n‐Propanol was found to be produced from propionate in a coupled ethanol oxidation/propionate reduction reaction, mediated by ethanol‐oxidizing organisms during high rates of ethanol utilization and elevated P. When P declined, n‐propanol was oxidized back to its precursor propionate. Both reaction energetics and intracellular diffusion of the electron carrier may effect transient mediation of this coupled reaction.
Urea hydrolysis by immobilized urease in a fixed‐bed reactor: Analysis and kinetic parameter estimation
Authors: Moynihan H. J. , Lee C. K. , Clark W. , Wang N.‐H. L. 
Published: December 2004

AbstractUrea hydrolysis by urease immobilized onto ion exchange resins in a fixed‐bed reactor has been studied. A modified Michaelis–Menten rate expression is used to describe the pH‐dependent, substrate‐ and product‐inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug‐flow reactor model has been developed. An effectiveness factor is used to describe the reaction–diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best‐fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the Vmax (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.
A comparison of three models for the diffusion of oxygen in electrolyte solutions
Authors: Holtzapple Mark T. , Eubank Philip T. , Matthews Michael A. 
Published: December 2004

AbstractDiffusion of oxygen through aqueous solutions is of great importance in biological systems. In this work, three models for the diffusion of oxygen through aqueous salt solutions are compared. One model uses mole fraction as the driving force (Fick's Law) and another uses chemical potential. The third model uses the gradient in oxygen activity as the driving force. This new model was chosen because of the availability of oxygen electrodes which directly measure oxygen activity in aqueous solution. These models have been used to reevaluate the technique of measuring O2 diffusivities. We show that Pick's Law diffusion coefficients do not vary strongly with salt concentration as was erroneously reported in the literature. In addition, we compare the predicted O2 fluxes of the three models over a wide range in O2 concentrations. For oxygen concentrations of biological interest, the three models give identical predictions of the flux.
Uptake of cadmium and zinc by the alga Chlorella vulgaris: Part 1. Individual ion species
Authors: Ting Y. P. , Lawson F. , Prince I. G. 
Published: December 2004

AbstractThe ability of algae and bacteria to accumulate heavy metals from the surrounding environment is a widely recognized phenomenon that has a number of important implications. This work reports on the development of a quantitative model that addresses the basic mechanisms inherent in many uptake processes. The model postulates two mechanisms: an initial rapid metal ion uptake due to attachment onto the cell wall followed by a relatively slow uptake due to membrane transport of the metal into the cell. The mathematical model has been tested using the alga Chlorella vulgaris in the presence of cadmium and zinc in solution under various experimental conditions.
Properties of a reversible soluble–insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose
Authors: Taniguchi Masayuki , Kobayashi Mikio , Fujii Michihiro 
Published: December 2004

AbstractCellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product—the soluble sugar solution—by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble–insoluble carrier for immobilizing enzyme is promising, not only for cellulose–cellulase systems, but also for other heterogeneous reaction systems.
Dynamics of plasmid maintenance in a CSTR upon square‐wave perturbations in the dilution rate
Authors: Weber Allison E. , San Ka‐Yiu 
Published: December 2004

AbstractThe effects of forced oscillations in the dilution rate on a population of Escherichia coli K12 harboring the plasmid pBR322 in a chemostat with a nonselective medium were studied. In the constant dilution rate control experiments, the percentage of plasmid‐containing cells decreased after a long lag time. Eventually, the culture approached a population consisting of 100% plasmid‐free ells. However, under forced perturbations of the dilution rate, the culture maintained a mixed population of plasmid‐free and plasmid‐carrying cells for a longer period of ime. An unstructured model was developed to describe the above observations. Our results indicate that transient conditions created by dilution‐rate perturbations provide a favorable environment for the plasmid‐carrying population. In addition, experiments with different cycling frequencies suggest that adaptation by the culture to these transient conditions will reduce or totally eliminate such an advantage.
Acetone–butanol–ethanol (ABE) production in a novel hollow fiber fermentor–extractor
Authors: Shukla R. , Kang W. , Sirkar K. K. 
Published: December 2004

AbstractAcetone–butanol–ethanol (ABE) production by immobilized C. acetobutylicum cells is studied in a novel microporous hollow fiber based tubular fermentor–extractor. The solvent 2‐ethyl‐l‐hexanol is used for in situ dispersion‐free extraction of products. A mathematical model for simultaneous fermentation and extraction of the products has been investigated. The predicted as well as experimental data follow the same trend. The experimentally observed value of total solvent productivity increased by more than 40% as a result of in situ solvent extraction.
Enzymatic oxidation of ethanol in the gaseous phase
Authors: Barzana Eduardo , Karel Marcus , Klibanov Alexander M. 
Published: December 2004

AbstractThe enzymatic conversion of gaseous substrates represents a novel concept in bioprocessing. A critical parameter in such systems is the water activity, Aw The present article reports the effect of Aw on the catalytic performance of alcohol oxidase acting on ethanol vapors. Enzyme activity in the gas‐phase reaction increases several orders of magnitude, whereas the thermostability decreases drastically when Aw is increased from 0.11 to 0.97. The enzyme is active on gaseous substrates even at hydration levels below the monolayer coverage. Enhanced thermostability at lower hydrations results in an increase in the optimum temperature of the gas‐phase reaction catalyzed by alcohol oxidase. The apparent activation energy decreases as Aw increases, approaching the value obtained for the enzyme in aqueous solution. The formation of a pread‐sorbed ethanol phase on the surface of the support is not a prerequisite for the reaction, suggesting that the reaction occurs by direct interaction of the gaseous substrate with the enzyme. The gas‐phase reaction follows Michaelis–Menten kinetics, with a Km value almost 100 times lower than that in aqueous solution. Based on vapor–liquid equilibrium data and observed Km values, it is postulated that during the gas‐phase reaction the ethanol on the enzyme establishes an equilibrium with the ethanol vapor similar to that between ethanol in water and ethanol in the gas phase.
Structure of activated sludge floes
Authors: Li Da‐Hong , Ganczarczyk Jerzy J. 
Published: December 2004

AbstractRelatively large activated sludge floes (larger than about 100 μm) were stabilized, using a histological tissue specimen preparation procedure, and then were sliced into sections of 3 to 6 μm thick. The study of these sections, after staining, revealed the internal structure of the activated sludge floes. No uniformity of this structure was found. The distribution of microorganisms and of extracellular polymers (EPs) in the floes varied randomly on the plane of the sections and along the dimension perpendicular to the plane, leaving large water channels and reservoirs in some of the floes. The lack of a characteristic size for the water gaps in the floes and a general self‐similar appearance of the sections suggested that the activated sludge floes might be characterized by the fractal concept within a certain size limit. Direct observation of the interior of the floes indicated an abundant presence of extracellular polymers (EPs) in amorphous forms, surrounding microorganisms in most of the floes.
Lipolytic activities of a lipase immobilized on six selected supporting materials
Authors: Shaw Jei‐Fu , Chang Rey‐Chang , Wang Fung Fang , Wang Yng Jiin 
Published: December 2004

AbstractSix different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase‐coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45‐55°C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half‐life of the PVC immobilized lipase operating at 30°C in a packed‐bed reactor was estimated to be about 400 h.