bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell growth and death rates as factors in the long‐term performance, modeling, and design of immobilized cell reactors
Authors: Dale M. C. , Chen C. , Okos M. R.
Published: December 2004

The viable fraction of immobilized cells in a bioreactor may be critical in predicting long‐term or steady‐state reactor performance. The assumption of near 100% viable cells in a bioreactor may not be valid for portions of immobilized cell reactors (ICRs) characterized by conditions resulting in appreciable death rates. A mathematical model of an adsorbed cell type ICR is presented in which a steady‐state viable cell fraction is predicted, based on the assumptions of no cell accumulation in the reactor and a random loss of cells from the reactor. Data on cell death rates, cell growth rates, and productivity rates as functions of temperature, substrate, and ethanol concentration for the lactose utilizing yeast K. fragillis were incorporated into this model. The steady‐state reactor viable cell fraction as predicted by this model is a strong function of both temperature and ethanol concentration. For example, a stable 20% viable fraction of the immobilized cells is predicted in ICR locations experiencing continuous conditions of either 30 g/L ethanol at 40°C, or 95 g/L ethanol at 25°C. Steady‐state ICR “plug flow” concentration profiles and column productivities are predicted at three operating temperatures, 20, 30, and 40°C using two different models for ethanol inhibition of productivity. These profiles suggest that the reactor operating temperature should be low if higher outlet ethanol concentrations are desired. Three reactor design strategies are presented to maximize the viable cell fraction and improve long‐term ethanol productivity in ICR's: (1) reducing outlet ethanol concentrations, (2) rotating segments of an ICR between high and low ethanol environments, and (3) simultaneous removal of the ethanol produced from the reactor as it is formed.
Modeling of enzyme‐potentiometric sensors involving acid‐ or base‐forming reactions
Authors: Ogundiran Sunday O. , Varanasi Sasidhar , Ruckenstein Eli
Published: December 2004

Enzyme‐potentiometric sensors in which the chemical conversion of the analyte leads to the formation of an acid and/or a base can often display complex response characteristics. In these sensors, the, electrochemically monitored species is usually the H+ ion (pH‐based sensors). However, in some cases the conjugate‐ion of the H+ (or OH−) ion of the acid (or base) produced‐can also be monitored—a specific example being the urease–NH4+ sensor. The response of both types of sensors is strongly affected by: (1) the degree of dissociation of the products and their transport properties in the enzymic film, (2) the amounts of pH‐buffers present in the test solution, (3) the test solution's pH, and (4) the diffusion coefficients of the various species. In this article, a previously developed theoretical model for pH‐based sensors—in which the differences in diffusivities of the various species were ignored—is generalized to accommodate for such differences, and extended to the latter of the above two types of sensors. It is shown that when the sensor operates under analyte diffusion‐controlled conditions, the response of either type of sensor can be predicted by a simple algebraic equation which is independent of the actual kinetics of the enzymic reaction.
Influence of dilution rate and induction of cloned gene expression in continuous fermentations of recombinant yeast
Authors: Da Silva Nancy Anderson , Bailey James E.
Published: December 2004

The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10‐CYC1 hybrid promoter directing expression of the E. coli lacZ gene. β‐Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced “steady states” were observed, the first 10–15 residence times after induction and the second after 40–50 residence times. The second induced steady state was characterized by greater biomass concentration and lower β‐galactosidase specific activity relative to the first induced “steady‐state.” β‐Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.
A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing
Authors: Goetz Victor , Remaud Magali , Graves David J.
Published: December 2004

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well‐known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5–80 m2/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.
Strategies for reducing solvent toxicity in extractive fermentations
Authors: Yabannavar V. M. , Wang D. I. C.
Published: December 2004

The toxicity of an Alamine 336/oleyl‐alcohol extraction system on Lactobacillus delbrueckii was investigated. It was shown that the solvent affected the cells through the water‐soluble portion and the immiscible portion of the solvent. While immobilization significantly protected the cells from the immiscible solvent phase, the water‐soluble part of the solvent still caused toxicity to the microorganisms due to diffusion of the solvent into the matrix. Adding soybean oil to the κ‐carrageenan matrix could trap the diffusing solvent molecules, and therefore reduce the toxic effect from the water soluble portion of the solvent. The protective ability of soybean oil was quantified through mathematical modeling and experimentation.
High‐concentration cultivation of Lactococcus cremoris in a cell‐recycle reactor
Authors: Bibal Bernard , Vayssier Yves , Goma Gérard , Pareilleux Alain
Published: December 2004

High‐cell concentration cultivation of Lactococcus cremoris, a homofermentative lactic acid producer, in a cell‐recycle fermentor is described. Cross‐flow filtration allowing continuous removal of the inhibitory metabollte, the influence of dilution rate on growth was investigated in total or partial cell‐recycle cultures. The dependence of growth characteristics on operating conditions was identified and quantified using lactose as the carbon source. Growth kinetics could be described by both lactate removal efficiency and nutrient availability. Based on physiological observations, biomass and lactic acid productivities were predicted in partial cell‐recycle cultures.
Characterization of immobilized cell growth rates using autoradiography
Authors: Stewart Philip S. , Karel Steven F. , Robertson Channing R.
Published: December 2004

The growth of immobilized Escherichia coli was analyzed by pulse‐chase radioisotope labeling of the cell mass with 35SO42− and subsequent liquid emulsion autoradiography of thin cross sections of the cell aggregate. Bacteria were retained in a planar aggregate on a microporous membrane and grown anaerobically on a phosphate‐buffered medium with glucose as the sole carbon and energy source. A mathematical model of immobilized cell growth and convection was used to predict the distribution of label in the cell mass and permit information about both the magnitude and variation in the intrinsic growth rate to be extracted. Growing zone dimensions ranging from 4 to 48 μm and growth rates from 0.28 to 0.5 h−1 were found. Data collected at low glucose concentrations were consistent with a zero‐order description of intrinsic growth kinetics. At high glucose concentrations, conditions under which the system was subject to significant pH inhibition, the data were best described by the prediction of a first‐order kinetic model. When coupled with a suitable analytical framework, the combination of radioisotope labeling and autoradiography provides a general method for characterizing immobilized cell growth rates.
Structured modeling of a microbial system: II. Experimental verification of a structured lactic acid fermentation model
Authors: Nielsen Jens , Nikolajsen Karin , Villadsen John
Published: December 2004

A two‐compartment model for the lactic acid fermentation with Streptococcus cremoris is experimentally verified. The seven parameters of the model are determined using steady‐state chemostat data at varying values of dilution rate, D, but with a constant feed concentration, sf, of a single carbohydrate source (glucose, lactose, or galactose), and a constant feed concentration of sNf of the N source. Steady‐state measurements of the RNA content at different exit concentrations, s, of the carbohydrate are included to calculate kinetic parameters that determine the cell composition for varying operating conditions. The model is tested using data from a large set of steady‐state and non‐steady‐state experiments: batch fermentations and step and pulse experiments in a chemostat. Both qualitatively and quantitatively the major features of the model are confirmed: the external substrates enter into intracellular high‐energy building blocks, and lactic acid is formed as a by‐product of these reactions. Cell growth depends on the fraction of active components (XA) of the cell and is not accompanied by lactic acid production. Possible model modifications are discussed, primarily to obtain a better description of lactic acid fermentation at nongrowth conditions.
Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures
Authors: Dussap C. G. , De Vita D. , Pons A.
Published: December 2004

Wild‐type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4‐L stirred‐tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic‐ and organic‐nitrogen concentrations were measured, and oxygen consumption and CO2 production rates were obtained by a gas‐balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in ±5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen‐limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking–Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.
Protease‐catalyzed peptide synthesis using inverse substrates: The influence of reaction conditions on the trypsin acyl transfer efficiency
Authors: Schellenberger Volker , Jakubke Hans‐Dieter , Zapevalova Nina P. , Mitin Yuri V.
Published: December 2004

Benzyloxycarbonyl‐L‐alanine p‐guanidinophenyl ester behaves as a trypsin “inverse substrate,” i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30°C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H‐Leu‐NH2 and H‐Val‐NH2 in deacylation is almost the same for “inverse” and normal type substrates.
Relative contributions of biological and chemical reactions to the overall rate of pyrite oxidation at temperatures between 30°C and 70°C
Authors: Boogerd F. C. , van den Beemd C. , Stoelwinder T. , Bos P. , Kuenen J. G.
Published: December 2004

The stoichiometry and kinetics of the spontaneous, chemical reaction between pyrite and ferric iron was studied at 30, 45, and 70°C in shake flasks at pH 1.5 by monitoring the ferrous iron, total iron, elemental sulfur, and sulfate concentration profiles in time. It was found that the sulfur moiety of pyrite was oxidized completely to sulfate. Elemental sulfur was not produced in detectable amounts. The iron moiety of pyrite was released as ferrous iron. All observed initial reaction rates could be fitted into an empirical equation. This equation includes the concentrations of ferric iron and pyrite, and a constant which is dependent on the temperature and the nature of the main anion present. It was observed that ferrous iron formed during the reaction slowed down the oxidation of pyrite by ferric iron. The extent of this effect decreased with increasing temperature. With the aid of the empirical equation, the contribution of the chemical oxidation of pyrite by ferric iron to the overall oxidation in a hypothetical plug‐flow reactor, in which biologically mediated oxdidation of pyrite and ferrous iron by oxygen also takes place, can be assessed. At 30, 45, and 70°C, respectively, 2, 8–17, and 43% of the pyrite was oxidized chemically by ferric iron. Therefore, it is expected that only in reactors operating at high temperatures with extremely thermophilic bacteria, will chemical oxidation cause a significant deviation from the apparent first order overall kinetics of biological pyrite oxidation.
NADH fluorscence in a carbon‐limited fermentation
Authors: Srivastava A. K. , Volesky B.
Published: December 2004

The continuous flow acetone–butanol fermentation conducted at lowered inlet feed sugar concentrations and at a constant dilution rate D =0.075 h−1 demonstrated a significant decreases in the availability of the cell population “reduction energy” (F/X), resulting in an exclusive accumulation of intermediate acids under those conditions. The cultures resumed its solvent production activity when the inlet sugar concentration in the feed stream was increased from 20 to 40 g/L at the same low growth/dilution rates. A linear correlation between the culture reduction content (F/X) and the specific butanol rate (qB) was observed under the present conditions, indicating the necessity of the NADH availability for the increased solvent production.
Recovery of bacteria from growth media by starch‐dependent floc formation
Authors: Ferenci Thomas , Lee Kin‐Sang
Published: December 2004

Addition of starch to suspensions of Escherichia coli K‐12 resulted in the formation of bacterial flocs. The flocculation was dependent on the high expression of a receptor for starch (maltoporin) on the surface of the bacterium. Factors influencing floc formation were investigated and optimal conditions for flocculation based on cell density, starch concentration, time, and pH established. As quantitated by a sedimentation assay, over 80% of bacteria in a culture could be removed by settling without centrifugation in 3 h under optimal conditions. Floc formation was evident with bacteria containing wild‐type maltoporin but was faster and occurred to a greater extent with strains expressing a high‐affinity allele (lamB1400) of the starch receptor. Bacteria could be harvested by floc formation directly in growth medium under defined conditions of maltoporin expression and medium composition. These results demonstrate the effectiveness of starch‐dependent aggregation in the harvesting of cells, using an inexpensive, biologically acceptable agent to induce flocculation.
Effect of chemically‐induced, cloned‐gene expression on protein synthesis in E. Coli
Authors: Wood Thomas K. , Peretti Steven W.
Published: December 2004

Earlier experiments in our lab investigated the metabolic limitations of cloned‐gene expression in bacterial cells (for over‐production of β‐lactamase). These experiments showed that the steady‐state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady‐state β‐lactamase mRNA level increased significantly. This appeared to indicate substantial limitation exist within the transnational machinery of the bacterial cell at high copy numbers. To establish the generality of this phenomenon, the impact increasing protein expression from pa plasmid by chemically inducing a strong promoter while maintaining constant copy number has been investigated. A plasmid has been constructed which contains the lacZ gene under control of the tac promoter and contains the parB stability locus to maintain plasmid stability. Using this vector, β‐galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h−1 was induced with IPTG such that enzyme activity was varied over a 460‐fold range. When fully induced β‐galactosidase protein production represented 14 wt % of total cell protein. As transcription was induced, the synthesis rate of the β‐galactosidase mRNA increased 42‐fold while the steady‐state level of β‐galactosidase mRNA increased only fourfold. This indicates stability may play a larger role for β‐galactosidase expression with a strong promoter than seen with β‐lactamase production in the elevated copy number system. Furthermore, rRNA synthesis rates increased at high expression rates as seen in the copy number experiments. However, unlike the amplified‐plasmid system, the steady‐state levels of rRNA increased as well. Since the total protein levels closely followed the steady‐state level of eRNA, transnational limitations are again suggested for the chemically induced transcription system.
Albumin denaturation during ultrafiltration: Effects of operating conditions and consequences on membrane fouling
Authors: Meireles Martine , Aimar Pierre , Sanchez Victor
Published: December 2004

Ultrafiltration of high‐purity grade bovine serum albumin has been carried out under various temperature between 5 and 30°C and at various cross‐flow velocities, pressures, and concentrations with the aim of studying protein denaturation and its consequences on the process. Three different pump heads have been tested. Denaturation of proteins in solution has been monitored by laser light scattering and size exclusion chromatography. The rate of protein denaturation increases with temperature, cross‐flow, and time. It is observed that membrane fouling is different whether denaturation has occurred or not. Under high‐concentration polarization, denaturation can occur in the boundary layer if the wall concentration exceeds 400 g/L. It is shown how the residence time, operating temperature, and pressure play an important part in membrane fouling. This can provide guidelines for process design and control.
Synthesis and secretion of α‐amylase by rice callus: Evidence for differential gene expression
Authors: Simmons Carl R. , Huang Ning , Cao Yicheng , Rodriguez Raymond L.
Published: December 2004

Rice seed callus expressed and secreted α‐amylase at high levels. Twenty percent of the protein secreted by the callus was αamylase. The callus secreted about 840 μg α‐amylase with 10.9 × 103 units of activity per gram dry weight callus per day. The α‐amylase from callus exhibited a more complex isoform pattern than the germinating seed α‐amylase. In addition, the level of mRNA expression by the five α‐amylase gene groups was markedly different between callus and the germinating seed. The rice callus culture has features which it attractive as a potential system for expression proteins in plant cell fermentation systems.
Gas holdup, power consumption, and oxygen absorption coefficient in a stirred‐tank fermentor under foam control
Authors: Yasukawa Masami , Onodera Masayuki , Yamagiwa Kazuaki , Ohkawa Akira
Published: December 2004

For a laboratory stirred‐tank fermentor (STF) with foaming system of 0.5M sulfite solution containing an anionic soft detergent, the performing of a foam‐breaking apparatus with a rotating disk (FARD)fitted to the STF was evaluated. The gas holdup in a mechanical foam‐control system (MFS), i.e., the stirred‐tank fermentor with the rotating disk foambreaker, was confirmed to be larger than that in a nonfoaming system (NS), i.e., the STF with an antifoam agent added. The agitation power in the mechanical foam control system was found to be smaller compared with the agitation power in the nonfoaming system, due to the increased gas holdup. Comparison of the oxygen absorption coefficient between the mechanical foam control system and the nonfoaming system in terms of the specific power input also demonstrated the superiority of the mechanical foam control system, not only in oxygen transfer performance but also in power input economy.
A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells
Authors: Bibila Theodora A. , Flickinger Michael C.
Published: December 2004

A three compartment model (ER → Golgi → extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG2a monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse‐chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.
Construction of a specialized‐ribosome vector or cloned‐gene expression in E. coli
Authors: Wood Thomas K. , Peretti Steven W.
Published: December 2004

An expression system utilizing specialized ribosomes has been constructed with β‐galactosidase as the product. Ribosomes specific for lacZ mRNA are generated due to a mutation within the anti‐Shine‐Dalgarno region of a plasmidborne 16S rRNA gene that is complementary to a mutation within the ribosome‐binding site of lacZ. Hence, a subpopulation of ribsomes specific for translation of the cloned gene mRNA is produced. Transcription of the lacZ gene is regulated by the tac promoter, while transcription of the mutated rrnB locus is controlled by the λPL promoter. Batch experiments indicate that full induction of both operons (2 mM IPTG, 42°C) leads to maximal β‐galactosidase activity per cell at levels 35% higher than that obtained using a wild‐type ribosome expression system. Using a novel, site‐directed mutagenesis technique, construction of the specialized ribosome vector is outlined, and the results of lacZ expression are presented as transcription of both the cloned‐gene and the specialized‐ribosome locus are induced.
Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports
Authors: Chetty Ashok S. , Burns Mark. A.
Published: December 2004

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid‐solids contact, mass‐transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion‐exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.
Continuous production of Ammonium lactate by Streptococccus cremoris in a three‐stage reactor
Authors: Mulligan Catherine N. , Safi Béchara F. , Groleau Denis
Published: December 2004

Batch and continuous fermentation studies were performed to optimize the production of ammonium lactate from whey to optimize the production of ammonium lactate from whey permeate. The product known as fermented ammoniated condensed whey permeate (FACWP) is a very promising animal feed. After an initial screening of four strains which produce predominantly L(+)‐ lactic acid, the desired isomer [D(−)‐lactic acid is toxic], Streptococcus cremoris 2487 was chosen for further study. In batch mode, pH between 6.0 and 6.5 and 35°C provided optimum incubation conditions. To stimulate a plug flow reactor, three CSTRs (continuous stirred tank reactors) were connected in tandem. For a 7.5‐h retention time, 1.6‐fold and 1.3‐fold higher productivities were obtained for three‐stage than for the single‐ and two‐stage reactors, respectively. Various retentions times were examined (5, 7.5, and 10 h; 5g/L yeast extract). Although maximum lactate productivity occurred at a 5‐h residence time (5.38 g/L H. 75% lactose utilization), lactose utilization was more complete at 7.5 h (4.38 g/L h productivity, 91% lactose utilization and a productivity, 91% lactose utilization). Retention time was increased to 15 h to obtain 95.9% lactose utilization and a productivity of 2.42g/L h for 2g/L yeast extract. Based on this lower yeast extract concentration, it was determined that ammonium lactate production and subsequent concentration by 11‐fold would yield a product (FACWP) 17% more than soybean meal (crude protein contents are equivalent, 44%) at current market prices.
Experimental simulation of dissolved oxygen fluctuations in large fermentors: Effect on Streptomyces clavuligerus
Authors: Yegneswaran P. K. , Gray M. R. , Thompson B. G.
Published: December 2004

An experimental Monte Carlo method was used to study the effect of fluctuations in oxygen concentration on the synthesis of antibiotics by Streptomyces clavuligerus. Air was supplied to the culture in a 2‐L fermentor in random cycles following the lognormal distribution in order to model the circulation within large production‐scale vessels. Each cycle consisted of air supply for 5 s followed by no aeration for the balance of the cycle time which ranged from 8 to 44 s, with a mean time of 20 s. Comparable experiments were also conducted with constant period cycling of air and with continuous supply of air. The yields of cephamycin C and its precursor, penicillin N, were suppressed by the Monte Carlo simulation of circulation in a large tank, as compared to constant period cycling. The concentration of dissolved oxygen remained at a low, ca. 5% of saturation, for 5–10 h longer during the Monte Carlo experiment than during the periodic aeration experiment. The biosynthetic enzymes, which are sensitive to oxygen levels, were likely affected not only by the mean time of cycling but also by the distribution of the cycles.
Biomass axial distribution in airlift bioreactor with yeast and plant cells
Authors: Assa Amir , Bar Raphael
Published: December 2004

This study was aimed at determining the degree of biomass homogeneity in the various parts of an internal loop airlift bioreactor, thus verifying the assumption, often made in bioreactor studies, of a well‐mixed liquid‐biomass system. Following characterization of the hydrodynamics of the vessel with water, the axial biomass distribution in the riser and downcomer was determined for plant and yeast cell suspensions of 5.8, 8.5, and 12.5 g DW/L Phaseolus vulgaris and of 30 and 46 g DW/L Saccharomyces cerevisiae. The airlift bioreactor with a surface ratio AD/AD of 1.04 and aspect ratio of 4.95 was investigated under various aeration rates. The yeast cells were found to be distributed practically uniformly throughout the vessel at the aeration rates of 0.1−1.45 vvm. However, in the case of the denser and cluster‐forming plant cells, a clear trend of a gradual bio‐mass accumulation in the downcomer, a slightly lower but uniform biomass loading in the riser, and a slightly higher biomass concentration in the gas‐liquid separator was observed at the lower aeration rates of 0.1−0.61 vvm. In the case of powderized calcium carbonate (55g/L) often used in fermentations of organic acids, a slight trend of a gradual accumulation of solids towards the bottom parts in both the downcomer and riser was observed. A better representative sampling location, in terms of solids and biomass loading, seems to be in the middle part of the vessel. It is suggested that airlift bioreactors with higher aspect ratios (>5) may be prone to a more significant inhomogeneity of solids (biomass and particles).
Rapid determination of plasmid‐carrying yeast cells by using an imaging sensor system
Authors: Endo Hideaki , Suzuki Masayasu , Sode Koji , Tamiya Eiichi , Karube Isao
Published: December 2004

A novel imaging sensor system for the determination of plasmid carrying yeast cells was developed. The sensor system consisted of an Silicon Intensifier Target (SIT) video camera, a fluorescent microscope, and a personal computer system equipped with an image memory board. This system was based on the fact that the membrane integrity of only plasmid‐carrying cells is lost following cell growth in 5‐fluoro‐orotic acid (5‐FOA) containing medium, and consequently these target cell can be stained with fluorescent probes and detected. In this study, plasmid‐carrying cells were detected and their fraction determined in a mixture of both plasmid‐carring and plasmid‐free cells. A good correlation was observed between the values determined by this sensor system and the conventional method in the 30%‐80% range, and one assay was possible within 4 h. This sensor system could be used for the monitoring of plasmid‐carrying fraction in recombinant yeast cells during cultivation.
An expert system approach for the control of a bioprocess. I: Knowledge representation and processing
Authors: Hitzmann Bernd , Lübbert Andreas , Schügerl Karl
Published: December 2004

For the control of bioprocesses, a priori knowledge exists which cannot be readily incorporated as a mathematical model but which can be represented by language‐based rules, such as “IF/THEN” conditions. This knowledge is usually implemented via a process operator for the automation of the fermentation. A knowledge‐based system has been developed which can use this kind of knowledge and which is complemented by algorithmic systems, such as statistical analyses and mathematical modeling, for the supervision and control of a bioprocess. In this article, a description of this system is presented. Furthermore, the following features of the system are discussed in detail which are especially important for the development of real‐time, on‐line, knowledge‐based systems: the representation of time‐dependent knowledge; processing of imprecise, uncertain, and incomplete knowledge; the combination of shallow reasoning with model‐based reasoning; informing the bioprocess operator about the inferences and decisions; the demands of the diversity of the knowledge handling; performance; and maintenance and extension of the system.
Influence of expression of the pet operon on intracellular metabolic fluxes of Escherichia coli
Authors: Diaz‐Ricci J. C. , Tsu M. , Bailey J. E.
Published: December 2004

Fermentation patterns of Escherichia coli HB101 carrying plasmids expressing cloned genes of Zymomonas mobilis pyruvate decarboxylase (PDC) and alcohol dehydrogenase li (ADH) were determined in glucose‐limited complex medium in pH‐controlled anaerobic batch cultivations. Time profiles of glucose, dry cell weight, succinate, formate, acetate, and ethanol were determined, as were the activities of ADH and PDC. Fluxes through the central carbon pathways were calculated for each construct utilizing exponential phase data on extracellular components and assuming quasi‐steady state for intermediate metabolites. Overall biomass yields were greatest for cells expressing both PDC and ADH activities. Yields of carbon catabolite end products were similar for all PDC‐expressing strains and different from those for other strains. Relative to its glucose uptake rate, the strain with greatest PDC and ADH activities produces formate and acetate more slowly and ethanol more rapidly than other strains. Strong influences of plasmid presence and metabolic coupling complicate detailed interpretations of the data.
Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase
Authors: Kim Dong‐Man , Kim Hak‐Sung
Published: December 2004

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose‐fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross‐linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin‐immobilized inulinase in alginate beads. The optimum amounts of both chitin‐immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h−1 and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed‐bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h−1 and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed‐bed reactor without any loss of activity, while half‐life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.
A computer‐aided measuring system for the characterization of yeast populations combining 2D‐image analysis, electronic particle counter, and flow cytometry
Authors: Huls P. G. , Nanninga N. , van Spronsen E. A. , Valkenburg J. A. C. , Vishcer N. O. E. , Woldringh C. L.
Published: December 2004

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D‐image processing program for rapid and accurate determination of volume distributions of the different sub‐populations. The program combines the capacity of image processing and volume calculation by contour‐rotation, with the potential of visual evaluation of the cells. High‐contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D‐image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2‐D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.
A proposed biparticle fluidized‐bed for lactic acid fermentation and simultaneous adsorption
Authors: Davison Brian H. , Scott Charles D.
Published: December 2004

A bioreactor configuration is proposed for simultaneous fermentation and separation of the desired product. The bioreactor consists of a columnar fluidized bed of immobilized microorganisms. Denser adsorbent particles are added to this column. These adsorbent particles fall through the bed, absorb the product, and are removed from the base of the columnar reactor. The system hydrodynamics and the separability of the two types of particles were confirmed for low‐density gel beads. The addition of the adsorbent, activated carbon, to a fermentation of Lactobacillus delbreuckii absorbed lactic acid. The addition of adsorbent enhanced the fermentation and controlled the pH.
Displacement effects in large‐scale chromatography?
Authors: Jungbauer Alois , Uhl Karola , Schulz Petra , Tauer Christa , Gruber Gerhard , Steindl Franz , Buchacher Andrea , Schoenhofer Wolfgang , Unterluggauer Florian
Published: December 2004

Displacement effects in large‐scale (total column volume vt = 150 L) and preparative ion‐exchange chromatography purifying human erythrocyte superoxide dismutase are described in the present article. The biomolecules are eluted in a very small peak elution volume (<0.2 vo) behind the salt wave using a step gradient. The theoretical peak width and retention behavior are calculated according to the model of Yamamoto. The theoretical values are then compared with the experimental data. There was a difference observed between the elution type I (also called fronting) and the experimentally obtained elution. Some instructions are given on how to achieve these phenomenona because a beneficial effect in respect to resolution and recovery of a biomolecule is observed.
Surface immobilization of anchorage‐dependent mammalian cells
Authors: Robert Joëlle , Côté Johanne , Archambault Jean
Published: December 2004

Anchorage‐dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75–125 and 40 μm, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 μm, respectively, in 100‐mL spinner flasks and 2‐L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (≤2–3 h) attachment of inoculated cells (≥95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate ∼0.03–0.04 h−1, maximum cell density of 8 × 106–9 × 106 cells · mL−1, and yields of 0.4 × 108 cells · mM−1 on glucose and 2 × 108−3 × 108 cells · mM−1 on glutamine). Scale‐up of this culture technique in a 2‐L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 × 106 cells · mL−1. Two other anchorage‐dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.
Application of a new structured model to tobacco cell cultures
Authors: Hooker Brian S. , Lee James M.
Published: December 2004

A new structured kinetic model has been formulated and applied to batch suspensions of Nicotiana tabacum. This model has been developed by representing culture interactions with pathways designated for structural component production, secondary metabolite synthesis, and cellular respiration. Additional provisions were made to distinguish growth‐competitive secondary metabolite production from non‐growth‐competitive secondary metabolite production. Parameters for kinetic rate expressions within the model were estimated based upon experimental observations utilized in conjunction with numerical optimization techniques. Using these parameters, culture growth, substrate uptake, cell respiration, and total phenolics production were all successfully correlated to experimenta data from shake flask cultures of N. tabacum.
Characterization of the growth and sporulation behavior of Penicillium roquefortii in solid substrate fermentation by material and bioenergetic balances
Authors: Larroche C. , Gros J. B.
Published: December 2004

When Pencillium roquefortii is grown on two kinds of buckwheat, exhibiting a low [0.85 g water/g dry matter (DM), buckwheat A] and a high [1.5 g water/g initial dry matter (IDM), buckwheat B] water content, a marked difference in the mode of development of the fungus is observed. Material balances for buckwheat A show that growth does not stop because of nutrient exhaustion. Analysis of water balance shows that active growth proceeds with a permanent limitation by the turgor potential which disappears when the water activity of the substrate is close to 0.96, thus arresting growth. This limitation causes intensive water excretion from the system due to the lowering of the water activity of the substrate. The water content of the mycelium thus decreases from 79% at the beginning of the cultivation to 74% when the growth stops. This is linked to a substantial oxidative metabolism and a high sporulation efficiency, close to 0.85. The spores obtained have a low dry weight and a reduced nitrogen content. In the case of buckwheat B, the active growth is shown to stop because of available mineral nitrogen depletion. No significant decrease in the water activity of the substrate is found during the protein synthesis, and the turgor potential remains high at the end of this period. Culture proceeds with new wall synthesis; the sporulation efficiency remains high and the spores obtained exhibit a high dry weight and a high nitrogen content. The bioenergetic balances show that the P/O ratio varies with the kind of substrate used; its value is close to 1.56 for the low water medium and to 2.21 for the high one. The ATP yield Z is always close to 1, and fungal development occurs with limitations of both anabolism and catabolism on buckwheat B and only of anabolism and catabolism on buckwheat B and only of anabolism on buckwheat A.
Affinity purification of insoluble recombinant fusion proteins containing glutathione‐S‐transferase
Authors: Hartman John , Daram Paru , Frizzell Raymond A. , Rado Thomas , Benos Dale J. , Sorscher Eric J.
Published: December 2004

Prokaryotic expression of polypeptides as fusion proteins with glutathione‐S‐transferase has recently been reported as a one‐step means of purifying recombinant protein. The usefulness of the glutathione‐S‐transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione‐S‐transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one‐step purification utilizing this scheme requires proper folding of recombinant glutathione‐S‐transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione‐agarose affinity purification. Low‐temperature induction of fusion protein synthesis, but not incubation with anion‐exchange resins, led to improved one‐step purification of glutathione‐S‐transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity‐purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione‐S‐transferase.
Comparison of two experimental methods for the determination of Michaelis–Menten kinetics of an immobilized enzyme
Authors: Hooijmans C. M. , Stoop M. L. , Boon M. , Luyben K. C. A. M.
Published: December 2004

For the application of immobilized enzymes, the influence of immobilization on the activity of the enzyme should be Known. This influence can be obtained by determining the intrinsic kinetic parameters of the immobilized enzyme, and by comparing them with the kinetic parameters of the suspended enzyme. This article deals with the determination of the intrinsic kinetic parameters of an agarose‐gel bead immobilized oxygen‐consuming enzyme: L‐lactate 2‐monooxygenase. The reaction rate of the enzyme can be described by Michaelis–Menten kinetics. Batch conversion experiments using a biological oxygen monitor, as well as steady‐state profile measurements within the biocatalyst particles using an oxygen microsensor, were performed. Two different mathematical methods were used for the batch conversion experiments, both assuming a pseudosteady‐state situation with respect to the shape of the profile inside the bead. One of the methods used an approximate relation for the effectiveness factor for Michaelis–Menten kinetics which interpolates between the analytical solutions for zero‐ and first‐order kinetics. The other mathematical method was based on a numerical solution and combined a mass balance over the reactor with a mass balance over the bead. The main difference in the application of the two methods is the computer calculation time; the completely numerical calculation procedure was about 20 times slower than the other calculation procedure.

The intrinsic kinetic parameters resulting from both experimental methods were compared to check the reliability of the methods. There was no significant difference in the intrinsic kinetic parameters obtained from the two experimental methods. By comparison of the kinetic parameters for the suspended enzyme with the intrinsic kinetic parameters for the immobilized enzyme, it appeared that immobilization caused a decrease in the value of Vm by a factor of 2, but there was no significant difference in the values obtained for Km.