bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of a human erythroid differentiation factor (EDF) and its large‐scale production
Authors: Tsuji T. , Eto Y. , Takano S. , Takezawa M. , Yokogawa Y. , Shibai H.
Published: December 2004

A novel human protein exhibiting erythroid differentiation activity was discovered in the culture fluids of phorbol ester‐stimulated human cells. The differentiation assay system involving Friend virus‐derived mouse leukemia cells was used. THP‐1 cells of myelomonocytic origin were typical producers. 4β‐Phorbol 12‐myristate 13‐acetate (PMA) was essential for inducible excretion of the erythroid differentiation factor (EDF). The factor was stable toward heat and pH (acidic or alkaline) but lost its activity on pronase treatment, which suggested its proteinous nature. After an optimization of the condition, production of EDF was performed on a 200‐L scale for purification of the protein.
Comparison of free and immobilized Pichia pastoris cells for conversion of ethanol to acetaldehyde
Authors: Duff Sheldon J. B. , Murray William D.
Published: December 2004

Free and immobilized cells of Pichia pastoris were used to convert ethanol to acetaldehyde in small‐scale batch reactors. Immobilized cells were less active than free cells (Vmax free = 7.81 g/L h, Vmax immobilized = 3.17 g/L h) due to a number of factors including end product inhibition and diffusional limitations. Immobilized cells were more resistant to heat denaturation both in the presence and absence of ethanol. Immobilized cells retained more of their activity during repeated batch cycles than did free cells.
Continuous fractionation of human plasma proteins by precipitation from the suspension of the recycling stream
Authors: Chang Chong E.
Published: December 2004

The conventional cold‐ethanol batch fractionation method of human plasma is converted to an automatically controlled continuous fractionation process. The selected protein fractions are precipitated by mixing in the recycled product stream of the suspension. Compared to the batch process, the continuous fractionation process generates less coprecipitation and less spontaneous nucleation, allowing efficient centrifugation of precipitates, and the yield and purity of albumin in the final fraction is significantly increased.
Biochemical limits to microbial growth yields: An analysis of mixed substrate utilization
Authors: Gommers P. J. F. , van Schie B. J. , van Dijken J. P. , Kuenen J. G.
Published: December 2004

A theoretical analysis has been made of carbon conversion efficiency during heterotrophic microbial growth. The expectation was that the maximal growth yield occurs when all the substrate is assimilated and the net flow of carbon through dissimilation is zero. This, however, is not identical to a 100% carbon conversion, since assimilatory pathways lead to a net production of CO2. It can be shown that the amount of CO2 produced by way of assimilatory processes is dependent upon the nature of the carbon source, but independent of its degree of reduction and varies between 12 and 29% of the substrate carbon. An analysis of published yield data reveals that nearly complete assimilation can occur during growth on substrates with a high energy content. This holds for substrates with a heat of combustion of ca. 550 kJ/mol C, or a degree of reduction higher than 5 (e.g. ethane, ethanol, and methanol). Complete assimilation can also be achieved on substrates with a lower energy content, provided that an auxiliary energy source is present that cannot be used as a carbon source. This is evident from the cell yields reported for Candida utilis grown on glucose plus formate and for Thiobacillus versutus grown on acetate plus thiosulfate. This evaluation of the carbon conversion efficiency during assimilation also made it possible to compare the energy content of the auxiliary energy substrate added with the quantity of the carbon source it had replaced. It will be shown that utilization of the auxiliary energy source may lead to extreme changes in the efficiency of dissimilatory processes.
Modelling ethanol and secondary inhibitions of ethanol fermentation in a multistage reactor
Authors: Chattaway Thomas , Goma Gérard , Renaud Pierre‐Yves
Published: December 2004

A kinetic model of ethanol fermentation conducted under a variety of conditions in a continuous four‐stage reactor is proposed. The expressions for specific growth and product formation rates are:

Parameters were identified by nonlinear programming and shown to fit data correctly for steady states of seven different experiments. The product inhibition constants were of 27 and 84 g/L, respectively. Secondary inhibitions were represented by the linear biomass term. The proposed model gave a better description of phenomena than one which only took ethanol inhibition into account. The same model also fitted batch fermentation data, with only some parameters altering significantly. The use of this model for on‐line purposes is discussed.
An immobilized hydrogenase from Alcaligenes eutrophus H‐16
Authors: Deloggio Theodore J. , Graves David J.
Published: December 2004

Alcaligenes Eutrophus H‐16 was grown in continuous culture under conditions which induced hydrogenase production. The hydrogenase enzyme was extracted, partially purified and immobilized on porous glass. This enzyme was then studied both in solution and in immobilized form as a possible candidate for a number of industrial applications. It proved to have a stability (storage and operational) which was highly temperature dependent. Temperatures near freezing caused the enzyme to retain its activity for long periods of time. Although its kinetics were more favorable at elevated temperatures of up to 40°C, the loss of stability outweighed this gain substantially. The effects of buffer type and pH on enzyme activity were also studied. This enzyme has only a modest sensitivity to destruction by oxygen during storage, in contrast to hydrogenases produced by several other microorganisms.
Effects of microorganisms on effective oxygen diffusion coefficients and solubilities in fermentation media
Authors: Ho Chester S. , Ju Lu‐Kwang
Published: December 2004

Effective oxygen diffusion coefficients and solubilities were measured for submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Both effective oxygen diffusion coefficients and solubilities were found to decrease with increasing cell concentrations in the fermentation media. Comparison of the experimental results of effective oxygen diffusion coefficients in fermentation media with values theoretically predicted on the assumption of unpenetrable microbial cells indicates that oxygen molecules diffuse through the cells during the diffusion process. Within the cell concentration range of typical submerged fermentations, the effective oxygen diffusion coefficient of the fermentation media can be described as De = A1f + A2f2. In this equation, fis the cell volume fraction and both A1 and A2 are functions of the shape of the cells and the ratio of effective oxygen diffusion coefficient in microbial cells to that in the medium.
Transient response simulations of recombinant microbial populations
Authors: Peretti S. W. , Bailey J. E.
Published: December 2004

An asynchronous bacterial population has been approximated using a finite number of “computer” cells, each based on a complex single‐cell model for Escherichia coli. This formulation correctly simulates the transient responses of protein and total cell mass synthesis rate to the sudden increase in the concentration of limiting energy source in the growth medium. Experimentally observed responses of rRNA and mRNA synthesis rates to growth rate shifts are qualitatively mirrored by the model. Simulation trends following those of a rel− mutant suggest that model modifications are needed to describe the dynamics of the stringent response. Simulations of the responses of recombinant populations to plasmid amplification or plasmid promoter induction also result in behavior similar to that determined experimentally. The calculated responses for recombinant populations subjected to constant promoter induction or cyclic induction‐noninduction lead to the conclusion that inducible systems give greater productivity than those with fixed promoter strength. This formulation may be utilized as a basis for exploring other aspects of recombinant population dynamics.
Nature and significance of oscillatory behavior during solvent production by Clostridium acetobutylicum in continuous culture
Authors: Clarke Kim G. , Hansford Geoffrey S. , Jones David T.
Published: December 2004

The concurrent production of acids and solvents and the production of acetone during continuous culture in a product‐limited chemostat indicated that the culture contained a mixture of acid‐ and solvent‐producing cells. Periodic oscillations in the yield of end products and the specific growth rate of the culture were ob served during undisturbed continuous culture at a constant dilution rate. The increased specific growth rate was associated with an increased acid yield and an increase in the rate of cell division and the proportion of short rods. The decreased specific growth rate was as sociated with an increase in the solvent yield and a decrease in the rate of cell division, resulting in the production of elongated rods. It is proposed that the oscillatory behavior observed during continuous culture is an inherent characteristic related to the shift from primary to secondary metabolism. A major consequence of the oscillation of the specific rates of growth and division in cultures containing acid‐ and solvent‐producing cells is that it precludes the attainment of a true steady state during continuous culture.
Properties of invertase immobilized on the poly(ethylene‐co‐vinyl alcohol) hollow fiber membrane
Authors: Shiomi Tomoo , Tohyama Masao , Satoh Mikitoshi , Miya Masamitsu , Imai Kiyokazu
Published: December 2004

Invertase was ionically immobilized on the poly(ethylene‐co‐vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2‐dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 μg/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.
Plasmid instability kinetics of the yeast S288C pUCKm8 [cir+] in non‐selective and selective media
Authors: Schwartz L. S. , Jansen N. B. , Ho N. W. Y. , Tsao G. T.
Published: December 2004

Plasmid loss kinetics for Saccharomyces cerevisiae transformed with the 2‐μm DNA‐based‐plasmid pUCKm8 were measured in nonselective and selective media. The plasmid pUCKm8 gives the organism two new phenotypes: resistance to the wide spectrum antibiotic G418 sulfate, and the ability to produce the enzyme, β‐lactamase. Plasmid stability was determined using the production of β‐lactamase as a marker. The effect of G418 on the growth rates of all organisms present in the culture and on plasmid stability was also determined. Mathematical models describing plasmid loss kinetics during exponential growth for both nonselective and selective conditions are used to simulate the experimental data. In nonselective medium, over 80% of the cells still exhibited the desired phenotype after 50 doublings. In medium containing G418, improvements in plasmid stability were only marginal due to the appearance of antibiotic‐resistant cells.
Variation in cellulase‐constituting components from Trichoderma reesei with agitation intensity
Authors: Mukataka Sukekuni , Kobayashi Nobuyuki , Sato Seigo , Takahashi Joji
Published: December 2004

Cellulase producing activity of Trichoderma reesei QM9414 was examined under various agitation intensities and at the dissolved oxygen concentration above 3 ppm. The producing activity greatly depended upon the agitation intensity, and the dependence on the agitation was different for each cellulase‐constituting component. The maximum producing activities of FPA, CM Case, and β‐glucosidase were obtained under different agitation conditions, 1.0, 0.7, and 1.4 m/s in tip velocity, respectively. Intensive agitation brought about remarkable reduction in all cellulase components. The mycelial transformation through agitation intensity was also observed. Comparatively mild agitation of 0.3–1.0 m/s caused pellet formation as the culture progressed, although the pelletization was delayed with increasing agitation intensity. The behavior of the pelletization did not occur at 1.3 and 1.7 m/s throughout the course of cultivation, and under the latter agitation condition hyphae were broken up into short fragments. The cellulase producing activity is discussed in relation to such morphological changes.
Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis
Authors: Frankena J. , Van Verseveld H. W. , Stouthamer A. H.
Published: December 2004

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46–1.73 mol glucose and 2.31–2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Ypsm' g/mol) are 80 < Ypsm < 95 when product is formed from glucose and 50 < Ypsm < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO2 production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.

Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Yspm. Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel‐status of B. Iicheniformis S1684.
Continuous fermentation of feed streams containing D‐glucose and D‐xylose in a two‐stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus
Authors: Slininger P. J. , Bothast R. J.
Published: December 2004

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,1,2,3 To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y‐2460 and Saccharomyces cerevisiae NRRL Y‐2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y‐2235 fermented glucose but not xylose. Y‐2460 consumed both sugars but fermented glucose inefficiently relative to Y‐2235, and it suffered a diauxic lag lasting 10‐20 h when given a sugar mixture. Immobilized Y‐2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y‐2460 exhibited an optimum productivity at 30–40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y‐2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y‐2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8‐g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y‐2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y‐2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two‐stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y‐2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y‐2235 plug flow reactor would enrich the hemicellulose‐derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.
Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy
Authors: Shanks Jacqueline Vanni , Bailey James E.
Published: December 2004

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon 31P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the 31P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3‐phosphoglycerate, beta;‐fructose 1,6‐diphosphate, fructose 6‐phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such‐as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same 31P spectrum. Extension of this strategy to other cellular systems should be straightforward.
Experimental multivariable adaptive optimization of the steady‐state cellular productivity of a continuous baker's yeast culture
Authors: Semomes Gary B. , Lim Henry C.
Published: December 2004

A multivariable adaptive optimization algorithm that uses transient data to improve the optimization speed was successfully implemented on‐line to maximize the steady‐state cellular productivity of a continuous culture of baker's yeast. The algorithm was shown to be stable even during periods of oscillatory growth and was able to reoptimize the culture when planned disturbances were introduced. Although adaptive tuning of the forgetting factor improved the performance, further refinements in the adaptive forgetting factor algorithm are necessary for completely satisfactory results.
Protein purification by affinity binding to unilamellar vesicles
Authors: Powers Johnny D. , Kilpatrick Peter K. , Carbonell Ruben G.
Published: December 2004

A novel purification technique is proposed which employs affinity‐ligand‐modified liposomes to specifically purify bioactive macromolecules from solution. This process is demonstrated with avidin as the model biomolecule and biotin as the affinity ligand. Biotin is covalently bound to the surface of small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylethanolamine (DMPE). The number of accessible binding sites on the liposomes is determined by titration with avidin, and the kinetics of binding are evaluated by monitoring the concentration of free avidin in solution after the addition of biotinylated liposomes. The specificity of the process is determined by following the affinity binding of avidin to biotinylated liposomes in the presence of model impurities (i.e., lysozyme and cytochrome C). Liposome‐bound avidin is separated from the impurities by ultrafiltration through a membrane which retains the liposomes.
A heuristic approach for the economic optimization of a series of CSTR's performing michaelis–menten reactions
Authors: Malcata F. Xavier
Published: December 2004

The estimation of the size of each reactor of a series of CSTR's performing a Michaelis–Menten reaction in the liquid phase can be obtained to advantage via an optimization technique leading to the minimum overall capital cost. The cost scaleup is assumed to be described by a power rule on the equipment capacity. Various contributions are lumped into the exponent, thus leading to values above unity. The analytical development leading to the optimal intermediate concentrations of substrate according to the foregoing criterion is presented. A short‐cut method based on an empirical expression that approximates the numerical solution is reported. This correlation is found to be exact at the asymptotic behaviors, and to give accurate results within an acceptable error level for the range with physical interest. Therefore, it is particularly useful during the predesign steps of equipment for the biochemical industry.
New sensor allowing continuous water activity measurement of submerged or solid‐substrate fermentations
Authors: Gervais P.
Published: December 2004

Water activity of the substrate is an important and acute factor for growth as well as for metabolic production of microorganisms or for biocatalyst systems. A sensor has been designed in order to control this parameter on‐line during submerged and solid‐substrate fermentations. This sterilizable sensor allows the measurement of the relative humidity of the atmosphere in a small chamber by means of a capacitive element separated from the medium by a thin ethylenepolytetrafluoride membrane. For high aw values (>0.90) a sequential circulation of a dried gas prevents the sensor saturation. Measurements are rapid and accurate, and control of the water activity of a fermentation medium has been carried out for 5 days using this sensor.
A bio‐mimetic cadmium adsorbent: Design, synthesis, and characterization
Authors: Yin John , Blanch Harvey W.
Published: December 2004

A cadmium‐binding adsorbent has been designed based on insights gained from the study of cadmium‐binding molecules isolated from living organisms. Cadmium‐chelating thiolate groups—common in proteins that bind cadmium—have been covalently attached to a commercially available ion‐exchange resin. The new adsorbent exhibits a favorable affinity (2 × 10−10M), selectivity (25‐fold greater affinity for Cd2+ than Zn2+), and capacity (1.4 mmol Cd2+/g dry resin) for cadmium ion. Adsorbed Cd2+ may be released with 20mM pyro‐phosphate at pH 2. The adsorbent also recovers Cd2+ in the presence of NH4CI and KCN.

The apparent adsorption rate at pH 7.0 (2M−1 min−1) increases 30‐fold as pH is increased to 11. The rate dependence on pH may be due to the inhibition of adsorbent‐metal association by intramolecular hydrogen bonding at neutral pH.
Kinetic aspects of the bioconversion of L‐tyrosine into L‐DOPA by cells of Mucuna pruriensL. Entrapped in different matrices
Authors: Pras Niesko , Hesselink Paul G. M. , ten Tusscher Jan , Malingré Theo M.
Published: December 2004

Plant cells of Mucuna pruriens L. entrapped In calcium alginate, calcium pectinate, agarose, or gelatine were able to convert L‐tyrosine to L‐DOPA, which was released Into the medium. Michaelis–Menten kinetics could be applied on the entrapped cells, based on the measurement of initial rates of L‐DOPA production. The calculated apparent affinity constants were comparable with the affinity constants obtained with enzyme preparations. Comparison of the apparent maximum rate of bioconversion of the entrapped cells and the maximum rate of bioconversion of a derived cell homogenate indicated that the systems were not operating optimally. Measurement of the effective diffusion coefficients of L‐tyrosine pointed out that this substrate could diffuse freely into the matrices. From the initial rates of bioconversion and the effective diffusion coefficients, the observable modulus was calculated for each system. The obtained values confirmed that the diffusional supply rate of L‐tyrosine was not the limiting factor. For oxygen, which was utilized for byconversion as well as for cell respiration, the calculated observable moduli was directed toward strong oxygen transfer limitations. The values found for the oxygen consumption indicated that the entrapped cells remained partly or totally viable in the four matrices tested. Based on the highest viability and the highest rates of bioconversion, it was concluded that alginate‐entrapped cells of M. pruriens formed the most suitable biocatalytic system for the production of L‐DOPA from L‐tyrosinre.
Cell mass synthesis and degradation by immobilized Escherichia coli
Authors: Karel Steven F. , Robertson Channing R.
Published: December 2004

Escherichia coli K‐12 cells were grown in a confined volume using microporous hollow fiber membranes. The local cell concentrations in the reactors were above 400 g dry mass/L, in excess of the predicted limit based on the specific volume of free cells determined by tracer exclusion. Cell mass synthesis and degradation rates in these reactors were measured using radioisotope labeling with 35S. Net accumulation of cell material persisted at these high cell densities. The rates of substrate uptake and cell growth were predicted from the theory of reaction and diffusion assuming that kinetics of cell metabolism are identical for free‐living and immobilized cells. This theory was tested by comparison of overall rates and by the size of the region in which cell growth occurred, measured by autoradiography. A yield coefficient of 4 ± 1 mol sulfur/mol glucose was measured, in agreement with the value determined for free‐living cells in similar conditions. Cell growth occurs in a thin layer (10–30 μm), at a rate similar to the growth rate for free cells. Volume expansion by the cells as a consequence of proliferation induces convection of cell mass out of the growth region into a region of the reactor filled with starving cells, which then accumulate in the reactor. The combination of mass‐balance and spatial distribution measurements made possible by the use of radioisotope labeling enables a direct test for mass transfer limitations, the determination of the intrinsic cell kinetics, and noninvasive measurements of cell growth in immobilized cell reactors.
Compartment model concept used in the design of fermentation with recombinant microorganisms
Authors: Nielsen J. , Emborg C. , Halberg K. , Villadsen J.
Published: December 2004

Simple models are of great value in order to simulate cell dynamics, and a class of potentially useful models are the so‐called compartment models, which are a simple extension of the unstructured approach. Compartment models have earlier been used with great success to describe cell dynamics. In this article the authors present a 4‐compartment model that is able to describe a majority of the observations that have been made through fermentations with recombinant microorganisms. The compartment model is especially suited for description of dynamic changes in plasmid copy number, e.g., runaway replication. Simulation of batch fermentation with runaway conditions corresponds very well with experimental results. Finally the model is used in the design of an optimal fermentation strategy in a CSTR.
Graphical determination of mean activation energy and standard deviation in a microheterogeneity model of enzyme deactivation
Authors: Henley James P. , Sadana Ajit
Published: December 2004

Traditionally, enzyme populations have been treated as if they were either homogenous, or heterogeneous with distinct and separable subpopulations. The microheterogeneity model, however, assumes that there is a continuous distribution of properties in the population. In the area of enzyme deactivation kinetics, this model describes the heterogeneous population as having a continuous distribution of activation energy of deactivation. This distribution is characterized by mean activation energy, and a standard deviation of activation energy. The microheterogeneity model contains two parameters, ϵ0 and σ. Parameter ϵ0 is the mean value of ϵ for a heterogeneous enzyme population; ϵ is the activation energy divided by absolute temperature and the ideal gas constant. Parameter σ is the standard deviation of the Gaussian distribution of ϵ values in the population. If the population is homogeneous, then ϵ = ϵ0 for all enzyme molecules and σ = 0. There are certain ratios which are independent of ϵ0 and dependent upon σ. Two important ratios are t1/4/t1/2 and t1/2/t, where t represents t1/2 for a homogeneous enzyme population with the same mean ϵ (ϵ0), as the heterogeneous population. If there is experimental deactivation data for the heterogeneous population which is well behaved, the first ratio, t1/4/t1/2, can be determined by estimating the time in minutes at which the enzyme has lost 25% of its activity (t1/4), and the time in minutes at which the enzyme has lost 50% of its activity (t1/2), and then taking the ratio t1/4/t1/2. The corresponding value of σ can be estimated from a graph. The ratio t1/2/t can be found directly as a function of t1/4/t1/2, and can be estimated from another graph. The value of ϵ0 can then be calculated from the formulasgiven in the article.
Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector
Authors: Schendel F. J. , Baude E. J. , Flickinger M. C.
Published: December 2004

On‐line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non‐secreted proteins. As an initial model for monitoring gene expression in intact cells, a non‐gene‐fusion enzyme‐based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real‐time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high‐copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target‐to‐indicator ratio by 10‐ to 40‐fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real‐time determination of the accumulation of proteins from cloned genes in E. coli.
How suspension cultures of Catharanthus roseus respond to oxygen limitation: Small‐scale tests with applications to large‐scale cultures
Authors: Snape J. B. , Thomas N. H. , Callow J. A.
Published: December 2004

The effect of oxygen supply on the growth of suspension cultures of Catharanthus roseus in Erlenmeyer flasks was investigated. Below a critical oxygen supply rate the culture could not survive. By increasing the oxygen supply, a point is reached where the culture survives but no growth is possible. At higher oxygen supply rates there is a regime where both growth rate and the maximum biomass concentration increase with oxygen supply. Eventually there comes a point where no further increase in biomass is achieved, probably due to the depletion of the sugars; however, the growth rate continues to increase with oxygen supply until a maximum growth rate is obtained. The ratio of fresh to dry weight at maximum fresh weight increased with shaker table speed of rotation accompanied by a greater rate of sugar depletion.
Cell adsorption and local accumulation of extracellular polysaccharide in an immobilized Acinetobacter calcoaceticus system
Authors: Wang Sy‐Dar , Wang Daniel I. C.
Published: December 2004

Acinetobacter calcoaceticus can be immobilized on Celite by adsorption. The salt concentrations suitable for immobilized cell fermentation are between 10 and 50 mM phosphate concentration. Low salt concentrations cause desorption of immobilized cells while high salt concentrations inhibit the adsorption of cells on Celite. It is also found that cell adsorption is better at lower pH than at higher pH. An airlift fermentation using immobilized cells at 300 g/L Celite loading shows that about 70% of the total polymer produced is accumulated in Celite pores at a concentration (15.4 g/L) almost threefold higher than that in the bulk liquid (5.7 g/L).
Production of ethanol by Clostridium thermosaccharolyticum: II. A quantitative model describing product distributions
Authors: Mistry Firoz R. , Cooney Charles L.
Published: December 2004

The study of the continuous culture of Clostridium thermosaccharolyticum on xylose showed multiple steady states and hysteresis. A quantitative model based on the biochemistry and physiology of xylose fermentation by C. thermosaccharolyticum was developed. The objective in developing this model was to bring together the observations both of this study and of other researchers on the fermentation of xylose. The model equations were written based on the metabolic pathway for xylose utilization by C. thermosaccharolyticum and the requirement that the carbon, ATP, and NADH within the cell be balanced. Given the specific growth rate μ and the specific xylose utilization rate qs, a set of product distributions (ethanol, acetate, and lactate) satisfying these balances was obtained. This set was plotted on a triangular plot and named the permitted region. The product distributions within this permitted region were shown to be affected by the environmental parameters such as iron concentration and hydrogen partial pressure. The model predicted trends in product distribution which correlate with experimentally observed phenomena. The model was also used to analyze the continuous‐culture data from our experimental work.
Membrane filtration affinity purification (MFAP) of dehydrogenases using cibacron blue
Authors: Ling T. G. I. , Mattiasson B.
Published: December 2004

The method for purification of biomolecules by a combination of affinity interactions and membrane filtration for separation of unwanted material has been found to be of interest for large‐scale work. This study examines the suitability of silica nanoparticles as carriers in the process. Alcohol dehydrogenase and lactate dehydrogenases were chosen as target molecules to be purified. The binding capacity was found to be comparative to what is obtained for high‐performance liquid chromatography (HPLC) packing material. Both binding and desorption of the enzymes were found to be effective. The limiting factor of the process was the filtration flow rate.
Cell counting and carbon utilization velocities via microbial calorimetry
Authors: Boe Irwin , Lovrien Rex
Published: December 2004

Bacteria rapidly metabolize sugars and produce heat accordingly (Escherichia coli, aerobic conditions, 25°C). Two kinds of heat output are gotten: (1) from excess cells and limiting carbon, 2 × 109 to 5 × 109 cells, 5‐50 nanomole glucose; (2) from limited cells and excess carbon, 0. 1 × 109‐1 × 109 bacteria and 200‐600 nmol glucose. The thermograms from heat conduction calorimetry under the first conditions measure velocities of sugar uptake and initial metabolic throughput in 1‐6‐min time spans before a growth cycle possibly can occur. Under the second conditions with limited cells, power output plateaus to a steady state proportional to cell biomass and number of cells. In order to evaluate the calorimetric means for measuring number of cells, six independent means including spectrophotometry (turbidity) were compared: microkjeldahl nitrogen, biuret protein, dry weight, microscopy direct counting in Petroff‐Hausser chambers, and viable colony counting. Using turbidity as a central standard, all methods including calorimetry under the second set of conditions agree within ±18% of one another. Spectrophotometry is the most rapid method but is seriously interfered with by pigments that absorb and foreign particles that also scatter. Calorimetry requires 10‐30 min but measures cell numbers in opaque samples impossible for optical means.
Oxygen transfer rates in a mammalian cell culture bioreactor equipped with a cell‐lift impeller
Authors: Johnson M. , André G. , Chavarie C. , Archambault J.
Published: December 2004

Measurements of kLa were carried out in 1. 5‐ and 5‐L New Brunswick Scientific CelliGen® bioreactors. The measured kLa in water were identical for both vessel sizes operated in similar condition. The mass transfer rate increased with temperature, mixing speed, and aeration rate, with this last parameter being the most significant. Surface aeration alone gave kLa values of 0. 4 to 1. 6 h−1. A 25% decrease in kLa was observed above an aeration rate of 1. 6 vvm. This was caused by the particular foam breaker of the CelliGen bioreactor. Measurements of kLa using a mammalian cell culture medium supplemented with 5% fetal calf serum (FCS) have confirmed the negative effect of the foam breaker on kLa The measured value in this medium was 1. 2 h−1 for all aeration rates, more than 60% of which was attributed to surface aeration.
Reasons for the apparent difference in the effects of produced and added ethanol on culture viability during rapid fermentation by Saccharomyces cerevisiae
Authors: Dasari G. , Worth M. A. , Connor M. A. , Pamment N. B.
Published: December 2004

By feeding ethanol at various high rates to low cell density cultures of Saccharomyces cerevisiae it was shown that the sharp fall in viability when ethanol is produced during rapid fermentations is in part a direct consequence of the high rate of change of extracellular ethanol concentration. Nevertheless, the fall in viability in high cell density rapid fermentations which produced 98 g L−1 ethanol in 3 h considerably exceeded that of control low cell density cultures to which ethanol was added at the same rate. This difference was shown to be not due to intracellular ethanol accumulation or to differences in glucose concentration between the cultures. The concentrations of a range of potentially toxic fatty acids, higher alcohols, and esters were measured during rapid fermentations, but when added at these concentrations to control cultures in the presence of ethanol they had no significant toxic effect. However, when rapid fermentations were conducted in rich medium containing 80 g L−1 yeast extract, the apparent difference in toxicity of produced and added ethanol virtually disappeared. Magnesium was shown to be the component of yeast extract primarily responsible for this effect. The high rate of fall of viability when ethanol is rapidly produced is suggested to be partly due to the inability of the cells to adapt quickly enough to the rising ethanol concentration and partly to an increased demand for magnesium at higher ethanol concentrations which cannot be met in Mg‐unsupplemented high cell density fermentations.
Enzyme loading in a solid support with nonuniform pore size distribution
Authors: Chiang C. L. , Wu T. C. , Wang Y. J.
Published: December 2004

The concept of pore size distribution is incorporated into the Clark model of enzyme immobilization in the present study. This refined model predicted that in the case of small harmonic pore radius with the same surface area and porosity of the support, more enzyme could be loaded in a support with nonuniform pores than that with uniform pores. In comparing the enzyme loading efficiency of the support with two different pore size distributions, the one with Gaussian distribution had the greater amount of enzyme immobilized than the other one with Rajagopalan's distribution. Furthermore, more enzyme could be loaded in a support with wider Gaussian pore size distribution than that with narrower distribution. The immobilized enzyme profile in the solid support with pore size distribution displayed a stepwise pattern which differed appreciably from the sigmoidal profile predicted for the support with uniform pore size. This stepwise enzyme distribution profile became sigmoidal with decreasing hT or increasing k. The new model could be used for designing protocols for an enzyme immobilization process.
New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium‐type resin
Authors: Kawabata Nariyoshi , Nishimura Sadaki , Yoshimura Tohru
Published: December 2004

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross‐linked poly(N‐benzyl‐4‐vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium‐type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed‐bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L‐aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L‐aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41–0.83 bed volumes per hour at pH 7.4–7.7 at 30°C.
Elucidation of the cytoplasmic and vacuolar components in the inorganic phosphate region in the 31P NMR spectrum of yeast
Authors: Shanks Jacqueline Vanni , Bailey James E.
Published: December 2004

Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon 31P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of Pi chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of poly‐phosphate (PP1) which reside in the vacuole. An in vivo correlation relating PP1 and P chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of 31P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate‐containing medium. Application of this method shows that both vacuolar and extracellular Pi were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrane was not correlated with the pH gradient across the membrane.
Production of L‐phenylacetyl carbinol by immobilized yeast cells: II. Semicontinuous fermentation
Authors: Mahmoud Wafaa M. , El‐Sayed Abdel‐Halim M. M. , Coughlin Robert W.
Published: December 2004

The cyclic, semicontinuous production of L‐phenylacetyl carbinol (L‐PAC) from a benzaldehyde substrate by Saccharomyces cerevisiae ATCC 834 immobilized in calcium alginate beads was substantially enhanced to about 4.5 g/L in a second cycle by reactivation in fresh medium for 24 h, following an earlier 24‐h period of production from substrate. Intermittent feeding of benzaldehyde was employed (four doses in 3 h). In subsequent similar cycles, however, the production returned to that produced in the first cycle, viz. L‐PAC concentration of 2–3 g/L in the medium. Production of L‐PAC was also increased by adaptation of the cells over 200 h of exposure to the benzaldehyde substrate (compared to wild‐type cells) and by continuous (as compared to intermittent) feeding of the substrate. A liter as great as 10 g/L was obtained with wild‐type cells by continuous feeding of benzaldehyde over 6 h. Immobilization not only protected the cells from toxic effects of substrate but also permitted them to be used during 7 cycles of semicontinuous operation over more than 200 h.
Calorimetric control of fed‐batch fermentations
Authors: Randolph T. W. , Marison I. W. , Martens D. E. , von Stockar U.
Published: December 2004

Measurements of the heat produced by Saccharomyces cerevisiae CBS 426 were used in conjunction with elemental and enthalpic balances to control fed‐batch fermentations. A proportional control using the ratio of carbon dioxide evolution rate to heat production resulted in high biomass yields and minimal ethanol production. For the special case where the carbon source contains no nitrogen, biomass production estimated from heat measurements agreed well with measured values. When the controller gain was set below the maximum specific cellular growth rate, stable control was achieved, even in response to large upsets in feed concentrations.
Population dynamics of a recombinant culture in a chemostat under prolonged cultivation
Authors: Weber Allison E. , San Ka‐Yiu
Published: December 2004

The dynamics of a chemostat culture of Escherichia coli K12 harboring plasmid pBR322 under prolonged cultivation with a nonselective complex medium were studied. The ability of the culture to form colonies on plates supplemented with different ampicillin concentrations was monitored. It was observed that almost all cells sampled were able to grow on a high concentration of ampicillin at the beginning of the experiment. However, a subpopulation which formed colonies on intermediate‐concentration (500–1000 mg/L) plates, but not on a high‐concentration (2000 mg/L) plate, was detected just before the appearance of the plasmid‐free cells. As time progressed, the percentage of this subpopulation increased, reached a maximum, then decreased toward the end of experiment. At this time the culture was dominated by a subpopulation which could not form colonies on the 100 mg/L ampicillin plates. These results indicate that three major processes may occur in the chemostat: a gradual shift of the higher plasmid copy number population toward a relatively lower copy number population; the complete shedding of the plasmid due to faulty segregation of plasmids during cell division; and growth competition among the subpopulations. A previously derived model is extended to account for all subpopulations. The model agrees qualitatively with the experimental results.