bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosorption of uranium and lead by Streptomyces longwoodensis
Authors: Friis N. , Myers‐Keith P.
Published: December 2004

Biosorption of uranium and lead by lyophilized cells of Streptomyces longwoodensis was examined as a function of metal concentration, pH, cell concentration, and culture age. Cells harvested from the stationary growth phase exhibited an exceptionally high capacity for uranium (0.44 g U/g dry weight) at pH 5. Calculated values of the distribution coefficient and separation factor indicated a strong preference of the cell mass for uranyl ions over lead ions. The specific uranium uptake was similar for the cell wall and the cytoplasmic fraction. Uranium uptake was associated with an increase in hydrogen ion concentration, and phosphorus analysis of whole cells indicated a simple stoichiometric ratio between uranium uptake and phosphorus content. It is proposed that metal ions are bound to phosphodiester residues present both in the cell wall and cytoplasmic fractions. Based on this model, it was shown that uranium accumulation exhibits a maximum at pH 4.6 that is supported by experimental data from previous investigations.
Role of water activity in ethanol fermentations
Authors: Jones Rodney P. , Greenfield Paul F.
Published: December 2004

A separate role for water activity in the conversion of sugars to ethanol by two strains of yeast is identified. During fermentation of both single and mixed sugar substrates, the water activity was shown to remain constant during the logarithmic growth phase. This is despite the changes in concentration of substrates and product, the constancy reflecting the fact that the greater influence of ethanol on the solution activity is counterbalanced, in the early stages of the fermentation, by its low yield. The end of the log phase of growth coincides with the start of a period of gradually decreasing water activity. For the more ethanol‐tolerant strain UQM66Y, growth was found to cease at a constant value of water activity while that for the less tolerant strain UQM70Y depended on both ethanol concentration and water activity. It is argued that water activity is a more appropriate variable than ethanol concentration for describing some of the nonspecific inhibitory effects apparent in ethanol fermentations. A straightforward method for the calculation of water activity during such fermentations based on the use of solution osmolality is presented.
The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles
Authors: Webb C. , Fukuda H. , Atkinson B.
Published: December 2004

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10‐L spouted bed fermentor. In this type of reactor‐recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady‐state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h−1 (nominal washout rate for freely suspended cells is 0.012 h−1), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L· h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.
Periodic optimization of continuous microbial growth processes
Authors: Abulesz E‐M , Lyberatos G.
Published: December 2004

Steady‐state operation of continuous bioreactors is not necessarily the optimum type of operation. The method of π‐criterion is used in this work to determine whether periodic variation of the dilution rate can enhance the performance of continuous fermentation processes. It is found that the presence of time delay in the dynamic response of the chemostat renders a periodic operation of bioreactors, used for biomass production, superior to any steady‐state operation. Also, employing Williams' structured model it is shown that cycling improves the average protein productivity.
Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate
Authors: Downing Kim M. , Ho Chester S. , Zabriskie D. W.
Published: December 2004

A two‐stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water‐soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification‐fermentation with Pestal‐otiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.

Water‐soluble cellulose acetate was successfully prepared from purified wood cellulose (Solka Floe) and chemical reagents. Enzyme pretreatment of WSCAto form metabolizable sugars was a necessary step in achieving practical conversion of WSCA to ethanol using yeast. The results showed that WSCA has a low enzyme requirement and a high convertibility to reducing sugars with enzymes from P. westerdijkii fungus. Pestalotiopsis westerdijkii enzymes were found to be superior to enzymes from Trichoderma viride in producing metabolizable glucose from WSCA. The yeast utilized 55–70% of the hydrolyzate sugars that were produced by P. westerrlijkii enzymes on WSCA and produced ethanol. The acetate that was liberated into solution by the action of acetyl esterase enzymes on WSCA was found to have a stimulatory effect on ethanol production in yeast. This is an important feature that can be used to advantage in manipulating the conversion to maximize the production of ethanol. Hence, the simultaneous saccharification‐fermentation of WSCA to ethanol using P. westerdijkii enzymes and yeast has features that are highly desirable for developing an economical cellulose conversion process.
Kinetics of ethanol fermentations in membrane cell recycle fermentors
Authors: Lee Chang Woo , Chang Ho Nam
Published: December 2004

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow‐fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.
Effects of cellobiose and glucose on cellulose hydrolysis by both growing and resting cells of Bacteroides cellulosolvens
Authors: Murray William D.
Published: December 2004

The cellulase system of Bacteroides cellulosolvens was subjected to both catabolite repression and feedback inhibition by cellobiose. Cellulose‐solubilizing activity was 50% inhibited at a cellobiose concentration of 2.6 g/L and completely inhibited by 12 g/L. Glucose at 12 g/L (the highest concentration tested) had no effect on cellulase activity. Supplementation of B. cellulosolvens cellulase with β‐glucosidase resulted in increased conversion of cellobiose to glucose; however, a constant cellobiose pool size of approximately 7 g/L was maintained.
Pullulan from peat hydrolyzate fermentation kinetics
Authors: Boa Jacques M. , LeDuy Anh
Published: December 2004

The Luedeking–Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, α, and β varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant‐feed fed‐batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed‐batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed‐batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.
Kinetics of enzymatic lysis and disruption of yeast cells: I. Evaluation of two lytic systems with different properties
Authors: Hunter J. B. , Asenjo J. A.
Published: December 2004

Many microorganisms produce enzymes which lyse the walls of yeasts, fungi, and bacteria. The proportions of different enzyme activities present in the lytic system, their action patterns, synergism, and dependence on inhibitors, constitute the activity profile of the lytic system. Taken together, the activity profile and process conditions for lysis determine the reaction rate and the distribution of products from lysis of any given type of cells. Kinetics of glucan hydrolysis, proteolysis, and lysis of brewer's yeast were compared for two extracellular yeast–lytic enzyme systems with different properties. The enzyme sources used were filtered culture broths from Cytophaga sp. NCIB 9497 grown in batch culture and from Oerskovia xanthineolytica LL‐G109, grown under carbon limitation in continuous culture. Rate and extent of cell hydrolysis, and the accumulation of soluble proteins, peptides, and carbohydrates from the lysed yeast cells, are discussed in terms of the activity profiles and potential applications of the two enzyme systems.
Kinetics of enzymatic lysis and disruption of yeast cells: II. A simple model of lysis kinetics
Authors: Hunter J. B. , Asenjo J. A.
Published: December 2004

A simple two‐step model is proposed to describe the kinetics of the two lytic systems examined in the preceding article. The model predicts concentrations of yeast solids, soluble proteins, peptides, and carbohyrates. In the first reaction step, yeast cell mass is solubilized; in the second, the released protein can be hydrolyzed to peptides. Kinetics for both yeast lysis and the subsequent protein breakdown are based on Michaelis–Menten expressions. Terms have been included for competitive inhibition of yeast lysis by substances in the Cytophaga enzyme preparation, and for incomplete hydrolysis of cells by the Oerskovia enzyme system. Parameters have been independently determined for all reactions except Oerskovia proteolysis, where they were fit by a leastsquares method to data from model test runs. The model has been verified for yeast concentrations between 0.7 and 70 g/L yeast (dry basis) and 4–40% crude enzyme solution.
Evaluation of the effectiveness factor along immobilized enzyme fixed‐bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase‐coated porous glass
Authors: Manjón A. , Iborra J. L. , Gómez J. L. , Gómez E. , Bastida J. , Bódalo A.
Published: December 2004

A design equation is presented for packed‐bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one‐intermediate Michaelis–Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase‐coated porous glass operating in a packed‐bed reactor and hydrolyzing both p‐nitrophenyl‐α‐L‐rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.
Mathematical analysis of two‐phase mass transfer in a batch reactor for the chemical transformation of a steroid
Authors: Pereira Vincent , Tigli Husevin , Gryte Carl C.
Published: December 2004

A reactor is described for the conversion of the slightly water‐soluble steroid testosterone (T) to 4‐androstene‐3, 17‐dione (4‐AD) by enzyme in the presence of excess cofactor. Since the enzyme is subject to substrate inhibition, reaction rates are strong functions of aqueous substrate concentration. High concentrations of the substrate, testosterone, per unit reactor volume are maintained within poly(dimethylsiloxane) beads that are suspended in the aqueous enzyme solution. Mass transfer (controlled by bead size, polymer to water volume ratio, enzyme loading) is used to control the degree and rate of conversion. The reactor dynamics are predicted over a wide range of reaction conditions. The product steroid is recovered in the polymeric beads from the enzyme solution.
Affinity partitioning: Development of mathematical model describing behavior of biomolecules in aqueous two‐phase systems
Authors: Cordes A. , Flossdorf J. , Kula M.‐R.
Published: December 2004

A mathematical model describing the affinity partitioning of macromolecules in aqueous two‐phase systems has been derived. The model was used to calculate binding parameters that were compared against values deter mined by means of ultracentrifugation and fluorescence titration. The mathematical model and its modifications were found to describe satisfactorily the partition behavior of macromolecules with differing numbers of binding sites. It could be shown that in solutions containing PEG the binding behavior of FDH is changed fundamentally. The dissociation constants of FDH with PEG‐blue in the presence and absence of PEG are different.
Feedback optimization of fed‐batch fermentation
Authors: Modak J. M. , Lim H. C.
Published: December 2004

The problem of feedback optimization of the feed rate for fed‐batch fermentation processes is formulated in the framework of singular control theory and switching hypersurfaces. Using four differential balance equations that describe a general class of fedbatch processes and a general objective function to be minimized, it is shown that under certain restrictions the feedback optimization of the feed rate can be realized as a nonlinear function of the state variables, such as the concentrations of cell mass, substrate and product, and the fermentor volume. The restrictions on the initial conditions, the fermentation kinetics and the objective function, that are needed for realization of the feedback optimization, are provided. Fed‐batch fermentation models of lysine and alcohol are used to construct switching curves and to illustrate the feedback optimization of the feed flow rates.
A fermentor for study of sauerkraut fermentation
Authors: Fleming H. P. , McFeeters R. F. , Humphries Ervin G.
Published: December 2004

A fermentor was designed and constructed for study of the physical, microbiological, and chemical changes that occur during the sauerkraut fermentation. The fermentor has some essential features that include restriction in volume of the sauerkraut bed, construction of clear plastic to permit visual determination of liquid‐level changes as a result of gas entrapment within the sauerkraut bed, and a gas‐lift device for use in nitrogen purging of the fermenting brine. Fermentations exhibited two distinct stages, the first one gaseous and the second non‐gaseous. The gaseous stage was characterized by rapid CO2 and acid production due to growth by hetero‐fermentative lactic acid bacteria with resultant gas entrapment within the sauerkraut bed and a rise in liquid level. Also, rapid disappearance of fructose and rapid appearance of mannitol occurred during this stage. The nongaseous stage was characterized by growth of homo‐fermentative lactic acid bacteria with little or no CO2 production and a gradual increase in lactic acid until all fermentable sugars were metabolized. Nitrogen purging appeared to offer several potential advantages, including a means for brine circulation, removal of CO2 from the brine, and anaerobiosis to ensure retention of ascorbic acid, desirable color, and other oxygen‐sensitive traits in sauerkraut.
Treatment of leachate from a solid waste landfill site using a two‐stage anaerobic filter
Authors: Wu Y. C. , Hao O. J. , Ou K. C. , Scholze R. J.
Published: December 2004

Raw leachate was treated using a two‐stage upflow anaerobic filter process. Leachate from a solid waste landfill site, which received both municipal and industrial wastes, contained high organic matter (17–21 g/L COD, 13–14 g/L BOD, and 3.5–4.6 g/L volatile acids), and low metal (Zn and Fe) concentrations. Depending on sampling time, leachate composition and characteristics varied considerably. At an organic loading up to 4 g COD/day2 media area, the BOD and COD removal percentages were 98 and 91%, respectively. The biofilters were also effective for metal removal. However, the filter effluent contained a high concentration of ammonia. System overloading was characterized by the accumulation of large quantities of volatile acids and by a now ratio of alkalinity/volatile acids, resulting in low COD removal and reduced gas production. Once the first filter was upset, the second stage could only partially respond to the volatile acids accumulated in the effluent of first filter.
High‐temperature alkaline α‐amylase from Bacillus iicheniformis TCRDC‐B13
Authors: Bajpai Pratima , Bajpai Pramod K.
Published: December 2004

A bacterial strain, Bacillus licheniformis, has been isolated and identified which produces high‐temperature alkaline α‐amylase. Cultural conditions, such as types of carbon and nitrogen sources, temperature, pH, and time of reaction, have been optimized for production of α‐amylase in shake flask and fermenter. The enzyme produced was quite active even at 100°C; however, it showed optimum activity at 90°C. It exhibited optimum activity in the broad pH range 5.5–10. The effects of Na+ and Ca2+ ions on enzyme activity was also studied.
A study of methanol transport/uptake by a methanol‐utilizing bacteriumL3in a batch bioreactor
Authors: Agrawal Pramod , Flores Gary E.
Published: December 2004

Two novel, nitrogen‐limited and oxygen‐limited, perturbed transient experiments were performed to examine the presence of active transport of methanol and study the effect on methanol uptake by a methanol‐utilizing bacterium,L3, in a batch bioreactor. Transient limitations of both ammonium ions and O2in batch cultures were found to cause methanol leakages out of the cells, suggesting the presence of an active transport of methanol inL3. Such experimental results were used to indirectly estimate the intracellular levels of methanol during a batch growth ofL3. The results of our analysis indicate that the intracellular methanol level is high and show an increasing trend during the unbalanced phase, but falls to a constant low level in the balanced phase of a typical batch growth8ofL3. A simple modelling analysis suggests that a four‐parameter “pump‐and‐extrusion” model could be used to adequately describe the transport of methanol between the extra cellular and intracellular phases of batch cultures ofL3.
On‐line titration method for monitoring buffer capacity and total volatile fatty acid levels in anaerobic digesters
Authors: Powell G. E. , Archer D. B.
Published: December 2004

The construction and use of an automatic on‐line titration unit for routine or event‐ initiated monitoring of alkalinity, buffer capacity, and volatile fatty acid (VFA) levels is presented. Under computer control a sample of digester liquor is pumped into the titration vessel and weighed. A sequence of titration, sparging, and back‐titration operations are then initiated during which the pH and weight are recorded continuously and a titration curve constructed. From the curve, estimates of the alkalinity, buffer capacity to any desired pH endpoint, and total VFA levels are computed. The data is stored to disk and output as hard copy together with the titration curve itself. Monitoring and control of the titration apparatus is effected by a microcomputer via two analog input lines and eight digital output lines, respectively. The system is suitable for downloading to a small, inexpensive dedicated microprocessor‐based system. The apparatus is constructed from standard and widely available equipment and the titration sequence, being under software control, is fully adaptable to particular requirements. The use of this facility in the on‐line monitoring, control and optimization of the anaerobic digestion process is discussed.
Measurement of oxygen solubility in fermentation media: A colorimetric method
Authors: Slininger P. J. , Petroski R. J. , Bothast R. J. , Ladisch M. R. , Okos M. R.
Published: December 2004

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10–15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in‐line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.
Removal of thorium from simulated acid process streams by fungal biomass
Authors: Gadd G. M. , White C.
Published: December 2004

Biomass from several fungal species removed thorium from solution in 1M HNO3, pH 0–1. Thorium uptake was saturable with increasing thorium concentration, although the equilibria did not correspond to a simple ad sorption isotherm. Thorium uptake was altered by the biomass concentration, the uptake per unit biomass being reduced at high biomass concentrations. The presence of Al3+ and Fe3+ only slightly inhibited uptake of thorium while Ca2+, Mg2+, and Na+ had no effect. Thus fungal biomass appears capable of removing thorium from solution under chemical conditions existing in acid waste liquors. Thorium uptake was increased by pretreatment using detergent and also, in the case of filamentous fungi, varied with the culture conditions, which implies that the thorium uptake characteristics of fungal biomass are able to be manipulated by these or similar means for optimum performance.
Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor
Authors: Demura Makoto , Asakura Tetsuo
Published: December 2004

Glucose oxidase (GOD) was immobilized in Bombyx mori silk fibroin membrane by only physical treatment, i.e., stretching without any chemical reagents. This is due to the structural transition of the silk fibroin membrane from random coil to antiparallel β‐sheet (Silk II) induced by the stretching treatment. Permeability coefficients of glucose and oxygen through the fibroin membrane were determined; the permeability of glucose decreased with increasing degree of stretching. The immobilized enzyme activity was characterized with apparent Michaelis constant K and maximal activity Vm. Optimum pH of the activity of the immobilized enzyme was shifted to the value around neutrality, and the activity was maintained to the higher values on both sides of the optimum pH compared with the case of free enzymes. Thermal stability was scarcely lost even at 50°C, although the free enzyme lost about 70% of the original activity. Thus, the stabilities of the enzyme vs. pH and heat were much improved by the immobilization with silk. Glucose sensor prepared with this GOD‐immobilized fibroin membrane was developed; the capabilities such as the response time, calibration curve, and repeating usage were determined.
Periodic operation of a continuous culture of Baker's yeast
Authors: Abulesz E‐M. , Lyberatos G.
Published: December 2004

The possibility of enhancing the biomass productivity of a continuous culture of Saccharomyces cerevisiae growing on a glucose‐limited medium is addressed. An unstructured Monod‐type model is first identified using steady‐state data. The culture is subjected to step changes in dilution rate, and it is seen that the Monod model is unable to predict even qualitatively the dynamic response of the culture. Incorporation of a time delay allows significant improvement in the transient fit. It is found that the culture has a time lag of about 3 h in adapting its growth rate. Cycling the dilution rate with a period of 3 h leads to substantial improvement in the average biomass productivity.
Study of gaseous substrate fermentations: Carbon monoxide conversion to acetate. 1. Batch culture
Authors: Vega J. L. , Clausen E. C. , Gaddy J. L.
Published: December 2004

Biological processes may be used to convert gas phase substrates, such as H2S, CH4, CO, H2, and CO2, to useful products. Utilization of these substrates is often a mass transfer limited process, first requiring absorption across the gas‐liquid interface and diffusion through the culture medium to the cell surface, prior to reaction. This article presents a method for determining fermentation parameters of a gaseous substrate in convenient batch vessels using a modified Monod model. The procedure is illustrated with experimental data for the conversion of carbon monoxide to acetate by the strict anaerobe Peptostreptococcus productus.
Activity and flexibility of alcohol dehydrogenase in organic solvents
Authors: Guinn R. Mark , Skerker Paul S. , Kavanaugh Pascale , Clark Douglas S.
Published: December 2004

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active‐site directed spin labels were used to examine solvent‐dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.
Influence of plasmid origin and promoter strength in fermentations of recombinant yeast
Authors: Da Silva Nancy Anderson , Bailey James E.
Published: December 2004

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of β‐galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2μ and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average β‐galactosidase specific activity was an order of magnitude below that with the 2μ‐based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10‐CYC1 promoters. The rate of increase in β‐galactosidase specific activity after induction in batch culture was 3–5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher β‐galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10‐CYC1 promoter.
An investigation into the role of initial KLa on the growth and alkaloid accumulation by cultures of Catharanthus roseus
Authors: Leckie F. , Scragg A. H. , Cliffe K. C.
Published: December 2004

The effect of initial KLa on the batch culture of Catharanthus roseus ID1 in a 12.5‐L bioreactor has been examined. Optimum KLa values were found for both growth and alkaloid accumulation. Different sparger designs were used to alter the KLa values. High KLa values caused increased aggregation of the cultures, depressed biomass yields, and altered patterns of alkaloid accumulation.
A kinetic model for methanogenesis from whey permeate in a packed bed immobilized cell bioreactor
Authors: Yang Shang‐Tian , Quo Meining
Published: December 2004

The fermentation kinetics of methane production from whey permeate in a packed bed immobilized cell bioreactor at mesophilic temperatures and pHs around neutral was studied. Propionate and acetate were the only two major organic intermediates found in the methanogenic fermentation of lactose. Based on this finding, a three‐step reaction mechanism was proposed: lactose was first degraded to propionate, acetate, CO2, and H2 by fermentative bacteria; propionate was then converted to acetate by propionate‐degrading bacteria; and finally, CH4 and CO2 were produced from acetate, H2, and CO2 by methanogenic bacteria. The second reaction step was found to be the rate‐limiting step in the overall methanogenic fermentation of lactose. Monod‐type mathematical equations were used to model these three step reactions. The kinetic constants in the models were sequentially determined by fitting the mathematical equations with the experimental data on acetate, propionate, and lactose concentrations. A mixed‐culture fermentation model was also developed. This model simulates the methanogenic fermentation of whey permeate very well.
Minimizing the errors associated with the determination of effective diffusion coefficients when using spherical cell immobilization matrices
Authors: Petersen James N. , Davison Brian H. , Scott Charles D.
Published: December 2004

Gel materials are often used to entrap biological catalysts. Several experimental methods have been proposed to estimate the diffusion coefficient of important chemical species within these materials. An error analysis for the bead method was performed and, contrary to previously reported results, when proper experimental conditions were employed, the error associated with the bead method was similar to that obtained using the other common methods.
Studies on the optimization of growth and indole alkaloid production by hairy root cultures ofCatharanthus roseus
Authors: Toivonen L. , Ojala M. , Kauppinen V.
Published: December 2004

Catharanthus roseushairy root cultures, genetically transformed withAgrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production ofC. roseushairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH2PO4· H2O/L and 1. 311 g KNO3/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.
The production of the enzyme dextransucrase using nonaerated fermentation techniques
Authors: Barker P. E. , Ajongwen N. J.
Published: December 2004

High yields of the enzyme dextransucrase have been produced repeatedly by fed‐batch fermentation techniques. Activities in excess of 21.9 U/cm3 have been obtained by culturing Leuconostoc mesenteroides NRRL B‐512(F) under nonaerated fed‐batch fermentation conditions. Aerobic fermentations carried out under identical conditions have consistently produced enzyme of less than 17 U/cm3, but with no difference in the final cell concentration in the broth. Different types of yeast extract have been found to have significant effect on the final cell concentration and more especially on the enzyme activity with enzyme yields varying by as much as 50% when different types of yeast extracts were used.
Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli
Authors: Birnbaum S. , Bailey J. E.
Published: December 2004

Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two‐dimensional gel electrophoresis patterns. The plasmids investigated had very similar sequences except for small domains encoding the represser of plasmid replication. At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased. At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed. Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation. Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses. These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.
Affinity chromatography as a rapid and convenient method for purification of fungal laccases
Authors: Rogalski J. , Wojtas‐Wasilewska M. , Apalovič R. , Leonowicz A.
Published: December 2004

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl‐ and vanillyl‐controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed.
Enzyme thermoinactivation in anhydrous organic solvents
Authors: Volkin David B. , Staubli Andrea , Langer Robert , Klibanov Alexander M.
Published: December 2004

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110‐145°C in nonaqueous media all three enzymes inactivate due to heat‐induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat‐induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (Tm values as high as 124°C) compared to aqueous solution. The thermostability of ribonuclease towards heat‐induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.
Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two‐phase system
Authors: Tjerneld Folke , Persson Ingrid , Lee James M.
Published: December 2004

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two‐phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two‐phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h−1 and productivities of 2.1 g/L h, which is approximately a 10‐fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two‐phase system. The conversion of the substrate was 86%.
Measuring kLa with randomly pulsed dynamic method
Authors: Gauthier Line , Thibault Jules , LeDuy Anh
Published: December 2004

This reports on the determination of the overall oxygen transfer coefficient in a mechanically agitated vessel using a randomly pulsed dynamic method. This method consists in exciting the system by randomly switching the inlet gas stream with air or nitrogen with an identical volumetric flow rate. A pseudo‐random binary sequence was used. This procedure is routinely used in process control for the identification of system's transfer function. The pulsed dynamic method gives good reliability (as compared with the traditional gassing‐out method) and reproducibility in water. However, further improvement is needed before it can be used to monitor on‐line thekLaduring a fermentation.
Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose
Authors: Watanabe Etsuo , Takagi Masatoshi , Takei Satoko , Hoshi Masakazu , Shu‐gui Cao
Published: December 2004

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: magnified image The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, β‐galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8‐diamino‐4‐aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or β‐galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60–180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2–16% sucrose, 2.8–8.3% lactose, or 0.1–1% starch. The relative errors were ±4% for sucrose and ±3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.
α‐Amylase adsorption on starch crystallites
Authors: Leloup V. M. , Colonna P. , Ring S. G.
Published: December 2004

The goal of this work was to characterize the adsorption of Bacillus subtills α‐amylase onto crystalline starchy materials of the B‐type polymorph. Monodisperse spherulitic particles (R ż6; 5.0 μm), essentially resistant to α‐amylolysis at 25°C were prepared from short amylose chains (DPn ≈ 15). The α‐amylase adsorbed specifically onto the spherulites, and adsorption was found to be a prerequisite step for hydrolysis. Adsorption was inhibited by the presence of maltose and maltotriose in the reaction mixture. Adsorption isotherm of the enzyme on the particles showed a well developed plateau of 1.62 μg/cm2 at 25°C corresponding to a monolayer adsorption process. The binding free energy calculated from the initial slope of the isotherm was ΔG ≈ −20.7 kJ/mol. This is smaller than published values for the binding of α‐amylase to soluble amylosic chains (ΔG < −30 kJ/mol).
Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: Part 1. Dynamic modeling
Authors: de Gooijer C. D. , Wijffels R. H. , Tramper J.
Published: December 2004

The modeling of the growth of Nitrobacter agilis cell immobilized in κ‐carrageenan is presented. A detailed description is given of the modeling of internal diffusion and growth of cells in the support matrix in addition to external mass transfer resistance. The model predicts the substrate and biomass profiles in the support as well as the macroscopic oxygen consumption rate of the immobilized biocatalyst in time. The model is tested by experiments with continuously operated airlift loop reactors containing cells immobilized in κ‐carrageenan. The model describes experimental data very well. It is clearly shown that external mass transfer may not be neglected. Furthermore, a sensitivity analysis of the parameters at their values during the experiments revealed that apart from the radius of the spheres and the substrate bulk concentration, the external mass transfer resistance coefficient is the most sensitive parameter for our case.
Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: Part 2. Model evaluation
Authors: Wijffels R. H. , de Gooijer C. D. , Kortekaas S. , Tramper J.
Published: December 2004

A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 μm, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 μm was 11 kg · m−3 gel.
Biodegradation of 3,4‐dichloroaniline in a fluidized bed bioreactor and a steady‐state biofilm Kinetic model
Authors: Livingston Andrew Guy
Published: December 2004

Mixed culture of microorganisms immobilized onto Celite diatomaceous earth particles were used to degrade 3,4‐dichloroaniline (34DCA) in a three‐phase draft tube fluidized bed bioreactor. Biodegradation was confirmed as the dominant removal mechanism by measurements of the concomitant chloride ion evolution. Degradation efficiencies of 95% were obtained at a reactor retention time of 1.25 h. A mathematical model was used to describe the simultaneous diffusion and reaction of 34DCA and oxygen in the biofilms on the particles in the reactor. The parameters describing freely suspended cell growth on 34DCA were obtained in batch experiments. The model was found to describe the system well for three out of four steady states and to predict qualitatively the experimentally observed transition in the biofilm kinetics from 34DCA to oxygen limitation.
Parametric studies of ethanol production form xylose and other sugars by recombinant Escherichia coli
Authors: Beall David S. , Ohta K. , Ingram L. O.
Published: December 2004

The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH‐controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.