bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger
Authors: Grous William , Converse Alvin , Grethlein Hans , Lynd Lee
Published: December 2004

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary1 to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C‐30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high‐ and low‐concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.
Isomaltulose production using immobilized cells
Authors: Chhetham Peter S. J. , Garrett Christine , Clark Jeremy
Published: December 2004

Three strains of Erwinia rhapontici especially suitable for use in the form of nongrowing immobilized cells were selected by screening strains of cells for high activity and operational stability in an immobilized form. Immobilization in calcium alginate gel pellets was easily the best method of immobilizing E. rhapontici. Much greater operational stabilities were obtained than when other immobilization methods were used. Conditions of operation which optimize the activity, stability, and yield and the ease of operation of the immobilized cell columns working in a steady state are described. These include the effects of substrate concentration, diffusional restrictions and water activity, the concentration of cells immobilized, and the type of reactor used. Thus, the immobilized cells produce about 1500 times their own weight of isomaltulose during one half‐life of use (ca. 1 year). Loss of activity was most closely correlated with the volume of substrate processed and so presumably is due to the presence of low concentrations of a cummulative inhibitor in the substrate. Methods for regenerating the activity of the immobilized cells by the periodic administration of nutrients, of forming isomaltulose by continuously supplying nutrients to growing immobilized cells, and of crystallizing isomaltulose from the column eluate are also described.
Purification and characterization of endo‐xylanases from Aspergillus niger. III. An enzyme of pl 3.65
Authors: Ricardo Fournier A. , Frederick Mary M. , Frederick James R. , Reilly Peter J.
Published: December 2004

An endo‐xylanase (1,4‐β‐D‐xylan xylanohydrolase, EC 3.2.1.8) from Aspergillus niger was purified to homogeneity by chromatography with Ultrogel AcA 54, SP‐Sephadex C‐25 at pH 4.5, DEAE‐Sephadex A‐25 at pH 5.4, Sephadex G‐50, and DEAE‐Sephadex A‐25 at pH 5.15. The enzyme was active on soluble xylan, on insoluble xylan only after arabinosyl‐initiated branch points were removed, and on xylooligosaccharides longer than xylotetraose. There was slight activity on carboxymethyl‐cellulose, arabinogalactan, glucomannan, and p‐nitrophenyl‐β‐D‐glucopyranoside. The main products of the hydrolysis of soluble and insoluble xylan were oligosaccharides of intermediate length, especially the tri‐ and pentasaccharides. The isoelectric point of the enzyme was 3.65. It had a molecular weight of 2.8 × 104 by SDS‐gel electrophoresis, and was high in acidic amino acids but low in those containing sulfur. Highest activity in a 20‐min assay at pH 5 was between 40 and 45°C, with an activation energy up to 40°C of 11.1 kJ/mol. The optimum pH for activity was at 5.0. The enzyme was strongly activated by Ca2+.
Kinetic characterization of the extracellular xylanases of Thermomonospora sp.
Authors: Ristroph D. L. , Humphrey A. E.
Published: December 2004

This study deals with characterizing the extracellular xylanases produced by a strain of the thermophilic bacterial genus Thermomonospora. Supernatant from centrifuged fermentation broth was used as a crude enzyme preparation. From pH 5.5 to pH 7.7 the temperature optimum based on a 10‐min assay of activity was 80°C. The crude enzyme had a half‐life of approximately 1 month when stored at 55°C at pH 6.5. The enzyme produced a mixture of xylose oligomers from xylan, with xylobiose occuring in greatest quantity on a molar basis. Only trace quantities of xylose were produced by this hydrolysis.
Application of L‐(+)‐Lactate electrode for clinical analysis and monitoring of tissue culture medium
Authors: Tsuchida T. , Takasugi H. , Yoda K. , Takizawa K. , Kobayashi S.
Published: December 2004

L‐(+)‐Lactate oxidase (EC 1.1.3.2) was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide. The lactate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. Properties of the enzyme membrane and characteristics of the lactate electrode were clarified for the determination of L‐(+)‐lactic acid. The lactate electrode responded linearly to L‐(+)‐lactic acid over the final concentration 0‐0.25 mmol/L within 30 s. When the enzyme electrode was applied to the determination of L‐(+)‐lactic acid in control serum, within‐day precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.4% with a mean value of 4.54 mmol/L, 98.0%, and 0.986, respectively. The lactate electrode was sufficiently stable to perform 1040 assays over 13 days operation for the determination of L‐(+)‐lactic acid. The dried immobilized enzyme membrane retained 84% of its initial activity after storage at 4°C for 12 months. Moreover, the enzyme electrode was applied to the monitoring of culture medium for human melanoma cells. L‐(+)‐Lactate production and D‐glucose consumption were closely related to cell numbers.
Fundamental studies of glucose oxidase immobilization on controlled pore glass
Authors: Hossain Md. M. , Do D. D.
Published: December 2004

Immobilization experiments have been performed with glucose oxidase as enzyme and controlled‐pore glass of different pore sizes as support for chemical coupling. The experimental results have been analyzed for comparison with the theoretical model predictions. Analysis of the initial stage of the process gives the fundamental characteristic of the immobilization reaction. These investigations allow us to study the influence of the degree of diffusional restriction on the evolution of the immobilization process and spatial distribution of immobilized enzyme. Nonuniformly distributed concentrations have been achieved within the porous matrix, and suggestions have been made in designing such profiles by choosing appropriate experimental parameters.
Liquid level control in an aerated continous‐flow fermentor
Authors: Anderson K. W. , Grulke E. , Gerhardt Philipp
Published: December 2004

An electronic liquid level control system was developed to maintain constant working volume in a continuously fed and aerated fermentor from which spent medium was continously withdrawn by microfiltration in an external recycle loop. The level control system was comprised of an admittance sensor in the fermentor and an external transmitter and controller, which together regulated the speed of a recycle pump and thus the rate of liquid withdrawal from the fermentor. During test bacterial culture the liquid level was maintained usually within ± 1% and always within ± 5%. The control system could be applied to various other types and scales of continuous‐flow fermentors.
Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri
Authors: Nam Doo H. , Kim Cheol , Ryu Dewey D. Y.
Published: December 2004

In enzymatic synthesis of cephalexin from D‐α‐phenylglycine methyl ester (PGM) and 7‐amino‐3‐deacetoxy‐cephalosporanic acid (7‐ADCA) using α‐acylamino‐β‐lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl‐enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.
Simulation of the growth pattern of a single cell of Escherichia coli under anaerobic conditions
Authors: Ataai M. M. , Shuler M. L.
Published: December 2004

A computer model based on a previous model for aerobic growth of Escherichia coli is described which simulates cell composition, size, and shape; length of C and D periods; cell yields; and the rate of product formation of anaerobically grown cells of E. coli B/r‐A on glucose‐limited minimal medium. To verify the simulation results, the values of cell volume, cell content of DNA, RNA, and protein, substrate yield, ATP yield, and fermentation products for various growth rates were obtained experimentally. Model predictions are in good agreement with experimental results. Such agreement supports a hypothesis that only those equations describing energy metabolism need to be modified and other cell functions are not grossly altered by a switch from aerobic to an aerobic growth. The model's ability to predict reasonable growth responses under anaerobic conditions by only modifying energy metabolism is a further indication of the robust nature of the description of cell physiology included in the development of the aerobic model.
Enzyme reactor with knitted fabric made of poly(vinyl alcohol) superfine filaments
Authors: Ichijo Hisao , Suehiro Tetsuro , Nagasawa Junichi , Yamauchi Aizo , Sagesaka Michiaki
Published: December 2004

Various supports and bio‐reactors have been proposed. Packed bed reactors with polymer material in granular shape are most often employed in both laboratory and industry. But they have a disadvantage related to an increase in pressure drop. We already developed filter paper composed of short cut pieces of superfine filaments (SFF). It shows high performance, but its hydrodynamic resistance increases when substrate solution passes through it. A new type of enzyme reactor equipped with knitted SFF has been proposed. In this reactor, substrate does not pass through the support but flows along the thin channel and parallel to the support. Therefore, it is able to maintain flow rate constant during a considerable period. The productivity of the reactor fairly increases by reducing the thickness of the channel because linear velocity increases with the reduction of the thickness and that contributes to the decrease in mass transfer resistance.
Effect of ultrasound treatment on the extraction of insulin
Authors: Zayas Joseph
Published: December 2004

Insulin production requires a prolonged extraction process. An ultrasound method of insulin extraction was developed to increase efficiency. Application of ultrasound resulted in a significant increase in the extraction process as a result of pancreas tissue dispergation, destruction of the pancreas cells, intensive mixing, separation of particles, and increase in the interphase pancreas‐extractive medium. Optimal technological parameters of insulin extraction were established: ultrasound frequency, 19.5 kHz, specific intensity of ultrasound, 3.3 W/cm2; time of ultrasound exposure, 5–10 min; and influence of cavitation process. Effectiveness of insulin extraction depends on the properties of the extractive medium. Optimal conditions were pH, 1.6–1.7, concentration of alcohol, 75%, temperature of extractive mixture, 13–17°C; ratio of ground pancreas to liquid extractive medium, 1 : 3.
Induction of Cellulases in chaetomium cellulolyticum by cellobiose
Authors: Leisola Matti S. A. , Ulmer Duane C. , Pitkänen Kari , Fiechter Armin
Published: December 2004

The production of extracellular cellulases by Chaetomium cellulolyticum could be induced by slow feeding of cellobiose to the cultures. Both the rate of production and the amount of activity were comparable to that obtained in batch cultivation on cellulose. The specific filter paper activity of 2.06 U per mg protein was almost two times higher than that obtained in cellulose medium. Cellulases were not induced when glucose was slowly fed to the cultures. Changing the feed stream from glucose to cellobiose resulted in a rapid accumulation of cellulases. Thus cellobiose has a similar role in cellulase induction in C. cellulolyticum, as earlier shown for Trichoderma reesei.
Urokinase bound to fibrocollagenous tubes: An in vitro kinetic study
Authors: Senatore F. F. , Bernath F. R.
Published: December 2004

Urokinase (UK) has been immobilized to the inner surfaces of fibrocollagenous tubes (FCT) in an attempt to develop a fibrinolytic biomaterial which may be suitable for use as a small diameter vascular prosthesis. The enzyme was bound by adsorption followed by glutaraldehyde crosslinking. An in virto kinetic study of immobilized urokinase was conducted by employing the tubular material as a flow through reactor operated in a batch recycle mode in which the esterolysis of the model substrate, N‐α‐acetyl‐L‐lysine methyl ester (ALME), was monitored as a function of substrate concentration, recycle flow rate, and temperature. Results were compared with data from the soluble enzyme reaction, which was conducted in the presence and absence of 10% swine skin gelatin, in order to identify the specific effects of a collagenous microenvironment. Observed rates for the UK‐FCT catalyzed reaction were observed to be dependent on recycle flow rates below 12 mL/min (Re = 107). Apparent Michaelis–Menten rate parameters were determined by a nonlinear search technique for two flow rates: one above the critical point for external diffusion effects (Re = 282) and one within the mass‐transfer‐limited region (Re = 71). When the latter data were corrected for external diffusion by applying the Graetz correlation for laminar flow in tubes to estimate themass transfer coefficient, the corrected Km of 6.45 ± 0.38 mM agreed very closely with the diffusion free parameter (i.e. 6.13 ± 0.63). Furthermore, this value was observed to be an order of magnitude higher than that of the soluble enzyme but approximately equal to the Km of the soluble enzyme in a 10% gelatin environment (8.13 ± 1.53 mM). It is postulated that the difference in kinetic parameters between soluble and collagen immobilized UK is due to an inherent interaction between collagen and enzyme rather than to mass transfer effects. Such aninteraction is supported by the effects of collagen on thermal stability and energy of activation.
Fermentation to ethanol of pentose‐containing spent sulphite liquor
Authors: Yu Shiyuan , Wayman Morris , Parekh Sarad K.
Published: December 2004

Ethanolic fermentation of spent sulphite liquor with ordinary bakers' yeast is incomplete because this yeast cannot ferment the pentose sugars in the liquor. This results in poor alcohol yields, and a residual effluent problem By using the yeast Candida shehatae (R) for fermentation of the spent sulphite liquor from a large Canadian alcohol‐producing sulphite pulp and paper mill, pentoses as well as hexoses were fermented nearly completely, alcohol yields were raised by 33%, and sugar removal increased by 46%. Inhibitors were removed prior to fermentation by steam stripping. Major benefits were obtained by careful recycling of this yeast, which was shown to be tolerant both of high sugar concentrations and high alcohol concentrations. When sugar concentrations over 250 g/L (glucose: xylose 70:30) were fermented, ethanol became an inhibitor when its concentration reached 90 g/L. However, when the ethanol was removed by low‐temperature vacuum distillation, fermentation continued and resulted in a yield of 0.50 g ethanol/g sugar consumed. Further improvement was achieved by combining enzyme saccharification of sugar oligomers with fermentation. This yeast is able to ferment both hexoses and pentoses simultaneously, efficiently, and rapidly. Present indications are that it is well suited to industrial operations wherever hexoses and pentoses are both to be fermented to ethanol, for example, in wood hydrolysates.
Deterministic interpretation of the temperature response of microbial growth
Authors: Fiolitakis Emmanuel , Grobbelaar Johan U. , Hegewald Eberhard , Soeder Carl J.
Published: December 2004

A deterministic approximation of the thermal deactivation process is undertaken by structuring growth kinetics of cell populations on the activity level. Growth data of the green microalga Scenedesmus obliquus under forced periodic conditions of light intensity have been used for model verification. The model considers a straightforward extension of normal growth kinetics in such a way that different growth activities can be adjusted in the context of a thermal damage and repair mechanism. The nonlinear fitting of the measured response renders parameter values as a function of the light availability per unit biomass.
Selectivity enhancement of an Escherichia coli bacterial electrode using enzyme and transport inhibitors
Authors: Corcoran C. A. , Kobos R. K.
Published: December 2004

The feasibility of using specific enzyme and transport inhibitors to minimize the glutamine response of a potentiometric microbial sensor is demonstrated. The glutamine response of a bacterial electrode prepared with Escherichia coli as the biocatalyst in conjunction with an ammonia gas‐sensing electrode was greatly reduced by treating the electrode with the enzyme inhibitor 6‐diazo‐5‐oxo‐L‐norleucine (DONL) and the transport inhibitor γ‐L‐glutamylhydrazide. Each inhibitor effectively decreased glutamine response to a level sufficiently low to be considered negligible in clinical studies. Although the sensor ultimately recovered from the effects of a single exposure to an inhibitor, continuous exposure at an optimum concentration maintained a low response to glutamine. Furthermore, the treatment of the sensor with both inhibitors simultaneously resulted in a negligible response to glutamine of <1 mV, indicating that both inhibitors are necessary for optimum inhibition of glutamine response. This approach is promising as a means of enhancing the selectivity of microbial sensors.
Production of biosurfactants from sugar alcohols and vegetable oils catalyzed by lipases in a nonaqueous medium
Authors: Chopineau Joel , McCafferty Frank D. , Therisod Michel , Klibanov Alexander M.
Published: December 2004

Porcine pancreatic and Chromobacterium viscosum lipases catalyze transesterification reactions between a number of sugar alcohols and various plant and animal oils in dry pyridine. The products of this process have been identified as primary monoesters of sugar alcohols and fatty acids. These enzymatically prepared sugar alcohol esters have been found to be excellent surfactants in terms of their ability to reduce interfacial and surface tensions and to stabilize emulsions.
Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads
Authors: Martinsen A. , Skjåk‐Bræk G. , Smidsrød O.
Published: December 2004

Calcium alginate gel beads were prepared from a range of well characterized alginates. The physical properties of beads depended strongly on the composition, sequential structure, and molecular size of the polymers. Beads with the highest mechanical strength, lowest shrinkage, best stability towards monovalent cations, and highest porosity were made from alginate with a content of L‐guluronic acid higher than 70% and an average length of the G‐blocks higher than 15. For these “high G” alginates the critical overlap intrinsic viscosities have been determined, and for molecular weight higher than 2.4 × 105, the gel strength was independent of the molecular weight.
An algorithm for operating a fed‐batch fermentor at optimum specific‐growth rate
Authors: Agrawal Pramod , Koshy George , Ramseier Michael
Published: December 2004

An algorithm for operating a fed‐batch fermentor at an optimum specific fermentation rate is proposed. It does not require on‐line measurement of nutrient concentration in the culture medium. An on‐line estimate of the specific fermentation rate is sufficient for implementation of this scheme. The algorithm is model independent and works well even with poor estimates of the product yields and the specific fermentation rate. Results of a detailed simulation study are presented for a simple case of optimization of cell‐mass production in a fed‐batch fermentor. The results clearly demonstrate the efficacy of this algorithm under a wide range of fermentation situations.
Effect of surface potential on consumption of charged substrates
Authors: Skowlund Christopher T. , Kirmse Dale W.
Published: December 2004

Due to the surface charge inherent on all microorganism cell surfaces, the consumption of charged molecules by these microorganisms will be affected by the electrostatic interactions between the substrate and the cell surface. This article derives an equation that shows that these electrostatic interactions result in a change in the Monod half‐velocity constant. The resulting rate of consumption is affected and can be either higher or lower than the rate of consumption of an uncharged substrate molecule.
Study of gaseous substrate fermentations: Carbon monoxide conversion to acetate. 2. Continuous culture
Authors: Vega J. L. , Antorrena G. M. , Clausen E. C. , Gaddy J. L.
Published: December 2004

The fermentation of gaseous substrates such as CO, H2, and CO2 may be performed in a continuous stirred tank reactor, as well as the traditional batch reactor. In this article, the conversion of carbon monoxide by Peptostreptococcus productus is demonstrated in a stirred tank reactor under both mass transfer‐controlled and nonmass transfer‐controlled conditions. Utilizing a non‐steady‐state procedure, intrinsic rates are evaluated under non‐mass transfer‐controlled conditions in a time period of only 5–6 hours. A steady‐state procedure was used to evaluate CSTR performance under mass transfer‐controlled conditions. The mass transfer coefficient was calculated, followed by the development of a model to predict CSTR behavior for this gas phase substrate.
Effects of pH and added metabolites on bioconversions by immobilized non‐growing Clostridium acetobutylicum
Authors: Reardon Kenneth F. , Bailey James E.
Published: December 2004

The bioconversion activity of calcium alginate‐immobilized Clostridium acetobutylicum ATCC 824 was investigated in a continuous reactor system utilizing a defined feed medium which did not support cell growth. The changes in biocatalytic activity with time were studied at different pH values as well as when different metabolites (butyric, acetic, and acetoacetic acids) were present in the feed stream. Although the nongrowing cells were metabolically active, the product distribution was shifted from solvent production to acidogenesis. Overall activity losses occurred due to cell lysis, sporulation, and the effects of nitrogen level on macromolecular turnover. These effects were minimized under some operating conditions (e.g., pH 6), resulting in significantly longer productivity lifetimes.
Effect of induction temperature on the production of malaria antigens in recombinant E. coli
Authors: Okita B. , Arcuri E. , Turner K. , Sharr D. , Del Tito B. , Swanson J. , Shatzman A. , Zabriskie D.
Published: December 2004

As part of a process development campaign, studies have been conducted to determine the influence of induction temperature on the expression of two different malaria antigens, RN1 and RT2. Single‐step temperature inductions, in which growth at 32.0°C is followed by a shift in temperature to a desired setpoint, show that there exists an optimum duration and temperature of induction which is product specific. Between an induction temperature of 39.5 and 44.5°C RN1 yield is constant at ca. 0.20 g/g total soluble protein (TSP). RT2 yield approaches 0.20 g/g TSP only at elevated induction temperatures. The optimum temperature of induction for RN1 production is 39.5°C, whereas, that for RT2 production is 41.0°C. Above the optimum temperature of induction antigen concentration decreases owing to decreases in biomass. Furthermore, the maximum concentration of these two antigens differ by a factor of four. With increasing temperature of induction the extent of proteolysis of the products also appears to increase.
Performance of the anaerobic baffled reactor under steady‐state and shock loading conditions
Authors: Grobicki A. , Stuckey D. C.
Published: December 2004

The anaerobic baffled reactor (ABR) contains a granulated, mixed anaerobic culture segregated into compartments. Operation of four reactors under a range of hydraulic retention times showed that this novel reactor design offers highly efficient performance in the conversion of carbon in the feed stream to methane and carbon dioxide. The design parameter varied was the number of compartments. COD removal at 20 h retention time was routinely over 95% in all reactors, with low washout of biomass. Very high specific reaction rates were achievable (although with a loss of efficiency) at low biomass concentrations and high loading rates. In order to optimize volumetric reaction rates, a tradeoff has to be made between high biomass concentration, granule size, and the resulting mass transfer limitations. Formate is shown to be an important intermediate in the process under conditions of high loading.
Measurement of bacterial random motility and chemotaxis coefficients: II. Application of single‐cell‐based mathematical model
Authors: Ford Roseanne M. , Lauffenburger Douglas A.
Published: December 2004

A quantitative description of bacterial chemotaxis is necessary for making predictions about the migratory behavior of bacterial populations in applications such as biofilm development, release of genetically engineered bacteria into the environment, and in situ bioremediation technologies. The bacterial chemotactic response is characterized by a mathematical model which relates individual cell properties such as swimming speed and tumbling frequency to population parameters, specifically the random motility coefficient and the chemotactic sensitivity coefficient. Our model includes a nonlinear dependence of the chemotactic velocity on the attractant gradient as well as a dependence of the random motility coefficient on the temporal and spatial attractant gradients, both of which previous analyses have neglected. As we will show, these aspects are critical for interpreting the results from experiments like those performed in the stopped‐flow diffusion chamber (SFDC) because the initial temporal and spatial gradients are very steep. Our analysis demonstrates that values for the random motility coefficient and chemotactic sensitivity coefficient can be obtained from experimental plots of net cell redistribution from initial conditions versus the square root of time. Values for these parameters are determined from experimental measurements of bacterial population distributions in the SFDC as described in the companion article. Using parameter values determined from independent experiments, μ = 1.1 ± 0.4 ± 10−5 cm2/s and χ0 = 8 ± 3 ± 10−5 cm2/s, excellent agreement is found between theoretically predicted bacterial density profiles and actual experimental profiles for Escherichia coli K12 responding to fucose over two orders of magnitude in initial attractant concentration. Thus, our model captures the concentration dependence of this behavioral response satisfactorily in terms of cell population parameters which are derived from individual cell properties and will therefore be useful for making predictions about the migratory behavior of bacterial populations in the environment.
Mass production of human epidermal growth factor using fed‐batch cultures of recombinant Escherichia coli
Authors: Shimizu Norio , Fukuzono Shinichi , Harada Yoshinori , Fujimori Kiyoshi , Gotoh Kenichi , Yamazaki Yoshio
Published: December 2004

Fed‐batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N‐terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N‐terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed‐batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate‐induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.
Cleaning of inorganic membranes after whey and milk ultrafiltration
Authors: Daufin G. , Merin U. , Labbé J. P. , Quémerais A. , Kerhervé F. L.
Published: December 2004

Cleaning of an inorganic ultrafiltration membrane has been quantified through hydraulic, physicochemical, and spectroscopic (infrared and x‐photoelectron spectroscopy) analyses. An efficient cleaning sequence of nitric acid followed by sodium hypochlorite has been proposed for cleaning of defatted whey protein concentrate and milk ultrafiltration membranes. The influence of reversed sequence and time reduction are discussed together with the action of both cleaning chemicals. In spite of residual fouling left after every cleaning sequence studied, hydraulic cleanliness of the membrane was achieved, particularly after the standard procedure.
Hepatocyte culture on biodegradable polymeric substrates
Authors: Cima Linda G. , Ingber Donald E. , Vacanti Joseph P. , Langer Robert
Published: December 2004

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L‐lactic‐co‐glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L‐lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen‐coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion–the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140–160 μg/106 cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen‐coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen‐coated polystyrene, a substrate optimal for DNA synthesis.
Comments on the communication: On the calculation of shear rate and apparent viscosity in airlift and bubble column bioreactors
Authors: Allen D. Grant , Robinson Campbell W.
Published: December 2004

The determination of the shear rate in bubble column and airlift bioreactors is an important question from both the perspective of cell damage and the correlation of hydrodynamic parameters in non‐Newtonian fluids in these contractors. In the context of correlating hydrodynamic parameters in non‐Newtonian fluids, a common approach involves assuming that there exists an average shear rate in the column that is proportional to the superficial gas velocity. This average shear rate is then used to evaluate an effective viscosity of the non‐Newtonian fluid that is subsequently used to quantify the fluid's rheological behavior in correlation. Contrary to a recent communication, this report illustrates that this approach, which has mainly been applied to bubble columns, can also be applied to external loop airlift contractors, replacing the superficial gas velocity by the superficial gas velocity by the superficial gas velocity supplied to the riser of the contractor. This extension is based upon consideration of the relevant characteristic velocity in the active zone (i.e., the riser section) of the reactor.
A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase
Authors: Park Je‐Kyun , Ro Hyeon‐Su , Kim Hak‐Sung
Published: December 2004

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose–fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor‐connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady‐state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35°C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady‐state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.
A model for light‐limited continuous cultures: Growth, shading, and maintenance
Authors: Evers E. G.
Published: December 2004

Light‐limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up‐take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light‐limited chemostats. The model fits well to experimental data from literature on light‐limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light‐limited continuous cultures.
Use of coimmobilized biological systems to degrade toxic organic compounds
Authors: Lin Jian‐Er , Wang Henry Y. , Hickey Robert F.
Published: December 2004

The concept of coimmobilizing cell mass (and/or enzyme) and adsorbent in a hydrogel matrix for biodegradation of toxic organic chemicals was introduced. Under defined experimental conditions, the coimmobilized system using activated carbon and Phanerochaete chrysosporium was compared with nonimmobilized systems for the degradation of pentachlorophenol (PCP). It was demonstrated that the coimmobilized system degraded PCP more effectively than the nonimmobilized system. A solid substrate included in the coimmobilized system could support the biodegradation. Isolation of the degrading agents from a model interrupting microorganism by the coimmobilized capsule membrane reduced the interference on the biodegradation. In simulated contaminated soil extract and sand, the coimmobilized system also exhibited higher degradative ability and stability than the nonimmobilized systems.
Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis
Authors: Park S. C. , Baratti J.
Published: December 2004

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, μ, and biomass yield, YX/S) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, qp, and ethanol yield, Yp/s) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.
Estimation of Growth Yield and Maintenance Coefficient of Plant Cell Suspensions
Authors: Schnapp Sherry Rae , Curtis Wayne R. , Bressan Ray A. , Hasegawa Paul M.
Published: December 2004

Methodology is presented for the determination of growth yield (Yg) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Yg and m requires measurements of specific growth rate (µ) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Yg and m because µ is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the Km for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the Km, the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Yg and m because various steady‐state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of µ and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters.
Solvent effects on biocatalysis in organic systems: Equilibrium position and rates of lipase catalyzed esterification
Authors: Valivety Rao H. , Johnston Grant A. , Suckling Colin J. , Halling Peter J.
Published: December 2004

Porcine pancreatic lipase immobilized on celite particles has been employed as a catalyst for the esterification of dodecanol and decanoic acid in a predominantly organic system. Solvent influence on the equilibrium position and on the catalyst activity has been studied using 20 solvents, including aliphatic and aromatic hydrocarbons, ethers, ketones, nitro‐ and halogenated hydrocarbons, and esters. The equilibrium constant for esterification correlates well with the solubility of water in the organic solvent, which in turn shows a good relationship with a function of Guttman's donor number and the electron pair acceptance index number of the solvent. This may be rationalized in terms of the requirements for solvation of water and of the reactants. The catalyst activity, measured as the initial rate of the esterification reaction, is best correlated as a function of both n‐octanol‐water partition coefficient (log P) and either the electron pair acceptance index or the polarizability.
Immobilization‐stabilization of α‐chymotrypsin by covalent attachment to aldehyde‐agarose gels
Authors: Guisán José M. , Bastida Agatha , Cuesta Carmen , Fernandez‐Lufuente Roberto , Rosell Cristina M.
Published: December 2004

We have developed a strategy for immobilization‐stabilization of α‐chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme‐support multi‐interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one‐point attached derivatives and were more than 60,000‐fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme‐support multi‐interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde‐agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme‐support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.
Continuous glycerolysis of olive oil by Chromobacterium viscosum lipase immobilized in liposome in reversed micelles
Authors: Chang Pahn Shick , Rhee Joon Shick , Kim Jae‐Jin
Published: December 2004

Chromobacterium viscosum lipase which has adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37°C in continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high‐performance liquid chromatography (HPLC). The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol‐water‐lipase solution on the glycerolysis reaction. The conversion to desirable products, mono‐ and di‐olein, was improved without a substantial production of oleic acid at lower water concentrations, i.e., below 8.0% (w/v) which corresponds to a wo value of 0.97. At water concentration higher than 8.0% (w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme reactor, and the half‐line of the enzyme continuous reaction was about 7 weeks.
Gas transfer characteristics of a novel membrane bioreactor
Authors: Beeton S. , Millward H. R. , Bellhouse B. J. , Nicholson A. M. , Jenkins N. , Knowles C. J.
Published: December 2004

We show the design features of a membrane bioreactor based on pulsatile flow across dimpled membranes. Results show an enhanced mass transfer of air of at least five‐fold magnitude as compared with flat membranes. An increased working volume form 20 mL to 120 mL reduced the kLA at a given Reynolds number because of axial mixing of fluid from the deoxygenated end chamber. The bioreactor was used to supply air to a hybridoma mammalian cell line, and the calculated oxygen uptake showed that high‐density cultures could be maintained in a 20mL, single‐dimpled cultures could be maintained in a 20 mL, single‐dimpled membrane system. Indirect aeration of a 2 L continuous stirred tank reactor, by a double‐membrane system, showed that air could be supplied to mammalian cells at cell densities of approximately 4 × 106 /mL.
Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles
Authors: Aires‐Barros M. R. , Cabral J. M. S.
Published: December 2004

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid‐liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase‐loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K2HPO4 with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6‐fold with a recovery of 86%, and lipase B by 1.5‐fold with a recovery of 76%.
Saccharification of steam‐exploded poplar wood
Authors: Excoffier Gérard , Toussaint Bertrand , Vignon Michel R.
Published: December 2004

Effects of time, temperature, and pH during the steam explosion of poplar wood were studied with the aim of optimize both pentoses recovery and enzymatic hydrolysis efficiency. Steam explosion of acid impregnated wood chips allowed the recovery of 70% of potential xylose as monomers (217°C, 120 s) Enzymatic hydrolysis of pretreated fiber with Trichoderma reesei CL‐847 cellulase system increased progressively with the severity of the steam treatment conditions. The best yield in term of glucose recovery after 24 h of enzymatic hydrolysis was 70% of potential glucose (225°C, 120 s). Deactivation by adsorption on lignin of Trichoderma reesei cellulases and inhibition of these enzymes by low‐molecular‐weight phenols and trihydroxybutyric acids were noticed.
Xanthan production in bubble column and air‐lift reactors
Authors: Suh Ii‐Soon , Schumpe Adrian , Deckwer Wolf‐Dieter
Published: December 2004

A bubble column (0.05 m3) and an air‐lift fermentor (1.2 m3) were used for the production of the exocellular microbial polysaccharide xanthan withXanthomonas campestrisin a synthetic medium. Upon oxygen depletion in the liquid, the xanthan production rate dropped sharply and then became a linear function of the oxygen transfer rate. The volumetric mass transfer coefficients for oxygen conformed to the correlation of Suh et al. Using this correlation in combination with the model for xanthan batch fermentation suggested by Peters et al., the xanthan fermentations in the bubble column were well described. The model also correctly predicted the time course of the molecular weight of the polysaccharide even when a complex medium was used. In the air‐lift fermentor, however, the xanthan production rate and the xanthan yields with respect to oxygen and glucose were lower than expected at the overall oxygen transfer rate. The poor performance of the air lift was traced back to the lack of any oxygen supply in the downcomer.