bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemostat cultivation of Candida blankii on sugar cane bagasse hemicellulose hydrolysate
Authors: Meyer P. S. , Du Preez J. C. , Kilian S. G.
Published: December 2004

A Candida blankii yeast isolate was grown in sugar cane bagasse hemicellulose hydrolysate at 38°C in carbon‐limited chemostat culture. The pretreatment of the acid hydrolysate prior to microbial cultivation consisted of partial neutralization with ammonia and sodium hydroxide, plus the addition of phosphorus, which was the only other growth‐limiting nutrient apart from nitrogen. The cell yield coefficient on nitrogen was 16.78. The critical dilution rate was higher (0.35 h−1) in diluted hydrolysate than in undiluted hydrolysate (0.21 h−1). In undiluted hydrolysate at a dilution rate of 0.1 h−1 and pH 4, where aseptic procedures proved unnecessary, the cell and protein yield coefficients were 0.53 and 0.26, respectively, and no residual carbon substrates (D‐xylose, L‐arabinose, D‐glucose, and acetic acid) were detected. The cell yield on oxygen increased linearly as a function of dilution rate. The cellular content of protein, carbohydrate, and RNA also increased with an increase in dilution rate, whereas the DNA content decreased slightly. C. blankii has considerable potential for the production of single cell protein from hemicellulose hydrolysate, because of its ability to utilize all of the major carbon substrates in the hydrolysate at a low pH and at a relatively high temperature with a high protein yield. © 1992 John Wiley & Sons, Inc.
The use of laminar tube flow in the study of hydrodynamic and chemical influences on polymer flocculation of Escherichia coli
Authors: Whittington P. N. , George N.
Published: December 2004

The optimization of microbial flocculation for subsequent biomass separation must relate the floc properties to separation process criteria. The effects of flocculant type, dose, and hydrodynamic conditions on floc formation in laminar tube flow were determined for an Escherichia coli system. Combined with an on‐line aggregation sensor, this technique allows the flocculation process to be rapidly optimized. This is important, because interbatch variation in fermentation broth has consequences for flocculation control and subsequent downstream processing. Changing tube diameter and length while maintaining a constant flow rate allowed independent study of the effects of shear and time on the flocculation rate and floc characteristics. Tube flow at higher shear rates increased the rate and completeness of flocculation, but reduced the maximum floc size attained. The mechanism for this size limitation does not appear to be fracture or erosion of existing flocs. Rearrangement of particles within the flocs appears to be most likely. The Camp number predicted the extent of flocculation obtained in terms of the reduction in primary particle number, but not in terms of floc size. © 1992 John Wiley & Sons, Inc.
Structure and sucrose hydrolysis activity of Saccharomyces cerevisiae aggregates
Authors: Fontana Angélique , Bore Christian , Ghommidh Charles , Guiraud Joseph P.
Published: December 2004

The sucrose hydrolysis activity of dense spherical yeast flocs, cultivated on a sucrose medium in a continuous reactor with internal settler, is nearly proportional to the particle surface. From computer simulation, in good agreement with experimental determinations, the calculated sucrose penetration depth is in the range 0.2–0.3 mm, a dimension smaller than the usual diameter of strongly flocculating yeast particles. From specific gravity determinations, the flocs can be considered as homogeneous and cannot exhibit a fractal structure, reported in the literature for a number of microbial aggregates. However, the analysis of the sucrose hydrolysis rates reveals that the cell density may be lower in the outer layer of the flocs. © 1992 John Wiley & Sons, Inc.
CHO cell responses to low oxygen: Regulation of oxygen consumption and sensitization to oxidative stress
Authors: Lin Andy A. , Miller William M.
Published: December 2004

We have investigated the regulation of oxygen consumption and modulation of glutathione levels in CHO‐K1 cells under oxygen‐limiting conditions. We report here suppression of oxygen consumption and alteration of the supply‐dependent relationship as a consequence of prolonged hypoxic or anoxic exposure. The suppression is characterized by an increase in the value of P (the oxygen tension at which oxygen consumption is half maximal). Under prolonged anoxia there is also a decrease in the cells' potential to use oxygen. Elevated glucose consumption under low oxygen conditions may contribute to the suppression in respiration. The glutathione concentration remains constant throughout hypoxic exposure but may decrease by as much as 40% under anoxia. The glutathione level in hypoxic and anoxic cells increases by two‐ and four‐fold, respectively, over that of the control cells when exposed to a cytotoxic level of oxygen (93%). This suggests that anoxic and hypoxic exposure sensitizes CHO cells to oxidative stress. © 1992 John Wiley & Sons, Inc.
Optimization in integrated biochemical systems
Authors: Voit Eberhard O.
Published: December 2004

As of yet, steady‐state optimization in biochemical systems has been limited to a few studies in which networks of fluxes were optimized. These networks of fluxes are represented by linear (stoichiometric) equations that are used as constraints in a linear program, and a flux or a sum of weighted fluxes is used as the objective function. In contrast to networks of fluxes, systems of metabolic processes have not been optimized in a comparable manner. The primary reason is that these types of integrated biochemical systems are full of synergisms, antagonisms, and regulatory mechanisms that can only be captured appropriately with nonlinear models. These models are mathematically complex and difficult to analyze. In most cases it is not even possible to compute, let alone optimize, steady‐state solutions analytically. Rare exceptions are S‐system representations. These are nonlinear and able to represent virtually all types of dynamic behaviors, but their steady states are characterized by linear equations that can be evaluated both analytically and numerically. The steady‐state equations are expressed in terms of the logarithms of the original variables, and because a function and its logarithms of the original variables, and because a function and its logarithm assume their maxima for the same argument, yields or fluxes can be optimized with linear programs expressed in terms of the logarithms of the original variables. © 1992 John Wiley & Sons, Inc.
Influence of mass transfer limitations on determination of the half saturation constant for hydrogen uptake in a mixed‐culture CH4‐producing enrichment
Authors: Giraldo‐Gomez Eugenio , Goodwin Steve , Switzenbaum Michael S.
Published: December 2004

There is strong evidence in the literature supporting the existence of significant mass transfer limitations on the kinetics of exogenous H2 consumption by methanogens. The half saturation constant for H 2 uptake by a mixed‐culture, CH4 producing enrichment was measured using an experimental protocol that avoided internal mass transfer limitations. The value obtained was two orders of magnitude smaller than any other previously reported. A mathematical model for acetogenic syntrophic associations was developed to check the capacity of H2 as electron transporter between syntrophic partners. It was found that H2 diffusion could account for the rate of transport of electrons between the syntrophic microorganisms and that formate is not a necessary intermediate. The possibility that formate may be an intermediate in this system was not ruled out. A Monod‐type kinetic equation was modified to include the observed H2 threshold effect. This modified equation was used to predict the CH4‐production rate in a batch‐fed digester. The results show that the external and internal H2 pools are kinetically coupled. © 1992 John Wiley & Sons, Inc.
Cell inactivation in the presence of sparging and mechanical agitation
Authors: Yang Jeng‐Dar , Wang Nam Sun
Published: December 2004

Microbial cells are more readily rendered nonviable by the combined action of air sparging and mechanical agitation than by either action along. A. bubble breakup/coalescence model that incorporates the cell–bubble encounter rate, bubble breakup rate, and death probability is proposed to describe cell inactivation in the presence of bubbles maintained through the joint action of agitation and air, which is continually fed into the impeller stream region via passive vortex entrainment from the surface above or via active sparging from below. Experimental results obtained from a fragile algal (Ochromonas malhamensis) culture are consistent with the model prediction. In particular, the specific cell death rate is linearly related to the specific bubble interfacial surface area. It is shown that cells exhibit sparging‐sensitive characteristics when agitation is mild, but become sensitive to surface vortexing when agitation turns vigorous enough to introduce air entrainment. Experimental data obtained from different stirrer sizes are in good agreement with the model © 1992 John Wiley & Sons, Inc.
Direct and homogeneous immunoassay for IgG analyses
Authors: Huang Ping Yu , Lee Cheng S. , Heath Carole , Banik Gautam
Published: December 2004

A concept involving the use of antibody conjugated with reporter molecules for direct sensing of subtle changes in the local electrostatic environment of an antigen–antibody complex due to the antigen binding is presented. The proposed direct sensing mechanism is studied using the protein A–immunoglobulin G (IgG) complex as the model system. The change in the local electrostatic potential and pH around the protein A–IgG complex is observed by measuring the fluorescence intensity of fluorescein molecules conjugated with protein A before and after the introduction of IgG. Factors affecting the efficiency of our direct sensing mechanism including the conjugation ratio of fluorescein per protein A molecule, Solution conditions, and the amount of protein A–fluorescein conjugate used in the experiment are investigated. The IgG2A concentrations in the cell supernatants are analyzed by using our direct sensing method and then compared with the results obtained from enzyme–linked immunosorbent assay (ELISA). The comparisons between our homogeneous assay and heterogeneous ELISA in the analysis time, analysis procedures, cost, and sensitivity are reported and discussed. © 1992 John Wiley & Sons, Inc.
Determination of equilibrium and individual rate constants for subtilisin‐catalyzed transesterification in anhydrous environments
Authors: Chatterjee Sudipta , Russell Alan J.
Published: December 2004

We report here the first determinations of individual rate constants and equilibrium constants for enzymatic reactions in essentially anhydrous organic solvents. Using the added nucleophile method we have measured the effect of changing solvent on the binding and catalytic steps for subtilisin‐catalyzed transesterification of N‐protected amino acid esters. The detailed information generated indicates that once the substrate has bound to the enzyme, the catalytic machinery can work at rates equivalent to those in water. The decreased overall rates for subtilisin suspended in anhydrous solvents are merely the result of extremely high values for Ks, in most cases, coupled with low concentrations of nucleophile (∼1.0M in organic solvents, and 55M in water). The method described, which is generally applicable, and straightforward experimentally, will, we believe, enable a clearer understanding of how changing solvent can predictably affect the activity and specificity of the enzyme. © 1992 John Wiley & Sons, Inc.
Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3‐butanediol
Authors: Zeng An‐Ping , Deckwer Wolf‐Dieter
Published: December 2004

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle β, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen‐limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3‐butanediol, a method is proposed to calculate β from measurement of metabolites and exhaust gas. In continuous culture β was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH2) released by fermentation under these conditions. The increase of β at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH2 as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter β proved to be a useful reactor design criterium for the microaerobic cultivation. © 1992 John Wiley & Sons, Inc.
Production of xylitol from D‐xylose by candida tropicalis: Optimization of production rate
Authors: Horitsu Hiroyuki , Yahashi Yuuichi , Takamizawa Kazuhiro , Kawai Keiichi , Suzuki Tohru , Watanabe Noriyasu
Published: December 2004

The effect of culture conditions on xylitol production rate was investigated using Candida tropicalis IFO 0618. From the variance analysis of xylitol production rate, it was found that initial yeast extract concentration was highly significant (99%), while the interaction between D‐xylose concentration and aeration rate was significant (95%). These results show the importance of initial yeast extract concentration and of the balance between D‐xylose concentration and aeration in the production of xylitol. It was also clearly shown that C. tropicalis needed more yeast extract concentration for efficient xylitol production than for its growth. In order to enhance xylitol production rate, culture conditions were optimized by the Box–Wilson method. In this respect, initial D‐xylose concentration, yeast extract concentration, and KLa were chosen as the independent factors in 23‐factorial experimental design. As the result of experiments, a maximum xylitol production rate of 2.67 g/L · h was obtained when initial D‐xylose concentration and yeast extract concentration were 172.0 and 21.0 g/L, respectively, and KLa was 451.50 h−1 by 90% oxygen gas. © 1992 John Wiley & Sons, Inc.
Biodesulfurization of water‐soluble coal‐derived material by Rhodococcus rhodochrous IGTS8
Authors: Kilbane John J. , Jackowski Kathleen
Published: December 2004

Rhodococcus rhodochrous IGTS8 was previously isolated because of its ability to use coal as its sole source of sulfur for growth. Subsequent growth studies have revealed that IGTS8 is capable of using a variety of organosulfur compounds as sources of sulfur but not carbon. In this article, the ability of IGTS8 to selectively remove organic sulfur from water‐soluble coal‐derived material is investigated. The microbial removal of organic sulfur from coal requires microorganisms capable of cleaving carbon‐sulfur bonds and the accessibility of these bonds to microorganisms. The use of water‐soluble coal‐derived material effectively overcomes the problem of accessibility and allows the ability of microorganisms to cleave carbon‐sulfur bonds present in coal‐derived material to be assessed directly. Three coals, two coal solubilization procedures, and two methods of biodesulfurization were examined. The results of these experiments reveal that the microbial removal of significant amounts of organic sulfur from water‐soluble coal‐derived material with treatment times as brief as 24 h is possible. Moreover, the carbon content and calorific value of biotreated products are largely unaffected. Biotreatment does result, however, in an increased hydrogen and nitrogen content and a decreased oxygen content of the coal‐derived material. The aqueous supernatant obtained from biodesulfurization experiments does not contain sulfate, sulfite, or other forms of soluble sulfur at increased concentrations in comparison with control samples. Sulfur removed from water‐soluble coal‐derived material appears to be incorporated into biomass. © 1992 John Wiley & Sons, Inc.
Effect of changes in the pH and carbon dioxide evolution rate on the measured respiratory quotient of fermentations
Authors: Royce Patrick N.
Published: December 2004

The total concentration of dissolved carbon dioxide in fermentation broths is one to two orders of magnitude greater than that of oxygen for pH > 6.5. The rate of change in this total concentration can be sufficiently large to produce a discrepancy between the carbon dioxide transfer rate (CTR) across the gas–liquid interface, available from gas analyses, and the carbon dioxide evolution rate (CER) of biomass in the fermentor. The CER is the variable of most interest to fermentation technologists but cannot be measured directly. The CTR is commonly used to yield the measured respiratory quotient (called here the TQ, or transfer quotient). Evaluation of the real underlying respiratory quotient (RQ), however, requiures the unmeasureable CER. Equations defining the problem are presented and are found to accurately predict the discrepancy between the TQ and the RQ in fed‐batch fermentations of Escherichia coli. During the exponential growth phase, the TQ is less than the RQ. A changing pH can cause the TQ to be bigger or smaller than the RQ, while pH fluctuations associated with on–off pH controller action make the CTR and hence the TQ noisy. The RQ is estimated on‐line during an E. coli fermentation and is shown to be constant during the fermentation, even though the TQ varies greatly. © 1992 John Wiley & Sons, Inc.
Protein immobilization to alumina supports: I. Characterization of alumina‐organophosphate ligand interactions and use in the attachment of papain
Authors: Hyndman David , Flynn T. Geoffrey , Lever Gordon , Burrell Robert
Published: December 2004

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. © 1992 John Wiley & Sons, Inc.
Packed bed bioreactor with porous ceramic beads for animal cell culture
Authors: Park Seujeung , Stephanopoulos Gregory
Published: December 2004

A packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single‐particle level through total flow rate control, this relationship is a useful scale‐up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab‐scale reactors underline the potential of this simple bioreactor configuration for large‐scale cell culture applications. © 1993 John Wiley & Sons, Inc.
Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology
Authors: Sureshkumar G. K. , Mutharasan R.
Published: December 2004

A method has been developed to continuously measure the intracellular pH (pHi) of cells cultivated in a bioreactor in an on‐line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pHi indicator 9‐aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pHi from measured 9AA‐fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pHi (7 μM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA‐fluorescence. The pHi change in yeast cell following the addition of a proton ionophore carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well‐established pHi, indicator (which is however unsuitable for on‐line applications in a bioreactor) bis‐carboxyethyl carboxy fluorescein (BCECF). The pHi of yeast under substrate starved conditions was 6.4 units. The responses of pHi of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pHi increased by 0.12 unit following a 100‐ppm glucose pulse addition and by 0.25 unit following a 300‐ppm ethanol pulse addition. Under anaerobic condition, pHi increased by 0.1 unit following a 500‐ppm glucose pulse addition. Comparison of pHi with other indicators of cellular metabolic state suggests that pHi is a cellular metabolic state indicator. © 1993 John Wiley & Sons, Inc.
Kinetics and modeling of hexavalent chromium reduction in enterobacter cloacae
Authors: Yamamoto Koji , Kato Junichi , Yano Takuo , Ohtake Hisao
Published: December 2004

Kinetics of bacterial reduction of toxic hexavalent chromium (chromate: CrO42−) was investigated using batch and fedbatch cultures of Enterobacter cloacae strain HO1. In fedbatch cultures, the CrO42− feed was controlled on the basis of the rate of pH change. This control strategy has proven to be useful for avoiding toxic CrO42− overload. A simple mathematical model was developed to describe the bacterial process of CrO42− reduction. In this model, two types of bacterial cells were considered: induced, CrO42−‐resistant cells and uninduced, sensitive ones. Only resistant cells were assumed to be able to reduce CrO42−. These fundamental ideas were supported by the model predictions which well approximated all experimental data. In a simulation study, the model was also used to optimize fed‐batch cultures, instead of lengthy and expensive laboratory experiments. © 1993 John Wiley & Sons, Inc.
The effect of aqueous surfactant solutions on alcohol dehydrogenase (LADH)
Authors: Creagh A. L. , Prausnitz J. M. , Blanch H. W.
Published: December 2004

Alcohol dehydrogenase (LADH) was studied in aqueous solutions of surfactants to determine its structural and catalytic characteristics. Fluorescence, circular dichroism (CD), and electron paramagnetic resonance (ERP) techniques were used to study structural changes to the enzyme. The activity of LADH in catalyzing the oxidation of ethanol was investigated. Short‐chain alkyl sulfonates and sulfates did not deactivate LADH or alter its structure. Longer and branched alkyl sulfates and sulfonates, as well as a cationic surfactant (CTAB), affected both LADH activity and conformation. © 1993 John Wiley & Sons, Inc.
Variation and modeling of the probability of plasmid loss as a function of growth rate of plasmid‐bearing cells of Escherichia coli during continuous cultures
Authors: Mosrati Ridha , Nancib Nabil , Boudrant Joseph
Published: December 2004

A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid‐carrying cell and plasmid‐free cells (δμ) and (ii) the frequency of plasmid loss by plasmid‐containing cells (prμ+). Until now, studies have been performed on the global expression prμ+ and δμ, whose value during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms pr and μ+, respectively, probability of plasmid loss and specific growth rate of the plasmid‐carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galk (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). From data obtained during continuous cultures, it is shown that during an assay, δμ, and prμ+ do not remain constant. An appropriate mathematical analysis of the expression of μ− (specific growth rate of the plasmid‐free cells) and μ+ has been built up. This allows the evaluation of the values of μ+ and μ− during the continuous cultures carried out at different dilution rates. Values of pr have been calculated from these data. Indeed our results show that pr increases with μ+. A modeling approach which allows correct simulation of this variation is also proposed. This model is derived from the Hill equation regarding cooperative binding of enzymic type reaction. © 1993 John Wiley & Sons, Inc.
Action of lipolytical enzymes in biphasic organic‐aqueous systems: Dynamics of the irreversible Michaelis–Menten reaction
Authors: Tsai Shau‐Wei , Wei Hwa‐Jou , Chiang Chen‐Li
Published: December 2004

Through simple model analysis, the mass action kinetic model for lipolytic enzymes in biphasic aqueous‐organic systems can be simplified using the quasi–steady state assumption (or the quasi–equilibrium state assumption) for the adsorbed enzyme E* or the enzyme–substrate complex E*S. Some parameter combinations leading to the above assumptions are derived confirmed by full numerical integration of the whole enzymatic process. The results may be classified into three categories: (1) the quasi–equilibrium state assumption for E*, (2) the quasi–steady state assumption for E*, and (3) the quasi–steady state assumption for E*S. Further simplification for both E* and E*S is also discussed. © 1993 Wiley & Sons, Inc.
An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media
Authors: Prazers D. M. F. , Garcia F. A. P. , Cabral J. M. S.
Published: December 2004

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. © 1993 Wiley & Sons, Inc.
Computation of pH evolution versus ionic products concentration in a fermentation broth
Authors: Kennes C. , Naveau H. , Nyns E. J.
Published: December 2004

An algorithm developed for pH computation has been tested to calculate the theoretical pH changes in a culture medium during the course of a fermentation. A divergence between the computed pH value and the value measured with the electrode allows us to highlight the presence of undetected ionic products. The calculation with the algorithm by means of a computer requires only the knowledge of the ionic properties of the substrates and detected products and existing thermodynamic constants. © 1993 Wiley & Sons, Inc.
Kinetic study of the lipase‐catalyzed synthesis of triolein
Authors: Lortie Robert , Trani Michael , Ergan Françoise
Published: December 2004

The kinetics of the synthesis of triolein catalyzed by immobilized Mucor miehei lipase were studied. Equilibrium constants for the synthesis of mono‐, di‐, and triolein were calculated from the equilibrium compositions for different initial ratios of glycerol and oleic acid by means of multiresponse regression. The 1,3‐specific lipase can catalyze the synthesis of triolein because the ester enzymatically formed with the primary alcohol isomerizes, through acyl migration, to an ester on the secondary hydroxyl. The freed primary hydroxyl may then undergo further enzymatic conversion. The rates of isomerization depend on the concentration of oleic acid. © 1993 Wiley & Sons, Inc.
Anaerobic waste‐activated sludge digestion–a bioconversion mechanism and kinetic model
Authors: Shimizu Tatsuo , Kudo Kenzo , Nasu Yoshikazu
Published: December 2004

The anaerobic bioconversion of raw and mechanically lysed waste‐activated sludge was kinetically investigated. The hydrolysis of the biopolymers, such as protein, which leaked out from the biological sludge with ultrasonic lysis, was a first‐order reaction in anaerobic digestion and the rate constant was much higher that the decay rate constant of the raw waste activated sludge. An anaerobic digestion model that is capable of evaluating the effect of the mechanical sludge lysis on digestive performance was developed. The present model includes four major biological processes–the release of intracellular matter with sludge lysis; hydrolysis of biopolymers to volatile acids; the degradation of various volatile acids to acetate; and the conversion of acetate and hydrogen to methane. Each process was assumed to follow first order kinetics. The model suggested that when the lysed waste‐activated sludge was fed, the overall digestive performance remarkably increased in the two‐phase system consisting of an acid forming process and a methanogenic process, which ensured the symbiotic growth of acetogenic and methanogenic bacteria. © 1993 Wiley & Sons, Inc.
Determination of kinetic parameters of fermentation processes by a continuous unsteady‐state method: Application to the alcoholic fermentation of D‐xylose by Pichia stipitis
Authors: Domínguez H. , Núñez M. N. , Chamy R. , Lema J. M.
Published: December 2004

A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D‐xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, μm and Ks can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of Ks and μm and a little over 4% for μm were the deviations under the latter ones. © 1993 Wiley & Sons, Inc.
Emulsion liquid membrane extraction of lactic acid from aqueous solutions and fermentation broth
Authors: Schöller C. , Chaudhuri J. B. , Pyle D. L.
Published: December 2004

Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n‐heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. © 1993 John Wiley & Sons, Inc.
Overexpression of cloned genes using recombinant escherichia coli regulated by a T7 promoter: II. Two‐stage continuous cultures and model simulations
Authors: Miao Fudu , Kompala Dhinakar S.
Published: December 2004

A two‐stage culture strategy was studied for continuous high‐level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid‐bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid‐bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two‐stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two‐stage continuous cultures. © 1993 John Wiley & Sons, Inc.
Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model
Authors: Bentley William E. , Quiroga Oscar E.
Published: December 2004

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a “downward selective pressure,” is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. © 1993 John Wiley & Sons, Inc.
A two‐stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system
Authors: Zhang Junli , Kalogerakis Nicolas , Behie Leo A. , Iatrou Kostas
Published: December 2004

A two‐stage bioreactor scheme was developed for the large‐scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 μg CAT/(106 cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high‐density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high‐density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high‐density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two‐stage bioreactor system was proven to be reliable for a long‐term operation beyond 600 h. © 1993 John Wiley & Sons, Inc.
Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction
Authors: Corry James P. , Reed William L. , Curtis Wayne R.
Published: December 2004

The integrated recovery of solavetivone from fungus elicited “hairy root” cultures of Hyoscyamus muticus is examined using volatile organic solvents and solid‐phase adsorbents in an external loop extraction configuration. Hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. Solid‐phase neutral adsorbents, XAD‐7 and XAD‐16, display higher capacity and better solavetivone partitioning capability than the hydrocarbons; however, their selectivity for the sesquiterpene solavetivone is poor in comparison with hexane. In both cases, the integration of product recovery through extraction resulted in a doubling of product formation by alleviating feedback repression. Implications of these results to the recovery of secondary metabolites from plant root cultures are discussed. © 1993 John Wiley & Sons, Inc.
Electric prompting and control of denitrification
Authors: Sakakibara Yutaka , Kuroda Masao
Published: December 2004

The effectiveness of a denitrification process which is driven and controlled by an electric current is demonstrated. Denitrifying microorganisms were immobilized on a carbon electrode and hydrogen was produced through the electrolysis of water. The hydrogen was utilized for the reduction of nitrate to N2. The denitrification rate was a linear function of the electric current, and it was shown that about 1 mol of electron reduces 0.2 mol of nitrate to N2 gas. These results exhibit that the proposed process is simple and feasible, especially for the treatment of low‐strength nitrate solutions. © 1993 John Wiley & Sons, Inc.
Gas phase acetaldehyde production in a continuous bioreactor
Authors: Hwang Soon Ook , Trantolo Debra J. , Wise Donald L.
Published: December 2004

The gas phase continuous production of acetaldehyde was studied with particular emphasis on the development of biocatalyst (alcohol oxidase on solid phase support materials) for a fixed bed reactor. Based on the experimental results in a batch bioreactor, the biocatalysts were prepared by immobilization of alcohol oxidase on Amberlite IRA‐400, packed into a column, and the continuous acetaldehyde production in the gas phase by alcohol oxidase was performed. The effects of the reaction temperature, flow rates of gaseous stream, and ethanol vapor concentration on the performance of the continuous bioreactor were investigated. © 1993 John Wiley & Sons, Inc.
Modeling lipolysis in a reversed micellar system: Part II—membrane reactor
Authors: Prazeres D. M. F. , Lemos F. , Garcia F. A. P. , Cabral J. M. S.
Published: December 2004

Olive oil hydrolysis using Chromobacterium viscosum lipase B in a reversed micellar media was investigated in a membrane reactor. The dynamic evolution of the product concentration both in the concentrate and permeate stream was analyzed using a mechanistic model previously developed by us and further modified in this work. A kinetic law with a second‐order dependence in the substrate concentration and nonlinear product inhibition was found to be the most adequate for the description of the hydrolysis data over an extensive range of time and substrate concentration. These findings are discussed in terms of the specific interactions occurring in the membrane reactor. © 1993 John Wiley & Sons, Inc.
Affinity‐based reversed micellar protein extraction: II. Effect of cosurfactant tail length
Authors: Kelley Brian D. , Wang Daniel I. C. , Hatton T. Alan
Published: December 2004

The selectivity of protein extraction by reversed micellar solutions can be improved by the addition of affinity cosurfactants bearing ligands which bind strongly to the target protein. The interactions between cosurfactant and protein, as well as the interfacial activity of both the free cosurfactant and the protein–cosurfactant complex, were accounted for in a model of the affinity‐partitioning process. The aqueous phase dissociation constant was used to describe the protein–ligand interactions. The interfacial partition coefficient for several cosurfactant families varied with tail length according to the well‐established hydrophobic effect. Control studies with alkylated chymotrypsin showed that when longer hydrophobic tails are irreversibly attached to the protein, the protein partitions more strongly to the reversed micellar phase. In contrast, for reversible protein–cosurfactant binding, the model predicts a maximum in protein uptake when the cosurfactant tail length is varied; the decrease at longer tail lengths is due to the lowered aqueous phase concentration of affinity cosurfactant, resulting in the formation of fewer protein–cosurfactant complexes. This behavior was confirmed experimentally. © 1993 John Wiley & Sons, Inc.
Estimation of melting curves from enzymatic activity–temperature profiles
Authors: Hei Derek J. , Clark Douglas S.
Published: December 2004

Measuring the reversible thermal unfolding of enzymes is valuable for quantifying the effects of environmental factors on the thermodynamic stability of proteins. The thermal unfolding behavior of enzymes is typically studied using calorimetry or optical techniques such as circular dichroism, fluorescence, or light scattering. These techniques often have practical limitations and usually require the protein to be electrophoretically pure. An alternative technique for analyzing the thermodynamic stability of enzymes is to estimate the melting curve from temperature–activity data. This technique does not require electrophoretically pure enzyme, provided the sample does not have competing enzymatic activities or proteins which can affect enzyme stability (e.g., proteases). Moreover, small amounts of contaminant proteins should not affect the results as long as enzymatic assays are performed at low protein concentrations where nonspecific protein–protein interactions are negligible. To illustrate this technique, the melting curve for β‐galactosidase from Escherichia coli in the presence of 1 mM EDTA, and the shift caused by adding 1 mM Mg+2, were calculated from activity–temperature data. Melting temperatures predicted from activity–temperature data compared closely with those obtained using other techniques. Application of this analysis to multisubstrate enzymes is illustrated by estimating the melting profiles for partially purified hydrogenases from several thermophilic Methanococcii. Limitations and important considerations for estimating melting profiles from activity–temperature data are discussed. © 1993 John Wiley & Sons, Inc.
Scaleup of spinfilter perfusion bioreactor for mammalian cell retention
Authors: Yabannavar V. M. , Singh V. , Connelly N. V.
Published: December 2004

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (107 mL−1). Filter screens with openings (25 μm) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12‐ and 175‐L (working volume) bioreactors is shown in support of the scaleup analysis. © 1994 John Wiley & Sons, Inc.
A new visualization chamber to study the transient volumetric response of yeast cells submitted to osmotic shifts
Authors: Berner J.‐L. , Gervais P.
Published: December 2004

A visualization chamber has been developed to analyze potential correlations between osmotic step increase on yeasts and the resultant cell volume decreases. Image analysis was used to characterize the step increases in the center of the chamber and to measure the changes in the cell volume. Step increases of different intensities have been performed on the yeast Saccharomyces cerevisiae. This device has allowed the kinetics of the volumetric evolution of the cells to be observed. The water exit flow rate from the cell was found to occur in the first 10 s following the hypertonic step change. Comparison of the time constants of the chamber and of the cell volume variations allowed to conclude that the time constant of the water transfer across the membrane was short (about 1 s). © 1994 John Wiley & Sons, Inc.
A prototype neural network supervised control system for Bacillus thuringiensis fermentations
Authors: Zhang Qin , Reid John F. , Bruce Litchfield J. , Ren Jinliang , Chang Shiuan‐Wu
Published: December 2004

This article discusses the development of a prototype neural network‐based supervisory control system for Bacillus thuringiensis fermentations. The input pattern to the neural network included the type of inoculum, operation temperature, pH value, accumulated process time, optical density in fermentation medium, and change in optical density. The output from the neural network was the predicted optical density for the next sampling time. The control system has been implemented in both a computer simulation and a laboratory fermentation experiment with promising results. © 1994 John Wiley & Sons, Inc.
Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen‐I by chinese hamster ovary cells in a pH‐dependent manner
Authors: Borys Michael C. , Linzer Daniel I. H. , Papoutsakis Eleftherios T.
Published: December 2004

The N‐linked glycosylation of the recombinant protein mouse placental lactogen‐I (mPL‐I) expressed by Chinese hamster ovary (CHO) cells under nongrowth conditions was inhibited by increasing levels of ammonium chloride (3 and 9 mM) in a serum‐free, protein expression medium. The effect of ammonia on glycosylation was dependent on the extracellular pH (pHe). In media containing 0 and 9 mM ammonium chloride, the percentage of the most heavily glycosylated forms of secreted mPL‐I decreased from ca. 90% to ca. 25% at pHe 8.0, and from ca. 90% to ca. 65% at pHe 7.6, respectively. However, at pHe 7.2, the most heavily glycosylated forms of secreted mPL‐I decreased from ca. 90% to ca. 80% in media containing 0 and 9 mM ammonium chloride, respectively. Inhibition of mPL‐I glycosylation was found to correlate with the calculated concentrations of the ammonia species (NH3). Control experiments showed that the ammonia effect on mPL‐I glycosylation could not be attributed to increased chloride concentration or osmolarity, or to extracellular events after secretion of the recombinant protein into the supernatant. Ammonium chloride, 9 mM, inhibited the expression rate of MPL‐I by CHO cells at low pHe. © 1994 John Wiley & Sons, Inc.