bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal production of glutathione by controlling the specific growth rate of yeast in fed‐batch culture
Authors: Shimizu Hiroshi , Araki Katshuiro , Shioya Suteaki , Suga Ken‐ichi
Published: December 2004

The optimal of the specific growth rate was obtained with simple mathematical model in a yeast fed‐batch cultures. The model was based on the mass balance around the fed‐batch system and the relationship between the specific growth rate, μ, and the specific production rate of glutathione, ρG. The optimal profile of μ was calculated as a bang–bang type, That is μ, should start from the maximum value, μmax and should be kept at μmax; then μ should be switched to μc, which gives a maximum value of ρG. It was proven from the maximum principle that switching was needed only once, with the switching time from μmax to μc depending on the final required glutathione content. Finally, this ideal profile of μ for the maximum production of glutathione was realized by manipulating the substrates feed rate in the fed‐batch culture. Using the extended Kalman filter and a programmed‐controller/feedback‐compensator (PF) system, μ could be controlled at the optimal profile obtained. As a result, the maximum production of glutathione was accomplished fairly successfully. However, further improvement in the controller performance for μ is desired. The control strategy employed here can be applied to other batch reaction processes.
Approximation of continuous growth of Cephalotaxus harringtonia plant cell cultures using fed‐batch operation
Authors: Westgate Paul J. , Curtis Wayne R. , Emery Alden H. , Hasegawa Paul M. , Heinstein Peter F.
Published: December 2004

In Cephalotaxus harringtonia plant cell cultures, periods of batch growth that are limited by hexose uptake are too short to make an accurate estimate of the Monod saturation constant. Continuous cultures are infeasible on a laboratory scale, and semicontinuous cultures require too frequent sampling. Fed‐batch operation, consisting of intermittent removal from a culture that is fed continuously, was investigated as a possible solution to these problems. For a constant feed rate, computer simulations showed that a steady state can be achieved which is useful for studying growth at different specific growth rates. In terms of the dilution rate it was confirmed that the operation is essentially equivalent to continuous culture when the samples represent a small fraction of the total culture volume. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed by a multichannel syringe pump. Results indicate that Monod kinetics based on medium glucose levels cannot adequately describe growth under these conditions. Monod's expression for specific growth rate using internal glucose concentration gives an improved correlation.
Two‐ and three‐phase mixing in a concentric draft tube gas‐lift fermentor
Authors: Kennard Malcolm , Janekeh Massoud
Published: December 2004

Two‐ and three‐phase mixing studies were carried out in a 44‐L concentric draft tube gas‐lift fermentor. It was proposed to use the fermentor for the production of solvents using immobilized bacteria. Bubble size, gas holdup, liquid velocities, circulation, and mixing times were determined for various superficial gas velocities in distilled water, starch, carboxymethyl cellulose, and ethanol solutions. The observed trends for two phase mixing were similar to other studies but the results were found to be more sensitive to liquid properties. This was possibly due to the large value of downcomer to riser area used in this study. Mixing in three phases highlighted the difficulty in predicting the effect of adding solids to the gas‐liquid system. Results showed that the gas‐lift fermentor was ideally suited to dealing with three phases but more work is necessary before accurate models can be developed to account for the effect of solids.
Characterization of an on‐line commercial fluorescence probe: Modeling of the probe signal
Authors: Wang Nam Sun , Simmons Michael B.
Published: December 2004

A biosensor model was developed for a commercial NADH fluorescence probe to describe the single‐frequency excitation and emission fluorescence behavior of an aqueous mixture of fluorophores. This model is essential in correlating the measured signals to the concentrations of fluorescent compounds in a bioreactor. In addition to the concentrations of fluorescent components, the relevant parameters of the model are the absorbance at both the excitation and the emission frequencies by the solvent and other absorbing species, the background signals, the light path length of the bioreactor vessel, the fluorescence yield, and the lampdetector configuration. Due to inner‐filter effects and other interferences, the probe signal is intrinsically nonlinear in both the fluorophore concentration and the path length. An important parameter in the model is the geometric constant, S, which accounts for variations in the monitoring efficiency throughout the sample because fluorescent light is emitted in all directions. Previous models, derived from an unrealistic assumption that fluorescent light is emitted only in one direction parallel to the probe axis, are shown to be seriously deficient. The validity of the model was verified experimentally for a single‐component solution in which both the fluorophore concentration and path length were varied.
Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli
Authors: Diaz‐Ricci J. C. , Regan L. , Bailey J. E.
Published: December 2004

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the “pet” operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild‐type strain (K‐12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.
Antigenic characteristics and stability of microencapsulated Mycoplasma hyopneumoniae vaccine
Authors: Lee C. J. , Tzan Y. L.
Published: December 2004

The in vitro stability (temperature, pH, and trypsin) of Mycoplasma hyopneumoniae antigen (MHA) with and without enteric‐coated microencapsulation were examined. Microencapsulation of MHA with cellulose acetate phthalate (CAP) is an effective route to produce enteric‐coated vaccine microspheres for oral administration. The effect of temperature on the rate of inactivation of MHA was studied by exposing MHA to various temperatures, such as 25, 37, 50 and 60°C. The MHA microspheres were thermally more stable than that of the unencapsulated MHA. The kinetic parameters were observed to follow an Arrhenius‐type temperature dependence. The MHA microspheres were also more stable in acidic regions (pH 1.2–4.0) than that of the free one. The enteric‐coated MHA microspheres exhibited an excellent enteric function to prevent acidic degradation. A model similar to the well‐known Michaelis‐Menten equation was formulated to describe the effect of trypsin on the antigenic degradation of MHA. The equilibrium constant KA and the maximum reaction velocity Vm were obtained from experimental data for both free and microencapsulated MHA. Both KA and Vm values of the microencapsulated MHA were smaller than that of the free one, i.e., the resistance to proteolytic enzyme such as trypsin was enhanced by microencapsulation. The storage stability of enteric‐coated MHA microspheres has been satisfactorily prolonged that they could preserve more than 90% of original antigenicity after 30 days, and over 80% of antigenicity of MHA was retained in the microspheres for 95 days when it was stored at 4°C.
Solute exclusion from cellulose in packed columns: Experimental investigation and pore volume measurements
Authors: Neuman R. P. , Walker L. P.
Published: December 2004

A packed column approach was used in this investigation to determine pore volume and surface area distributions of several celluloses. Specific surface areas for Avicel PH 102, Solka Floc BW 300, and two size fractions of corn cobs that have been pretreated to remove lignin and hemicellulose were measured using this technique. In addition to measuring pore volume and specific surface area, the molecular diameters of several PEGs (polyethylene glycol) were estimated using viscosity measurements. Also, the influence of cellulose particle size, molecular diameter of PEGs, and PEG solution velocity on dispersion and tailing were investigated. Molecular diameter estimates from this investigation were 30%–35% lower than those reported in the literature. This discrepancy is due to earlier investigators using an inappropriate relationship for estimating molecular diameter from viscosity measurements. The precision of the column approach to solute exclusion was higher than that obtained by investigators using a batch approach. Dispersion increased with increasing particle size. Tailing of the elution curve was increased with increasing solute molecular diameter and elution rate. For a cellulase with a molecular diameter of 5.1 nm, estimated specific surface area ranged from 7.2 to 10.5 m2 g−1.
Calculation of thermodynamic properties of protein in Escherichia coli K‐12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis
Authors: Battley Edwin H.
Published: December 2004

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K‐12 protein. These resulted in values fro ΔGf of −38.09 kJ (−9.10 kcal)/ UCFW, for ΔHf of −68.18 kJ (−16.29 kcal)/UCFW, and for ΔSf of −94.2 J (−22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K‐12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for ΔG, 0.75 kJ (0.18 kcal)/UCFW for ΔH, and −4.09 J (−0.98 cal)/UCFW deg for ΔS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.
Automatic containment sampling of recombinant escherichia coli fermentations
Authors: Strudsholm Kim , Emborg Claus , Sigsgaard Poul
Published: December 2004

A containment sampling system for shake flasks and fermentors has been developed from a blood collection system used in hospitals. The core of the system is a collection vial with a vacuum inside. When a needle connected to the fermentation fluid penetrates a rubber seal on the vial, a sample is withdrawn. The system has been developed in two versions, a manual method for shake flasks, and an automated version for fermentors including cool storage of samples. The sampling system offers the same safety for fermentation containment as the original system offers safety for patients and hospital staff. © 1992 John Wiley & Sons, Inc.
A theoretical analysis of cellulase product inhibition: Effect of cellulase binding constant, enzyme/substrate ratio, and β‐glucosidase activity on the inhibition pattern
Authors: Gusakov Alexander V. , Sinitsyn Arkady P.
Published: December 2004

Theoretical analysis of cellulase product inhibition (by cellobiose and glucose) has been performed in terms of the mathematical model for enzymatic cellulose hydrolysis. The analysis showed that even in those cases when consideration of multienzyme cellulase system as one enzyme (cellulase) or two enzymes (cellulase and β‐glucosidase) is valid, double‐reciprocal plots, usually used in a product inhibition study, may be nonlinear, and different inhibition patterns (noncompetitive, competitive, or mixed type) may be observed. Inhibition pattern depends on the cellulase binding constant, enzyme concentration, maximum adsorption of the enzyme (cellulose surface area accessible to the enzyme), the range in which substrate concentration is varied, and β‐glucosidase activity. A limitation of cellulase adsorption by cellulose surface area that may occur at high enzyme/substrate ratio is the main reason for nonlinearity of double‐reciprocal plots. Also, the results of calculations showed that material balance by substrate, which is usually neglected by researchers studying cellulase product inhibition, must be taken into account in kinetic analysis even in those cases when the enzyme concentration is rather low. © 1992 John Wiley & Sons, Inc.
Functional differentiation and primary metabolism of mouse mammary epithelial cells in extended‐batch and hollow‐fiber culture
Authors: Sharfstein Susan T. , Gaillard Béatrice , Blanch Harvey W. , Clark Douglas S.
Published: December 2004

Growth, expression of functional differentiation (as characterized by synthesis and secretion of milk proteins), and primary metabolism were studied for a mouse mammary epithelial cell line, COMMA‐1D, in extended‐batch and hollow‐fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, allowing basal and apical exposure to medium. Protein production was induced in both batch and hollow‐fiber cultures in hormonesupplemented medium. In batch cultures, high levels of protein production and secretion were maintained for 18 days. Once differentiation was induced, the rate of deinduction was low, even in medium containing epidermal growth factor (EGF) and serum; cells continued to express and secrete proteins for at least 12 days after prolactin and hydrocortisone were removed. Cells in both batch and hollow‐fiber cultures were highly glycolytic and exhibited low rates of glutaminolysis. In batch culture on membrane inserts, cells showed polarized metabolism between the apical and basal side, maintaining significant gradients of glucose and lactate. Medium hormonal composition and subsequent differentiation affected both glucose uptake and lactate yield for COMMA‐1D in batch culture. © 1992 John Wiley & Sons, Inc.
Determination of intraparticle immobilized enzyme distribution under moderate diffusion conditions
Authors: Hossain Md. M. , Do D. D.
Published: December 2004

The method Presented earlier by Hossain and Do determine the active enzyme distribution and relevant rate parameters under the condition of strong diffusional resistance is extended in this article to cover the cases of comparable diffusion and reaction rates (3 < ϕ < 20). The theory proposed herein is tested wtih the experimental data of hydrogen perioxide‐catalase immobilized on controlled‐pore glass (CPG) particles of small size (150 μm). © 1992 John Wiley & Sons, Inc.
Application of computer to monitoring and control of fermentation process: Microbial conversion of ML‐236B Na to pravastatin
Authors: Hosobuchi Masahiko , Kurosawa Kazuyuki , Yoshikawa Hiroji
Published: December 2004

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML‐236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3‐hydroxy‐3‐methyglutaryl–coenzyme A reductase (HMG–CoA reductase) used as cholesterol‐lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on‐line monitoring method, which mainly consisted of a cross‐flow filtration module, high‐performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed‐forward control where conversion rate after Δt was estimated by using regression analysis of the five latest values of conversion rate. In a fed‐batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. © 1993 John Wiley & Sons, Inc.
Lipase immobilized on hydrophobic porous polymer supports prepared by concentrated emulsion polymerization and their activity in the hydrolysis of triacylglycerides
Authors: Ruckenstein E. , Wang X.
Published: December 2004

Microporous polymer supports for the immobilization of lipase have been prepared by the polymerization of a concentrated emulsion precursor. The concentrated emulsion consists of a mixture of styrene and divinyl‐benzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. The volume fraction of the latter phase was greater than 0.74, which is the volume fraction of the dispersed phase for the most compact arrangement of spheres of equal radius. The lipase from Candida rugosa has been immobilized on the internal surface of the hydrophobic microporous poly(styrene‐divinyl benzene) supports and used as biocatalysts for the hydrolysis of triacylglycerides. The effects of the amount of surfactant, of the molar ratio of divinylbenzene/styrene in the continuous phase, and of the aquaphilicity of the supports on the adsorption, activity, and stability of the immobilized lipase have been investigated. The microporous poly(styrene‐divinylbenzene) adsorbents constitute excellent supports for lipase because both the amount adsorbed is large and the rate of enzymatic reaction per molecule of lipase is higher for the immobilized enzyme than for the free one. © 1993 John Wiley & Sons, Inc.
Application of continuous zone electrophoresis to preparative separation of proteins
Authors: Nath Sunil , Schütte Horst , Hustedt Helmut , Deckwer Wolf‐Dieter
Published: December 2004

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of β‐galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion‐exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion‐exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 × 10−5 cm2/V · s. © 1993 John Wiley & Sons, Inc.
TCE degradation in a methanotrophic attached‐film bioreactor
Authors: Fennell Donna E. , Nelson Yarrow M. , Underhill Sheila E. , White Thomas E. , Jewell William J.
Published: December 2004

Trichloroethene was degraded in expanded‐bed bioreactors operated with mixed‐culture methanotrophic attached films. Biomass concentrations of 8 to 75 g volatile solids (VS) per liter static bed (Lsb) were observed. Batch TCE degradation rates at 35°C followed the Michaelis–Menten model, and a maximum TCE degradation rate (qmax) of 10.6 mg TCE/gVS · day and a half velocity coefficient (KS) of 2.8 mg TCE/L were predicted. Continuous‐flow kinetics also followed the Michaelis–Menten model, but other parameters may be limiting, such as dissolved copper and dissolved methane—qmax and KS were 2.9 mg TCE/gVS · day and 1.5 mg TCE/L, respectively, at low copper concentrations (0.003 to 0.006 mg Cu/L). The maximum rates decreased substantially with small increases in dissolved copper. Methane consumption during continuous‐flow operation varied from 23 to 1200 g CH4/g TCE degraded. Increasing the influent dissolved methane concentration from 0.01 mg/L to 5.4 mg/L reduced the TCE degradation rate by nearly an order of magnitude at 21°C. Exposure of biofilms to 1.4 mg/L tetrachloroethene (PCE) at 35°C resulted in the loss of methane utilization ability. Tests with methanotrophs grown on granular activated carbon indicated that lower effluent TCE concentrations could be obtained. The low efficiencies of TCE removal and low degradation rates obtained at 35°C suggest that additional improvements will be necessary to make methanotrophic TCE treatment attractive. © 1993 John Wiley & Sons, Inc.
Bioenergetics and end‐product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation
Authors: Hill Paul W. , Klapatch Taryn R. , Lynd Lee R.
Published: December 2004

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation qs(1 − Y)GATP = (μ/Y) + m, with qs the specific rate of xylose consumption (moles xylose/hour · g cells), Y the carbon based cell yield (g cell carbon/g substrate carbon), GATP the ATP gain (moles ATP produces/mol substrate catabolized), μ the specific growth rate (1/h), Y the ATP‐based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour · g cells). Y was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour · g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased GATP by initiating production of the secondary metabolites, propanediol, and in some cases, D‐lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen‐limited continuous free‐cell cultures maintained a constant cell yield, whereas phosphate‐limited continuous free‐cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus‐limited continuous free‐cell cultures exhibited an altered GATP in response to mineral nutrient limitation, and neither produced secondary metabolites. © 1993 John Wiley & Sons, Inc.
Perfusion strategy for rosmarinic acid production by Anchusa officinalis
Authors: Su Wei Wen , Lei Fei , Su Ling Yuan
Published: December 2004

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two‐stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4‐dichlorophenoxyacetic acid (2,4‐D), and 0.1 mg/L kinetin (2,4‐D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone‐free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two‐stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4‐D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single‐stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. © 1993 John Wiley & Sons, Inc.
A two‐plane tubular photobioreactor for outdoor culture of Spirulina
Authors: Torzillo Giuseppe , Carlozzi Pietro , Pushparaj Benjamin , Montaini Elena , Materassi Riccardo
Published: December 2004

A photobioreactor in the form of a 245‐m‐long loop made of plexiglass tubes having an inner diameter of 2.6 cm was designed and constructed for outdoor culture of Spirulina. The loop was arranged in two planes, with 15 8‐m‐long tubes in each plane. In the upper plane, the tubes were placed in the vacant space between the ones of the lower plane. The culture recycle was performed either with two airlifts, one per plane, or with two peristaltic pumps. The power required for water recycle in the tubular photobioreactor, with a Reynolds number of 4000, was 3.93 × 10−2 W m−2. The photobioreactor contained 145 L of culture and covered an overall area of 7.8 m2. The photobioreactor operation was computer controlled. Viscosity measurements performed on Spirulina cultures having different biomass concentrations showed non‐Newtonian behavior displaying decreasing viscosity with an increasing shear rate. The performance of the two‐plane photobioreactor was tested under the climatic conditions of central Italy (latitude 43.8° N, longitude 11.3° E). A biomass concentration of 3.5 g L−1 was found to be adequate for outdoor culture of Spirulina. With a biomass concentration of 6.3 g L−1, the biomass output rate significantly decreased. The net biomass output rate reached a mean value of 27.8 g m−2 d−1 in July; this corresponded to a net photosynthetic efficiency of 6.6% (based on visible irradiance). © 1993 John Wiley & Sons, Inc.
Adsorption of Clostridium thermocellum cellulases onto pretreated mixed hardwood, avicel, and lignin
Authors: Bernardez Timothy D. , Lyford Kimberly , Hogsett David A. , Lynd Lee R.
Published: December 2004

Adsorption of Avicel‐hydrolyzing activity was examined with respect to: mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220°C (PTW220), lignin prepared from PTW220 by either acid or enzymatic hydrolysis, and Avicel. Experiments were conducted at 60°C for all materials, and also at 25°C for PTW220. Based on transient adsorption results and reaction rates, times were selected at which to characterize adsorption at 60°C as follows: PTW220, 1 min; lignin, 30 min; and Avicel, 45 min. Similar results were obtained for adsorption of cellulase activity to PTW220 at 25 and 60°C, and for lignin prepared by enzymatic and acid hydrolysis. For all materials, adsorption was described well by a Langmuir equation, although the reversibility of adsorption was not investigated. Langmuir affinity constants (L/g) were: PTW220, 109; lignin, 17.9; Avicel, 4.3; cellulose from PTW220, ≥187. Langmuir capacity constants were 760 for PTW220 and 42 for Avicel; the cellulase binding capacity of lignin appeared to be very high under the conditions examined, and could not be determined. At low and moderate cellulase loadings at least, the majority of cellulase activity adsorbed to PTW220 is bound to the cellulosic component. The results indicate that PTW220, and its cellulose component in particular, differ radically from Avicel with respect to adsorption. Avicel‐hydrolyzing activity and CMC‐hydrolyzing activities were found to bind to Avicel with a constant ratio of essentially one, consistent with adsorption of a multi‐activity complex. © 1993 John Wiley & Sons, Inc.
Solvent effects on lipase‐catalyzed esterification of glycerol and fatty acids
Authors: Janssen Anja E. M. , Van der Padt Albert , Riet Klaas Van't
Published: December 2004

The lipase‐catalyzed acylglycerol synthesis with fatty acids of different chain length is studied. Measured ester mole fractions at equilibrium are compared with calculated mole fractions. For these calculations the computer program TREP (Two‐phase Reaction Equilibrium Prediction) is used. This program is based on the UNIFAC group contribution method and is developed for nondilute two‐phase reaction systems.

With one set of equilibrium constants, namely 1.3, 0.8, and 0.6 for monoester, diester, and triester synthesis, respectively, the equilibrium position of the reaction between glycerol and all saturated fatty acids with a chain length from 6 to 18 and oleic acid (cis‐9‐octadecenoic acid) can be calculated. Deviations, expressed as the ratio between calculated and measured ester mole fractions, usually were between 0.7 and 1.2. In the presence of solvents, the deviations of the monoester mole fractions were higher and rose up to 3. Without addition of a solvent, the ester mole fractions at equilibrium are dependent on the fatty acid chain length. With the short‐chain hexanoic acid, the monoester mole fraction is the highest ester mole fraction, while for the long‐chain oleic acid, the diester mole fraction is the highest one. The ester mole fractions become independent on the chain length of the fatty acid with a solvent added in a sufficient high concentration. Both reactions, with saturated and unsaturated C18 fatty acids, lead to the same equilibrium position. The program TREP is found to make good predictions of the equilibrium amounts of ester and fatty acid. However, systematic deviations arise between measured and calculated amounts of water and glycerol in the organic phase. The calculated water and glycerol amounts are always lower than the measured ones. These deviations seem to be highest in nonpolar media and are probably due to deficiencies in the UNIFAC calculation method. Some preliminary experiments show the effect of the choice of solvent on the reaction rates. In polar solvents, the monoester production rate is enhances by a factor of 1.5 as compared to the reaction rate in a system without solvent. © 1993 John Wiley & Sons, Inc.
Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon
Authors: Mermelstein Lee D. , Papoutsakis Eleftherios T. , Petersen Daniel J. , Bennett George N.
Published: December 2004

The ability to genetically alter the product‐formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the “ace operon”) in which the three homologous acetone‐formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA‐transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid‐free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22‐fold lower mass of residual carboxylic acids. At all pH values, the acetone‐formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid‐control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid‐borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA‐transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid‐control strain. © 1993 John Wiley & Sons, Inc.
Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling
Authors: Colomban Agnès , Roger Loïc , Boyaval Patrick
Published: December 2004

This article deals with the production by fermentation of a mycostatic and aromatic food additive based on propionic acid. Membrane bioreactors have been used from laboratory scale up to pilot and industrial production plants. Due to the high cell densities achieved by the sequential recycling mode of operation, a mixed acids solution was rapidly produced from whey permeate. The sterile fermented broth obtained was subsequently concentrated at different levels by evaporation and spray drying according to the projected use. Concentrated Propionibacterium cells (200 g · L−1 DW) were obtained from the process by periodic bleeds and could be used to good effect as cheese starters, silage preservatives, or probiotics. Propionic acid concentrations from 30 to 40 g · L−1 were easily achieved with no residual lactose. The highest volumetric productivity was 1.6 g · L−1 · h−1 for total acid and 1.2 g · L−1 · h−1 for propionic acid with a specific productivity of 0.035 h−1. © 1993 John Wiley & Sons, Inc.
Protein adsorption in polysulfone hollow fiber bioreactors used for serum‐free mammalian cell culture
Authors: Patkar Anant Y. , Bowen Bruce D. , Piret James M.
Published: December 2004

The recovery of serum‐free medium proteins from poly‐sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre‐adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA‐coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA‐coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30‐kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen. © 1993 John Wiley & Sons, Inc.
Linear constraint relations in biochemical reaction systems: I. Classification of the calculability and the balanceability of conversion rates
Authors: van der Heijden R. T. J. M. , Heijnen J. J. , Hellinga C. , Romein B. , Luyben K. Ch. A. M.
Published: December 2004

Measurements provide the basis for process monitoring and control as well as for model development and validation. Systematic approaches to increase the accuracy and credibility of the empirical data set are therefore of great value. In (bio)chemical conversions, linear conservation relations such as the balance equations for charge, enthalpy, and/or chemical elements, can be employed to relate conversion rates. In a pactical situation, some of these rates will be measured (in effect, be calculated directly from primary measurements of, e.g., concentrations and flow rates), as others can or cannot be calculated from the measured ones. When certain measured rates can also be calculated from other measured rates, the set of equations, the accuracy and credibility of the measured rates can indeed be improved by, respectively, balancing and gross error diagnosis. The balanced conversion rates are more accurate, and form a consistent set of data, which is more suitable for further application (e.g., to calculate nonmeasured rates) than the raw measurements. Such an approach has drawn attention in previous studies. The current study deals mainly with the problem of mathematically classifying the conversion rates into balanceable and calculable rates, given the subset of measured rates. The significance of this problem is illustrated with some examples. It is shown that a simple matrix equation can be derived that contains the vector of measured conversion rates and the redundancy matrix R. Matrix R plays a predominant role in the classification problem. In supplementary articles, significance of the redundancy matrix R for an improved gross error diagnosis approach will be shown. In addition, efficient equations have been derived to calculate the balanceable and/or calculable rates. The method is completely based on matrix algebra (principally different from the graph‐theoretical approach), and it is easily implemented into a computer program. © 1994 John Wiley & Sons, Inc.
Linear constraint relations in biochemical reaction systems: II. Diagnosis and estimation of gross errors
Authors: van der Heijden R. T. J. M. , Romein B. , Heijnen J. J. , Hellinga C. , Luyben K. Ch. A. M.
Published: December 2004

Conservation equations derived from elemental balances, heat balances, and metabolic stoichiometry, can be used to constrain the values of conversion rates of relevant components. In the present work, their use will be discussed for detection and localization of significant errors of the following types:

At least one of the primary measurements has a significant error (gross measurement error).

The system definition is incorrect: a component

is not included in the system description.

has a composition different from that specified.

The specified variances are too small, resulting in a too‐sensitive test.

The error diagnosis technique presented here, is based on the following: given the conservation equations, for each set of measured rates, a vector of residuals of these equations can be constructed, of which the direction is related to the error source, as its length is a measure of the error size. The similarity of the directions of such a residual vector and certain compare vectors, each corresponding to a specific error source, is considered in a statistical test. If two compare vectors that result from different error sources have (almost) the same direction, errors of these types cannot be distinguished from each other. For each possible error in the primary measurements of flows and concentrations, the compare vector can be constructed a priori, thus allowing analysis beforehand, which errors can be observed. Therefore, the detectability of certain errors likely to occur can be insured by selecting a proper measurement set. The possibility of performing this analysis before experiments are carried out is an important advantage, providing a profound understanding of the detectability of errors. The characteristics of the method with respect to diagnosis of simultaneous errors and error size estimation are discussed and compared to those of the serial elimination method and the serial compensation strategy, published elsewhere. © 1994 John Wiley & Sons, Inc.
Protein fractionation using fast flow immobilized metal chelate affinity membranes
Authors: Serafica Gonzalo C. , Belfort Georges , Pimbley Joseph
Published: December 2004

A new group‐specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified “active surfaces” were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu–IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.

The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.

The results presented here represent the development of a new fast flow affinity membrane process–immobilized metal affinity membranes (IMAM). © 1994 John Wiley & Sons, Inc.
Mini‐review: Osteoblasts: An in vitro model of bone‐implant interactions
Authors: Bizios Rena
Published: December 2004

Well‐characterized osteoblasts provide a successful in vitro model to study bone‐biomaterial interactions. Knowledge of the events occurring at this tissue‐biomaterial interface could lead to the design of improved orthopedic/dental biomaterials which elicit specific and desirable responses from surrounding cells/tissues, optimize function of osteoblasts (the boneforming cells), and enhance long‐term bone‐implant bonding. © 1994 John Wiley & Sons, Inc.
Homogenization theory and digital imaging: A basis for studying the mechanics and design principles of bone tissue
Authors: Hollister S. J. , Kikuchi N.
Published: December 2004

Bone tissue is a complex multilevel composite which has the ability to sense ad respond to its mechanical environment. It is believed that bone cells called osteocytes within the bone matrix sense the mechanical environment and determine whether structural alterations are needed. At present it is not known, however, how loads are transferred from the whole bone level to cells. A computational procedure combining representative volume element (RVE) based homogenization theory with digital imaging is proposed to estimate strains at various levels of bone structure. Bone tissue structural organization and RVE based analysis are briefly reviewed. The digital image based computational procedure was applied to estimate strains in individual trabeculae (first‐level microstructure). Homogenization analysis of an idealized model was used to estimate strains at one level of bone structure around osteocyte lacunae (second‐level trabecular microstructure). The results showed that strain at one level of bone structure is amplified to a broad range at the next microstructural level. In one case, a zeor‐level tensile principal strain of 495 μE engendered strains ranging between ‐1000 and 7000 μE in individual trabeculae (first‐level microstructure). Subsequently, a first‐level tensile principal strains of 1325 μE within an inidividual trabecula engendered strains ranging between 782 and 2530 μE around osteocyte lacunae. Lacunar orientation was found to influence strains around osteocyte lacunae much more than lacunar ellipticity. In conclusion, the computational procedure combining homogenization theory with digital imaging can proveide estimates of cell level strains within whole bones. Such results may be used to bridge experimental studies of bone adaptation at the whole bone and cell culture level. © 1994 John Wiley & Sons, Inc.
Fed‐batch operation of recombinant Escherichia coli containing trp promoter with controlled specific growth rate
Authors: Yoon Sung Kwan , Kang Whan Koo , Park Tai Hyun
Published: December 2004

A feb‐batch operation for the production of bovine somatotropin (bST) under the control of tryptophan promoter in Escherichia coli was investigated. The plasmid used contains a two‐cistron system and altered codon usage for higher expression of bST. Specific growth rate is an important parameter in the fermentation, because it affects the production of growth‐inhibitory organic acids and the expression of recombinant protein. The feeding rate was adjusted to keep the specific growth rate constant in this study. The variable growth yield expressed as a function of time was used for the calculation of the feeding rate. The growth yield decreases during the fermentation as product expression is induced. The specific growth rate was well controlled; however, intracellular bST concentration decreased at high cell concentrations. This is considered to be due to degradation by proteases. The decrease was prevented by an exponential feeding of the yeast extract as an organic nitrogen source. © 1994 John Wiley & Sons, Inc.