bluuebunny/crossref_metadata_2025 huggingface dataset:
Adsorption equilibrium in immunoaffnity chromatography with antibodies to synthetic peptides
Authors: Kondo Akihiko , Takamatsu Hiroyuki , Katoh Shigeo , Sada Eizo
Published: December 2004

The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr‐activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep‐tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide‐antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides.
Production of glycerides from glycerol and fatty acid by immobilized lipases in non‐aqueous media
Authors: Ergan F. , Trani M. , André G.
Published: December 2004

Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid. No organic solvent is necessary to solubilize the substrates, which allows for the use of a reaction medium solely composed of the necessary substrates. Water produced in the reaction evaporates due to the high temperature used for the process. A conversion of 86% of oleic acid into triolein is obtained when using the substrates in stoichiometric amounts. Varying the ratio of glycerol over oleic acid allows for the preferential synthesis of one of the glycerides. Some batch reactors have been set up using different means of removing the water: spontaneous evaporation, molecular sieves, vacuum, and dry air bubbling.
Extractive acetonobutylic fermentation by coupling ultrafiltration and distillation
Authors: Minier M. , Grateloup R. , Blanc‐Ferras E. , Goma G.
Published: December 2004

An extractive acetonobutylic fermentation process is developed by integrating bioproduction, Ultrafiltration, and distillation, providing simultaneous retention of biomass, selective removal of inhibitors from the permeate, as well as separation and purification of acetone–butanol–ethanol solvents. Successive batch fermentations were performed with normal pressure distillation (98°C), which permitted prolonging and enhancing (by a factor of 3) solvent production, with very few volume exchanges of medium (average dilution rate ws 0.002 h−1), and recovering on‐line concentrated solvents. Different operating conditions were also tested in order to study the presence of extracellular autolytic enzymes as inhibition factors: It was shown that, (1) extracellular autolytic activity remains low during the larger part of fermentations, even without enzyme‐inactivating thermotreatment in the distillation boiler, and (2) high‐temperature distillation causes deleterious effects to the culture medium for long duration treatments. Progressive improvements of the process were achieved, first, by managing continuous runs, providing a minimum renewal of the culture medium and, mainly, by decreasing temperature and pressure of distilation. Solvent productivity then reached 2.6 g/L h for a 0.036 h−1 average dilution rate, corresponding to a feed concentration of 156 g/L glucose actually consumed.
The effect of agitation on the morphology and penicillin production ofPenicillium chrysogenum
Authors: Smith J. J. , Lilly M. D. , Fox R. I.
Published: December 2004

Penicillium chrysogenumstrain P1 was grown on complex media in 10 and 100 L agitated fermenters at various aeration rates and stirrer speeds. Samples were removed at intervals for measurements of the culture morphology. At high stirrer speeds (1000 and 1200 rpm) in 10‐L fermentations the rate of decrease in the mean effective hyphal length was faster and the rate of penicillin production was lower than fermentations done at 800 rpm. At similar power inputs per unit volume in 100‐L fermentations, the change in mean effective hyphal length was less and higher penicillin production rates were observed. This work comparing the results at two scales has shown that neither of the concepts of impeller tip speed or the dissipation rate of turbulence have general validity as a measure of hyphal damage. Our results are reasonaby well correlated by groups similar to circulation rate (ND/V) with lower circulation rates being beneficial. An adaptation of the van Suijdam and Metz relationship, expressed asP/Dtc, was most successful. Our data are insufficient to demonstrate the generality of the relationship but do support the concept of a dispersion zone around the impellers in which mycelia may be damaged. The greater the frequency of circulation of mycelia through the zone the greater the damage and the lower the rate of penicillin synthesis by the culture.
Effects of spatial heterogeneity on the dynamics of a microbial feeding interaction
Authors: Tsangaropoulou Esperia , Pavlou Stavros
Published: December 2004

A mathematical model for an ideal chemostat in which one microbial population feeds on another and where Monod's model is used for the specific growth rates of both populations predicts a less stable behavior for the system than the one observed experimentally. Various factors have been proposed as being the reason for the increased stability of such systems. In this work, the effect of spatial heterogeneity on the dynamics of the microbial feeding interaction is studied. It is concluded that spatial heterogeneity has a stabilizing effect on the system. This effect combined with other factors could be the reason for the increased stability observed in systems where a microbial feeding interaction occurs.
Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production
Authors: McQueen Anne , Bailey James E.
Published: December 2004

The effects of NH4Cl addition on batch hybridoma cell growth at different external pH values (pHe) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pHe over the range 6.8–7.6 had minor effects on growth. Addition of 3 mM NH4Cl had little effect on cell growth while 10 mM NH4Cl caused a substantial growth inhibition, Measurements of the effects of pHe and NH4Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pHe decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pHe on glycolysis and previous measurements of its effects on intracellular pH (pHi). Increases in NH4Cl concentration were previously determined to decrease pHi and are shown here to decrease the integral cell yield on glucose. At all pHe values in the absence of NH4Cl, glutamine is depleted at the time the maximum cell density is reached. Both pHe decreases and NH4Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pHe and in the NH4Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH4Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.
Production of S‐adenosyl‐L‐methionine by Saccharomyces cerevisiae cells carrying a gene for ethionine resistance
Authors: Shiomi Naofumi , Fukuda Hideki , Fukuda Yasuki , Murata Kousaku , Kimura Akira
Published: December 2004

A gene for ethionine resistance isolated from the yeast Saccharomyces cerevisiae DKD‐5D‐H conferred on the yeast cells resistance to seleno‐L‐methionine and capability to produce S‐adenosyl‐L‐methionine in the cells. An enzymatic study of the L‐methionine synthetic pathway of L‐methionine proto‐ and auxotrophs and in dried yeast cells with or without the gene suggested that the cloned gene for ethionine resistance is responsible for the activity of S‐adenosyl‐L‐methionine synthase. To produce S‐adenosyl‐L‐methionine by yeast cells transformed with the ethionine resistance gene, some culturing conditions were determined.
Action pattern of alpha‐amylase from Aspergillus oryzae in concentrated media
Authors: Graber Marianne , Combes Didier
Published: December 2004

The influence of concentrated maltotetraose solutions on fungal alpha‐amylase activity and specificity has been determined. It has been found that the enzyme is not inhibited by 500 g/L substrate concentration and that transglycosylation reactions are increased with rising substrate concentration. The amount of oligosaccharides of polymerization degree higher than maltotetraose reaches 20% (w/w). Moreover, the effect of polyhydric alcohols, known as water activity depressors has been analyzed: the presence of polyols does not modify the amount of transglycosylation products, but changes the hydrolysis pattern by favoring the formation of low polymerization degree oligosaccharides.
Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: II. Analysis of cell synchronization and metabolism
Authors: Chen Ching‐I , McDonald Karen A.
Published: December 2004

Sustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures of Saccharomyces cerevisiae under controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5–3 h at a pH of 5.5 and 2–2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002–0.085 g/L) in the bioreactor.

The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.

The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time‐varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations as well.
Production of L‐phenylacetyl carbinol by immobilized yeast cells: I. Batch fermentation
Authors: Mahmoud Wafaa M. , El‐Sayed Abdel‐Halima M. M. , Coughlin Robert W.
Published: December 2004

Immobilization of Saccharomyces cerevisiae ATCC 834 within alginate beads enhances microbiological conversion of benzaldehyde to L‐phenylacetyl carbinol (L‐PAC), a precursor employed for synthesis of L‐ephedrine. Yields of 90% L‐PAC on benzaldehyde (initially 0.6% in medium) were obtained with immobilized cells, in contrast to about 10% with free cells which tend to form pellets in the presence of benzaldehyde. The predominant favorable action of immobilization appears to be a reduction in the toxic or inhibitory effects of benzaldehyde. With an initial benzaldehyde concentration of about 0.6% in the medium the optimum cell mass concentration was observed to be about 28 g cell mass (immobilized) per liter of medium.
Effect of regulatory mechanism on hyperbolic reaction network properties
Authors: Majewski R. A. , Domach M. M.
Published: December 2004

The notion that the regulated and flux‐controlling enzyme in a metabolic network need not correspond suggests that the purpose of regulation may not be flux homeostasis under all physiological circumstances. Additionally, the fact that diversity in the function of intact metabolic networks exists suggests that in addition to time constant separation, other kinetic structure/regulatory mechanism patterns exist. In order to compliment and expand prior work on identifying kinetic structure–property relationships in networks, the present work explores in a general way how the control, dynamic, and energetic properties of metabolic networks depend on operating point, kinetic structure, and regulatory mechanism. The basic feature of trade‐offs between properties is illustrated and used as a basis for indicating how particular subsets of structure, regulatory mechanism, and operating point emphasize certain properties that can be associated with a physiological function. Examples of scavenging trace metabolites and amphibolite coordination are proposed. Microstructure logic in terms of turnover number distributions as well as a potential mixed polynomial network analysis approach are also discussed.
Kinetic analysis of the growth of Chlorella vulgaris
Authors: Lehana Mitsunori
Published: December 2004

The energy of the total transmitted light was subtracted from that of the incident light in a culture vessel and the difference was divided by the weight of cells. The value thus obtained was defined as the amount, Ex, of light energy absorbed per unit cell weight per unit time.

Batch and continuous cultures of Chlorella vulgaris were carried out at 30°C in the pH range of 6.4–6.7 while restricting illumination. Next the specific growth rate, μ, in the batch culture and the fixed dilution rate, D, in the continuous culture were plotted against Ex. The results showed that the relation between D and Ex can be expressed in a Michaelis–Menten equation, where the maximal specific growth rate is 0.24 h −1 and the saturation constant is 6.58 kcal/g · h.

Cell concentration calculated by substituting the apparent concentration, Xe, of incubated cells and the apparent maintenance constant, Me, for this equation agreed with that observed in almost all growth phases. Furthermore, from the change of chlorophyll productivity and the relationship between D and Ex expressed in this equation, it is assumed that Ex involves the light energy directly utilized in photosynthesis in the cells and that which is converted into, e.g., heat. This equation also indicated that a maximum in the growth yield existed. Then the growth yield of 0.029 g/kcal obtained at the incident light of 1.46 or 2.63 cal/cm2 · h was maximum (maximal conversion efficiency of light energy, 15.6%).

These results indicate that this method of deriving the equation for the growth rate from this study is a useful procedure for obtaining bioengineering findings.
Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory‐scale reactor
Authors: Vos Henk J. , Zomerdijk Max , Groen Dick J. , Luyben Karel Ch. A. M.
Published: December 2004

In Part I of this series,1 we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory‐scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.

The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.
Control of an affinity purification procedure using a thermal biosensor
Authors: Flygare Lilian , Larsson Per‐Olof , Danielsson Bengt
Published: December 2004

Lactate dehydrogenase (LDH) was recovered from a solution by affinity binding to an N6‐(6‐aminohexyl)‐AMP‐Sepharose gel. An enzyme thermistor unit was employed to continously measure the activity of the unbound LDH. The enzyme activity signal from the enzyme thermistor was used in a PID controller to regulate the addition of AMP‐Sepharose gel to the LDH solution. In another type of experiment, a desktop computer was utilized to control the addition of the adsorbent. Both systems worked satisfactorily, and enabled a rapid and accurate assessment of correct addition of adsorbent.
Enhanced enzymatic activity and stability of trypsin by reductive alkylation in solid phase
Authors: Tsuji Ryohei F.
Published: December 2004

Amino groups of trypsin (EC 3.4.21.4) were reductively alkylated in solid phase to obtain a surface‐active and biologically active enzyme in an o/w emulsion system. Trypsin adsorbed on a benzamidine–sepharose column was reductively alkylated with n‐octanal in the presence of sodium borohydride, i.e., trypsin‐C8. Activity of trypsin‐C8 against Nα‐benzoyl‐L‐arginine‐p‐nitroanilide was three times higher than that of native trypsin. Activities of trypsin and trypsin‐C8 against casein were almost the same. After incubating the trypsin solution at 40°C for 1 h, residual activities in the emulsion and solution systems were 64.2 and 57.4%, respectively. On the other hand, residual activities of native trypsin following incubation were 21.8% in the emulsion system and 33.2% in the solution system. Enhancement of trypsin‐C8 stability in the emulsion system may derive from interaction between the hydrophobic areas of trypsin‐C8 molecules and the hydrophobic phase of the emulsion.
Group contributions for estimating standard gibbs energies of formation of biochemical compounds in aqueous solution
Authors: Mavrovouniotis Michael L.
Published: December 2004

A method is presented for the estimation of the standard Gibbs energies of formation of biochemical compounds (and hence the Gibbs energies and equilibrium constants of biochemical reactions) from the contributions of groups. The method employs a large set of groups and special corrections. The contributions were estimated via multiple linear regression, using screened and weighted literature data. For most of the data employed, the error is less than 2 kcal/mol. The method provides a useful first approximation to Gibbs energies and equilibrium constants in biochemical systems.
Decrease in iron oxidizing activity of Thiobacillus ferrooxidans adsorbed on activated carbon
Authors: Kai T. , Takahashi T. , Shirakawa Y. , Kawabata Y.
Published: December 2004

It was observed that about 90% of free‐swimming Thiobacillus ferrooxidans in 9 K medium was adsorbed on added activated carbon when the concentration of the cultivated bacteria reached about 4 × 1013 cells m−3. The oxidation of ferrous iron and the leaching of copper ore were carried out in shake flasks and in aerated columns. The rates of oxidation and leaching increased when bacteria adsorbed on activated carbon were used. However, the evaluation of the reaction rates by eliminating the catalytic effect of activated carbon showed that the contribution to the reaction by the adsorbed microorganism was very small.
Continuous cell suspension processing using magnetically stabilized fluidized beds
Authors: Terranova Brenda E. , Burns Mark A.
Published: December 2004

The behavior of a cell suspension in a continuous magnetically stabilized fluidized bed (MSFB) was investigated both experimentally and theoretically. The low, constant pressure drop and fluidity of the solids phase in the MSFB allowed a continuous countercurrent separator to be constructed. The magnetic field eliminated all motion of the solids phase (nickel spheres) and produced a device similar to a packed‐bed depth filter. Yeast cells were used as the suspended solids and the performance of the MSFB filter was assessed as a function of the bed height, solids velocity, cell concentration, and liquid composition. Removal rates could be adjusted by controlling the cell/support interaction and were found to be as high as 99%. A mathematical model was used to aid in understanding this filtration and was found to agree qualitatively with all experimental observations. Comparison of the model with the data suggests that both cell/cell binding and cell shadowing are occurring.
Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation
Authors: Fröhlich S. , Lotz M. , Korte T. , Lübbert A. , Schügerl K. , Seekamp M.
Published: December 2004

Saccharomyces cerevisiae was cultivated in a 4‐m3 pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m3 volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse‐shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.
Semicontinuous enzymatic hydrolysis of lignocelluloses
Authors: Ishihara Mitsuro , Uemura Shoko , Hayashi Noriko , Shimizu Kazumasa
Published: December 2004

Lignocelluloses (steamed hardwood and hardwood kraft pulp) were semicontinuously hydrolyzed on a large scale [2‐2. 5 kg of substrate vs. 20, 000 IU filter paperase (FPase)] using a 10‐L hydrolysis reactor with an ultrafiltration unit for the recovery and reuse of cellulases. The substrate was added to the reactor at appropriate intervals to keep a concentration of approximately 5% (w/v). All of the enzyme was added at the beginning and no further addition was done. The ultrafiltration unit was operated intermittently rather than continuously due to its enough capacity (dilution rate of 2.5 h−1) and making the enzyme durable. The enzyme required to produce one gram of reducing sugar in this reactor was 27.3 FPase IU/g RS for steamed hardwood and 7.4 FPase IU/g RS for hardwood kraft pulp. The sugar composition of hydrolyzate was unaltered virtually from beginning to end of the hydrolysis in spite of the progressive loss of enzyme activities. The analysis of the enzyme composition in the hydrolyzate during hydrolysis revealed that an exo‐β‐D‐glucanase component was adsorbed selectively at the stages of advanced hydrolysis extent.
Control of an Escherichia coli mixed culture via affinity binding
Authors: Roos Joseph W. , Hjortso Martin A.
Published: December 2004

A chemostat with population specific recycle was employed to alter the dynamics of a competitive mixed culture of Escherichia coli. Based on differential expression of a functional maltoporin, the two populations were separated by specific adhesion on starch–Sepharose. The slower growing population was the recycled to the reactor. The specific recycle was successful in maintaining the slower growing population at a high level than in comparable reactors without recycle.
An alternate method of calculating the heat of growth of Escherichia coli K‐12 on succinic acid
Authors: Battley Edwin H.
Published: December 2004

The ΔH unit weight of a complex substance such as a biological macromolecule is almost always obtained by means of combustion analysis. In theory, this can also be done by summing the ΔH values for the monomers comprising the macromolecule plus the enthalpic energies involved in their polymerization. The enthalpy of formation of one unit‐carbon formula weight of dried Escherichia coli K‐12 cells was determined by summing the values of the enthalpies of formation of the quantities of monomers in the major classes of macromolecules substances comprising the cellular biomass and the enthalpic energies involved in their polymerizations. To this value was added the enthalpy of formation of the cellular ions in their aqueous standard states, per unit‐carbon formula weight of cellular substance and the enthalpy change with respect to the ionization of the protein amino acid side chains. If it is assumed that the cellular fabric is insoluble and that the ions are soluble, the sum of the enthalpies of formation of all the cellular components should closely approximate the enthalpy of formation of one unit‐carbon formula weight equivalent of living cells. Using this value, a calculation of the enthalpy change accompanying anabolism shows this latter to be effectively zero, indicating that the heat of growth (anabolism plus catabolism) is equal to that calculated for catabolism alone. This conclusion is in accord with those of several investigators who have used manometry or direct calorimetry.
Continuous culture system for production of biopolymer levan using erwinia herbicola
Authors: Keith J. , Wiley B. , Ball D. , Arcidiacono S. , Zorfass D. , Mayer J. , Kaplan D.
Published: December 2004

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and 13C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low‐angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.
Linear relations in microbial reaction systems: A general overview of their origin, form, and use
Authors: Noorman H. J. , Heijnen J. J. , Ch. A. M. Luyben K.
Published: December 2004

In microbial reaction systems, there are a number of linear relations among net conversion rates. These can be very useful in the analysis of experimental data. This article provides a general approach for the formation and application of the linear relations. Two type of system descriptions, one considering the biomass as a black box and the other based on metabolic pathways, are encountered. These are defined in a linear vector and matrix algebra framework. A correct a priori description can be obtained by three useful tests: the independency, consistency, and observability tests. The independency are different. The black box approach provides only conservations relations. They are derived from element, electrical charge, energy, and Gibbs energy balances. The metabolic approach provides, in addition to the conservation relations, metabolic and reaction relations. These result from component, energy, and Gibbs energy balances. Thus it is more attractive to use the metabolic description than the black box approach. A number of different types of linear relations given in the literature are reviewed. They are classified according to the different categories that result from the black box or the metabolic system description. Validation of hypotheses related to metabolic pathways can be supported by experimental validation of the linear metabolic relations. However, definite proof from biochemical evidence remains indispensable.
A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum‐free medium
Authors: Hiller G. W. , Aeschlimann A. D. , Clark D. S. , Blanch H. W.
Published: December 2004

The steady‐state metabolic parameters for a murine hybridoma cell line have been determined in continuous suspension culture over a wide range of dilution rates. Long‐term adaption occurred over seven months in culture and resulted in lower glucose consumption rates, reduced lactate production, higher cell viability, and, consequently, growth rates more nearly matching the dilution rate. Antibody production rates decreased over the first two months and then remained stable for at least 75 days. The antibody production rate was not found to be growth associated. Steadystate amino acid uptake rates are presented for a wide range of growth rates.
Continuous ethanol production by immobilized yeast reactor coupled with membrane pervaporation unit
Authors: Shabtai Y. , Chaimovitz S. , Freeman A. , Katchalski‐Katzir E. , Linder C. , Nemas M. , Perry M. , Kedem O.
Published: December 2004

A system comprised of an immobilized yeast reactor producing ethanol, with a membrane pervaporation module for continuously removing and concentrating the produced ethanol, was developed. The combined system consisted of two integrated circulation loops: In one the sugar‐containing medium is circulated through the membrane pervaporation module. The two loops were interconnected in a way allowing for separate parameter optimization (e.g., flow rate, temperature, pH) for each loop.

The fermentation unit was 2.0 L bioreactor with five equal segments, packed with 5‐mm beads of immobilized yeasts. The bead matrix was a crosslinked polyacrylamide hydrazide gel coated with calcium alginate. The fast circulation loop of the bioreactor allowed for efficient liberation of CO2 at the top of the immobilized yeast reactor. Continuous operation of the uncoupled reactor for over 50 days with inflowing defined medium or dilute molasses at a residence time of 1.25 h yielded ethanol at a rate of about 10 g/L h.

The pervaporation unit was constructed from four 60‐cm‐long tubular membranes of silicone composite on a polysulfone support. The output from the fermentor was circulated through the inside of the tubes of a unit with a total surface area of 800 cm2, having an average flux of 150 mL/h, and selectivities to ethanol vs. water up to 7. A vacuum of 30 mb was applied to the outside of the tubes, removing 20–30 g of ethanol per hour, which was collected in condensors. The continuous removal of ethanol, avoiding inhibition of the fermentation process, resulted in an improved productivity and allowed the use of high sugar concentrations (40% wt/vol) offering the potential of a compact system with reduced stillage.

The combined system of ethanol production and removal enabled an operative steady state at which the liquid volume of the system, and the concentrations of ethanol within the reactor (˜4% wt/vol), as well as within the flux crossing the pervaporation membrane (17%–20% wt/vol) were kept constant. At the steady state, a 40% wt/vol sugar solution could be continuously added to the fermentor when 12%–20% wt/vol clear ethanol solution was continuously removed by the pervaporation unit. Membrane fouling was reversed by short washing steps, and continuous step operation was maintained by working with two different modules that were interchanged. In this manner, long term continuous operation (over 40 days) was achieved with a productivity of 20–30 g/L h, representing over a twofold increase relative to the continuously operated reactor uncoupled from the membrane and a fivefold increase in comparison with the value obtained fro a corresponding batch fermentation.
Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus
Authors: Malmberg Li‐Hong , Hu Wei‐Shou
Published: December 2004

A kinetic model describing the cephalosporin biosynthesis in Streptomyces clavuligerus was developed. Using previously reported kinetic data of biosynthetic enzymes, we examined the kinetics of cephalosporin production. The predicted time profile of the specific production rate during a batch culture parallels that of experimental observation. Sensitivity analysis reveals that δ‐(L‐α‐aminoadipyl)‐L‐cysteinyl‐D‐valine (ACV) synthetase is the rate‐limiting enzyme. The effect of amplifying ACV synthetase on the specific production rate was analyzed theoretically. Increasing ACV synthetase enhances the production rate initially until ACV synthetase enhances the production rate initially until deacetocycephalosporin C hydroxylase becomes rate‐limiting. Such kinetic analysis can provide a rational basis for modifying the biosynthetic machinery of cephalosporin through gene cloning.
Diffusion of lactose in k‐carrageenan/locust bean gum gel beads with or without entrapped growing lactic acid bacteria
Authors: Arnaud J. P. , Lacroix C.
Published: December 2004

Effective diffusion coefficients (De) of lactose in κ‐carrageenan (2.75% wt/wt)/locust bean gum (0.25% wt/wt) (LBG) gel beads (1.5–2.0‐mm diameter)with or without entrapped lactic acid bacteria (LAB) were determined at 40°C. The effects of lactose concentration, bacteria strain (Streptococcus salivarius subsp. thermophilus and Lactobacillus casei subsp. casei) and cell content at various steps of the fermentation process (after immobilization, pre‐incubation of the beads and successive fermentations) were measured on De as a first step for process modelling. Results were obtained from transiend concentration changes n well‐stirred lactose solutions in which the beads were suspended. A mathematical model of unsteady‐state diffusion in a sphere was used, and De was obtained from the best fit of the experimental data. Diffusivity of lactose in cell‐tree beads was significantly lower than in pure water mainly because of the obstruction effect of the polymer chains and the hydration region. Furthermore, effective diffusivity and equilibrium partition factor were independent of lactose concentration in the range from 12.5 to 50 g/L. No significant difference was found for De (effective diffusivity) and Kp (partition) coefficients between beads entrapping S. thermophilus (approximately 5 × 109 CFU/mL) and cell‐free beads. On the other hand higher cell counts obtained with L. casei (close to 1.8 × 1011 CFU/mL) increased mass transfer resistance resulting in lower effective diffusivities and Kp. Finally, the effects of the type of bacteria and their distribution in the beads on the diffusivity were also discussed.
Design and evaluation of a two‐stage, cyclic, recombinant fermentation process
Authors: Curless C. , Fu K. , Swank R. , Menjares A. , Fieschko J. , Tsai L.
Published: December 2004

A two‐stage, cyclic fed‐batch fermentation process to produce recombinant human lymphokine was designed. The organism used in the study was Escherichia coli K‐12 containing a temperature‐sensitive walkaway plasmid bearing an insert which codes for a human lymphokine. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature‐sensitive product of the cl857 gene at 30°C, but at 42°C the promoter is derepressed. The first or growth, stage of the process was maintained at 28°C and operated in the fed‐batch mode. The vessel was fed at a rate which gives a constant specific growth rate using a media designed to maintain a constant optical density OD600 of 50. After the volume in the first stage reached the maximum working volume of the vessel (12 L), a portion of the vessel contents was transferred to the second stage. The second, or induction/product formation, stage also operated in the fed‐batch mode, was kept at 42°C, and was fed with a media that is conducive to recombinant human lymphokine synthesis. An optical density of more than 100 was consistently achieved in the second stage. Thirty cycles were completed with a consistent yield of human lymphokine and cell density in each cycle. The process was used to produce 200 L of OD600 50 material from the first stage in 10 days. The volumetric productivity (g lymphokine/L. day) of the two‐stage, cyclic fed‐batch process is twice that of a single‐stage, fed‐batch fermentation process.
Catalytic action of L‐2‐halo acid dehalogenase on long‐chain L‐2‐haloalkanoic acids in organic solvents
Authors: Hasan A. K. M. Quamrul , Takada Harumi , Esaki Nobuyoshi , Soda Kenji
Published: December 2004

A Lyophilized preparation of L‐2‐halo acid dehalogenase was not only stable but also catalytically active in anhydrous dimethyl sulfoxide, toluene, and other organic solvents. 2‐Halo acids with long alkyl (C5‐C16) or aromatic (phenyl and benzyl) side chains were inert in water but dehalogenated effectively in anhydrous dimethyl sulfoxide by the lyophilized enzyme. Long chain 2‐haloalkanoic acids such as 2‐bromohexadecanoic acids were better as substrate than short‐chain halo acids (e.g., 2‐chloropropanoic acid). The dehalogenation proceed with inversion of C2 configuration to produce the corresponding (2R)‐2‐hydroxy acids in anhydrous dimethyl sulfoxide in the same way as found in water.
Product enhancement and recovery from transformed root cultures of Nicotiana glauca
Authors: Green K. D. , Thomas N. H. , Callow J. A.
Published: December 2004

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full‐strength Gamborg's B5 medium proved the best for biomass yield while half‐strength, or low‐salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.

Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD‐2 and XAD‐4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.
Measurement of the change in aerosol size distribution with bacterial growth in a pilot scale fermentor
Authors: Szewczyk Krzysztof W. , Lehtimäki Matti , Pan Man‐Juan , Krishnan Usha , Willeke Klaus , Elliott Diane D. , Muth William L.
Published: December 2004

A number of industrial processes require the addition of materials to the fermentation broth that are hazardous to health and environment. Agitation of broths inoculated with microorganisms can potentially release aerosols large enough to carry the microorganisms. The influence of agitation, air flow, and bacterial growth on aerosol size distribution, air flow, and bacterial growth on aerosol size distribution was investigated in an industrial pilot scale fermentor. A decrease in particle concentration was observed with increase in bacterial growth; this change was more pronounced in the size range above 2 μm. The aerosol size distribution was found to be practically independent of air flow rate and agitation rate for sizes less than 2 μm. However, for particles largar than 2 μm, the concentration was found to increase with an increase in air flow rate and agitation rate.
Design and evalution of control strategies for high cell density fermentations
Authors: O'Connor Gregory M. , Sanchez‐Riera Fernando , Cooney Charles L.
Published: December 2004

A methodology for the design and evalution of bioprocess control strategies is presented. The strategies manage nutrient supply with demand and vary with the metabolic condition and phase of fermentation operation. Six carbon source addition strategies are based on different combinations of available measurements; they are described and evaluated under different operating conditions for yeast cultivation. It is concluded that a single control strategy is not the most appropriate under all possible operating conditions. An oxygen uptake rate‐based control strategy performs better with a mean respiratory quotient (RQ) value less than 1.1 during an oxygen limitation than an ethanol control strategy which had a mean RQ of 14. The designed strategies and an approach of applying the strategy that best matches fermentation conditions consistently enables achievement of high cell densities 78.7 g DCW/L and yields 0.50 g DCW/g glucose as the mean values for three fermentations.
Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous‐organic solvent biphasic systems
Authors: Nikolova P. , Ward O. P.
Published: December 2004

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous‐organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell‐free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two‐phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.
Development and experimental evaluation of a steady‐state, multispecies biofilm model
Authors: Rittmann Bruce E. , Manem Jacques A.
Published: December 2004

A steady‐state model for quantifying the space competition in multispecies biofilms is developed. The model includes multiple active species, inert biomass, substrate utilization and diffusion within the biofilm, external mass transport, and detachment phenomena. It predicts the steady‐state values of biofilm thickness, species distribution, and substrate fluxes. An experimental evaluation is carried out in completely mixed biofilm reactors in which slow‐growing nitrifying bacteria compete with acetate‐utilizing heterotrophs. The experimental results show that the model successfully describes the space competition. In particular, increasing acetate concentrations causes NH4+‐N fluxes to decrease, because nitrifiers are forced deeper into the biofilm, where they experience greater mass‐transport resistance.
Polyoxazoline‐Peptide adducts that retain antibody avidity
Authors: Velander William H. , Madurawe Rapti D. , Subramanian Anuradha , Kumar Guneet , Sinai‐Zingde Gurudas , Riffle Judy S. , Orthner Carolyn L.
Published: December 2004

Synthetic polymers have long been used to modify various properties of proteins such as activity and solubility. Polyethylene glycol (PEG) has been widely used to form adducts with enzymes and antibodies. In this study, the polyoxazoline family of water‐soluble polymers was used to synthesize adducts containing a synthetic peptide recognized by a monoclonal antibody (MAb) directed against human protein C (hPC). This is the first application of direct conjugation of unterminated or “living” polymer to a peptide. The avidity of the antibody for the various adducts was characterized with respect to size and hydrophilicity of methyl‐ and ethyl‐substituted polyoxazoline polymers (POX). Avidity of the adducts was not found to be dependent upon the hydrophilicity and was slightly decreased due to polymer modification. The methyl‐POX‐peptide adducts were found to be highly water soluble, while the ethyl‐POX‐peptide adducts showed sporadic problems with aqueous solubility. Because the polymer‐peptide adducts retained avidity for the antibody, polyoxazoline polymers may have potential application to protein‐adduct chemistry.
Factors affecting 2‐hydroxypropiophenone formation by benzoylformate decarboxylase from Pseudomonas putida
Authors: Wilcocks R. , Ward O. P.
Published: December 2004

Benzoylformate (100 mM) was quantitatively converted to the acyloin compound, 2‐hydroxypropiophenone (61.76 mM) and benzaldehyde (38.2 mM) by an enzyme extract from Pseudomonas putida ATCC 12633 in the presence of 1.6M acetaldehyde. Biotransformations were carried out at pH 6.0 and 30°C with an incubation time of 60 min. Activity of the acyloin forming enzyme, benzoylformate decarboxylase, was 1.23 units/mL in the biotransformation mixture. Acyloin formation increased dramatically with pH in the range 4–5 and had a broad activity plateau in the pH range 5–8. A broad temperature optimum for acyloin formation was also observed in the range 20–40°C.
Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: I. Optimization of enzyme environment
Authors: Kamat Sanjay , Barrera Juliet , Beckman J. , Russell Alan J.
Published: December 2004

Biocatalytic transesterification of methylmethacrylate is possible in many different solvents. The reaction rate is readily controlled by variation in solvent physical properties. The reaction proceeds better in hydrophobic solvents, and activity can be restored in hydrophilic solvents by the addition of water. We have now demonstrated that supercritical carbon dioxide is not a good solvent for the reaction between 2‐ethlhexanol and methylmethacrylate. It apperars that the supercritical carbon dioxide may either alter the pH of the microaqueous environment associated with the protein or reversibly form covalent complexes with free amine groups on the surface of the enzyme. Although supercritical carbon dioxide is a poor solvent for acrylate transesterification, many other supercritical fluids (ethane, ethylene, sulfur hexafluoride, and fluoroform) are better than most conventional solvents. In supercritical ethane it is possible to control the activity of the enzyme by changing pressure, and the enzyme appears to follow Michaelis–Menten Kinetics. We find that sulfur hexafluoride, the first anhydrous inorganic solvent in which biocatalytic activity has been reported, is a better solvent than any conventional or supercritical organic fluid tested.