bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic analysis of bioconversion of cellulose in attrition bioreactor
Authors: Jones Evan O. , Lee James M.
Published: December 2004

The attrition bioreactor (ABR) combines wet ball milling and enzymatic hydrolysis in one process step. It was found that the ABR did not accelerate enzyme deacti‐vation. Interfacial forces, not shear forces, caused the most deactivation. Elimination of the air–liquid interface by covering the reactor substantially increased enzyme stability. A simple exponential kinetic model was tested to predict the cellulose conversion in an ABR. Kinetic parameters were estimated from batch runs performed at various enzyme and substrate concentrations.
Escherichia coil growth dynamics: A three‐pool biochemically based description
Authors: Joshi Abhay , Palsson Bernhard O.
Published: December 2004

A three‐pool growth model of an individual Escherichia coli cell is described herein. The model is based on a previously developed chemically structured complex single cell growth model. The reduction in model complexity and the identification of the essential modes of motion, over the time scale of growth, is achieved by temporal decomposition and analysis of hierarchy in relaxation times. The three‐pool model faithfully simulates the changes in cell size, cell shape, cell macromolecular composition, DNA initiation and termination periods, and the dependence of cell growth under abiotic glucose limitation. The predictions made by the reduced model compare favorably with both the experimental data and those of the full single cell model (SCM) without any parameter adjustments. The three‐pool model has very few significant parameters and has the potential to find immediate practical use in bioreactor design and process control strategies. The model development illustrates the use of modal analysis to yield reduced physiologically realistic dynamic model of complex microbial system such as E. coll.
In situ extractive fermentation of acetone and butanol
Authors: Roffler S. R. , Blanch H. W. , Wilke C. R.
Published: December 2004

The productivity of the acetone–butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed‐batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter‐currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale‐up.
Influence of some physicochemical parameters on bacterial activity of biofilm: Ferrous iron oxidation by Thiobacillus ferrooxidans
Authors: Karamanev D. G. , Nikolov L. N.
Published: December 2004

The influence of temperature, pH, and substrate and product concentrations on the oxidation rate of ferrous iron by biofilm of Thiobacillus ferrooxidans was determined. The experiments were performed in an inverse fluidized‐bed biofilm reactor in which the biofilm thickness was kept constant at 80 μm. Oxygen concentration and diffusion through the biofilm did not limit the oxidation rate. The oxidation rate was almost unaffected by temperature between 13 and 38°C, pH between 1.3 and 2.2, ferric iron concentration up to 14 g/L, or ferrous iron concentration from 4 to 13 g/L. The kinetics of the process was described by the Monod equation with respect to the mass of the biofilm and with ferrous ions as the limiting substrate.
Hydrodynamics and oxygen transfer in pneumatic bioreactor devices
Authors: Chisti M. Y. , Moo‐Young M.
Published: December 2004

Gas holdup and oxygen transfer studies in non‐Newtonian suspensions of cellulose fibres conducted in two large (0.098 m3 each) reactors are described. Both reactors—a bubble column and a similar internal loop airlift—were unusual in that they had rectangular cross‐sections. In all cases gas holdups and kLaL declined with increasing solid concentration and, under identical conditions, the bubble column performed better than the airlift. The fluid systems used were carefully selected to represent mould fermentation broths.

The behavior of true mass transfer coeffcient kL with changes in bubble size is discussed for these systems.
Restricted diffusion effect on the binding of proteins to porous polymer resins as studied by repetitive injection method
Authors: Kitano Hiromi , Nakamura Katsunori , Hirai Youhei , Kaku Takashi , Ise Norio
Published: December 2004

A solution of bovine serum albumin (BSA) is repeatedly injected into a column packed with highly porous and hydrophobic polymer resins at appropriate intervals. The injected BSA is thoroughly retained in the column for 10 injections and, afterwards, starts to be eluted from the column gradually. Taking into consideration the restricting effect of already bound BSA upon the diffusion of newly injected BSA into the pores of the polymer resins, we can interpret the BSA elution profile from columns packed with polymer resin of various pore sizes and porosities. The effects of the binding rate constant and BSA concentration upon the elution profiles of BSA are also analyzed. Formyl groups are introduced into the polymers as a binding site with proteins, and the elution profiles of BSA from the column packed with the formylated resin are also analyzed.
Limitations of methods of determining effective diffusion coefficients in cell immobilization matrices
Authors: Itamunoala G. F.
Published: December 2004

Various techniques are available for determining the effective diffusivity De of solutes such as glucose in cell immobilization matrices. Nearly all, if not all, are subject to errors and limitations as regards the ranges of temperature, pressure, and/or concentration over which they give reliable results. It is the purpose of this article to compare three of these methods, designated (a) thin disc, (b) cylinder, and (c) beads types, and to show by means of a sensitivity and error analysis of the equation used in each method that the thin‐disc and cylindrical techniques give more accurate results of De than does the bead method.
Lumping analysis of biochemical reaction systems with time scale separation
Authors: Liao James C. , Lightfoot Edwin N.
Published: December 2004

Due to the complexity of the systems, successful modelling of intracellular reaction networks must rely on lumping techniques which systematically reduce the number of variables and parameters. Fortunately, the time scale separation characteristics of biochemical systems provide opportunities for eliminating unnecessary details. Through the proper interpretation of eigenvalues and eigenvectors, this article presents a theoretical basis for systematic model reduction. Results are generalized as a semiheuristic basis for lumping systems without complete kinetic information. It is also illustrated that the simplified system can yield new insight which is otherwise unavailable.
Characterization of aqueous two‐phase systems based on polydisperse phase forming polymers: Enzymatic hydrolysis of starch in a PEG–starch aqueous two‐phase system
Authors: Larsson Mats , Mattiasson Bo
Published: December 2004

A new method for determination of the binodial of an aqueous two‐phase system, using spectrophotometric measurements of the turbidity, is described in this article. The method is especially designed to characterize phase systems composed of polydisperse phase components. It gives information about the area representing the transition from homogeneous solution to a two‐phase system. The two‐phase systems studied were first a conventional Dextran T40‐polyethylene glycol 20M (PEG 20M) system, then a less‐well‐defined phase system based on PEG 20M and partially hydrolyzed starch. The PEG 20M‐starch system could be changed with respect to the volume ratio between the phases with time using hydrolytic enzymes, and the possibility of using the glucose released from the starch polymer is pointed out. Then the system is transformed to an extractive biconversion where the bottom phase polymer also served as the substrate.
Catalytic properties and active‐site structural features of immobilized horse liver alcohol dehydrogenase
Authors: Skerker Paul S. , Clark Douglas S.
Published: December 2004

Alcohol dehydrogenase from horse liver was immobilized by covalent attachment to CNBr‐Sepharose and by adsorption to octyl‐Sepharose CL‐4B, a hydrophobic analog of Sepharose. In each case, rate constants for the binding and release of coenzyme and for the oxidation of substrates were measured based on the concentration of accessible active‐site zinc atoms determined by titration with a paramagnetic inhibitor. All rate constants were substantially reduced upon immobilization; however, the rate constant of immobilized enzyme for ethanol oxidation was independent of the immobilization method, whereas the rate constant for cyclohexanol oxidation was lower for enzyme immobilized to octyl‐Sepharose. Consequently, the substrate specificity of the two immobilized enzyme samples differed by an order of magnitude. Moreover, EPR spectroscopy studies and computer graphic analyses of spin labels occupying three defined regions of the active‐site domain indicated that the active‐site conformation adjacent to the catalytic zinc atom was similar in the two samples while the conformation slightly further from the zinc atom was different. This result may explain why the two immobilized enzyme preparations exhibited the same rate constant toward a small substrate (ethanol) yet different rate constants toward a larger substrate (cyclohexanol), whose rate constant is expected to be sensitive to a larger portion of the active site.
Packed‐ and fluidized‐bed biofilm reactor performance for anaerobic wastewater treatment
Authors: Denac M. , Dunn I. J.
Published: December 2004

Anaerobic degradation performance of a laboratory‐scale packed‐bed reactor (PBR) was compared with two fluidized‐bed biofilm reactors (FBRs) on molasses and whey feeds. The reactors were operated under constant pH (7) and temperature (35°C) conditions and were well mixed with high recirculation rates. The measured variables were chemical oxygen demand (COD), individual organic acids, gas composition, and gas rates. As carrier, sand of 0.3–0.5 mm diameter was used in the FBR, and porous clay spheres of 6 mm diameter were used in the PBR. Startup of the PBR was achieved with 1–5 day residence times. Start‐up of the FBR was only successful if liquid residence times were held low at 2–3 h. COD degradations of 86% with molasses (90% was biodegradable) were reached in both the FBR and PBR at 6 h residence time and loadings of 10 g COD/L day. At higher loadings the FBR gave the best performance; even at 40–45 g COD/L day, with 6 h residence times, 70% COD was degraded. The PBR could not be operated above 20 g COD/L day without clogging. A comparison of the reaction rates show that the PBR and FBR per formed similarly at low concentrations in the reactors up to 1 g COD/L, while above 3 g COD/L the rates were 17.4 g COD/L day for the PBR and 38.4 g COD/L day for the FBR. This difference is probably due to diffusion limitations and a less active biomass content of the PBR compared with the fluidized bed.

The results of dynamic step change experiments, in which residence times and feed concentrations were changed hanged at constant loading, demonstrated the rapid response of the reactors. Thus, the response times for an increase in gas rate or an increase in organic acids due to an increase in feed concentration were less than 1 day and could be explained by substrate limitation. Other slower responses were observed in which the reactor culture adapted over periods of 5–10 days; these were apparently growth related. An increase in loading of over 100% always resulted in large increases inorganic acids, especially acetic and propionic, as well as large increases in the CO2 gas content. In general, the CO2 content of the gas was very low, due to the large amount of dissolved CO2 that exited with the liquid phase at low residence times. The performance of the FBR with whey was comparable to its performance with molasses, and switching of molasses to whey feed resulted in immediate good performance without adaptation.
Preparation of polyelectrolyte‐coated pH‐sensitive poly(styrene) microcapsules and their application to initiation‐cessation control of an enzyme reaction
Authors: Kokufuta Etsuo , Shimizu Noboru , Nakamura Isei
Published: December 2004

Poly(styrene) microcapsules, prepared by depositing the polymer around emulsified aqueous droplets, were coated with a synthesized polyelectrolyte; i.e., copolymer of maleic acid (MA) with methyl vinyl ether (MVE), co‐poly(MA, MVE), or with styrene (St), copoly(Ma, St). The permeability of the capsule membrane was investigated under various pHs of the outer medium usingn‐propyl alcohol as a permeant. It became apparent that either copoly(MA, St)‐ or copoly(MA, MVE)‐coated microcapsules function as a pH‐sensitive capsule. In particular, the former showed a dramatic change of the permeability in response to small differences in pH (5–6). By reference to the viscometric and electrophoretic studies of both copolymers, these were interpreted as being due to a pH‐induced alteration of the configuration of the copolymer coating on the surface of the capsule membrane. When sucrose was hydrolyzed in an aqueous suspension of the copoly(MA, St)‐coated capsules into which invertase was loaded, the hydrolytic reaction was initiated at pH 5. 5 and stopped at pH 4. 5. Such initiation‐cessation control was repeated reversibly without damaging the capsules.
External‐circulation‐loop airlift bioreactors: Study of the liquid circulating velocity in highly viscous non‐Newtonian liquids
Authors: Popović Milan , Robinson Campbell W.
Published: December 2004

In order to obtain further information on the behavior and optimal design of external‐circulation‐loop airlift (ECL‐AL) bioreactors, the liquid circulating velocity, gas holdup and average bubble diameter in the downcomer were studied using highly viscous pseudoplastic solutions of various types of CMC. A few comparative measurements also were made using a viscous Newtonian aqueous sucrose solution. For the liquid velocity measurements, an ultrasonic flow meter (Doppler frequency shift principle) was applied for the first time to the gas/non‐Newtonian liquid dispersion in downward flow and satisfactory results were obtained. For viscous liquids, the circulating liquid velocity in the riser section of an ECL‐AL (uLR) is shown to be dependent mainly on the downcomer‐to‐riser cross‐sectional area ratio (Ad/Ar), the effective viscosity (ηeff) and the gas superficial velocity (uGR) as described by the following equation

The circulating liquid velocity exerts opposing effects on the mass transfer and liquid‐phase mixing performances of ECL‐AL fermentors. Therefore, it is proposed that the optimum operating conditions for a given fermentation may be best achieved by means of independently regulating the circulating liquid velocity.
Improved pseudoanalytical solution for steady‐state biofilm kinetics
Authors: Sáez Pablo B. , Rittmann Bruce E.
Published: December 2004

Simple algebraic expressions for the flux of substrate into a steady‐state biofilm are developed. This pseudoanalytical solution, which eliminates the need for repetitiously solving numerically a set of nonlinear differential equations, is based on an analysis of the numerical results from the numerical solution of the differential equations. The critical advantage of this new pseudoanalytical solution is that it is highly accurate for the entire range of substrate concentrations and kinetic parameters. The article also illustrates that previous pseudoanalytical solutions for steady‐state biofilm kinetics are seriously inaccurate for certain ranges of substrate concentration and kinetic parameters.
Extended monod kinetics for substrate, product, and cell inhibition
Authors: Han Keehyun , Levenspiel Octave
Published: December 2004

A generalized form of Monod kinetics is proposed to account for all kinds of product, cell, and substrate inhibition. This model assumes that there exists a critical inhibitor concentration above which cells cannot grow, and that the constants of the Monod equation are functions of this limiting inhibitor concentration. Methods for evaluating the constants of this rate form are presented. Finally the proposed kinetic form is compared with the available data in the literature, which unfortunately is very sparse. In all cases, this equation form fitted the data very well.
Economic evaluation of enzymatic hydrolysis of phenol‐pretreated wheat straw
Authors: Zacchi Guido , Skoog Kerstin , Hahn‐Hägerdal Bärbel
Published: December 2004

Experimental data from the enzymatic hydrolysis of phenol‐pretreated Swedish wheat straw have been used to evaluate the cost fractions of capital and utility, enzyme, and raw material. Two different raw material prices and varying enzyme prices have been used. The evaluation is based on an empirical model for the enzymatic hydrolysis and a computer program where utility and equipment, enzyme, and raw material prices can be varied. The optimal residence time for the enzymatic hydrolysis is in the range of 70–110 h. A fed‐batch procedure with substrate concentrations higher than 10% oven‐dried material (ODM) and enzyme concentrations in the range (6–10) · 106 FPU/ton ODM should be used.
Dearomatization of lignin derivatives by fungal protocatechuate 3,4‐dioxygenase immobilized on porosity glass
Authors: Wojtaś‐Wasilewska M. , Luterek J. , Leonowicz A. , Dawidowicz A.
Published: December 2004

Protocatechuate 3,4‐dioxygenase (see protocatechuate: oxygen 3,4‐oxidoreductase, EC 1.13.11.3) was isolated from the mycelium of Pleurotus ostreatus (induced with p‐hydroxybenzoic acid) and immobilized on controlled porosity glass beads. Four fractions of Na–lignosulfonates (varying in Mr, after chromatography on Sephadex G‐50) were treated with the immobilized enzyme. The products after incubation showed the same Mr as the untreated fractions, but their light absorption at 280 nm considerably decreased. These studies indicate that dioxygenase causes partial dearomatization of lignin macromolecule.
Batch kinetics of microbial polysaccharide biosynthesis
Authors: Mulchandani A. , Luong J. H. T. , Leduy A.
Published: December 2004

A modified form of logistic equation has been proposed to quantity the batch kinetics of microbial growth during the biosynthesis of extra‐ and intracellular polymers. Based on the experimental data developed in this study, the proposed model appeared to provide adequate growth and fermentation kinetics of Aureobasidium pullulans. The model was also applicable for representing the reported data on pullulan, xanthan, and poly‐β‐hydroxybutyricacid. In comparison to the logistic and Monod kinetics, this model fitted the data better and more accurately described the overall fermentation, both concentrations and fermentation time.
Ultrasound enhanced bioprocesses: Cholesterol oxidation by Rhodococcus erythropolis
Authors: Bar Raphael
Published: December 2004

Cholesterol oxidation to cholestenone by resting cells of Rhodococcus erythropolis ATCC25544 was investigated under a computer‐controlled ultrasonic irradiation at a frequency of 20 kHz. The optimization of the ultrasound intensity and its mode of application to a stirred bioreactor was first established at a level which preserved the structural integrity of the cells and enabled their metabolic activity. A significant enhancement in the kinetic rates of the biotransformation was observed in microbial slurries of 1.0 and 2.5 g/L cholesterol when sonicated for 5 s every 10 min with a power output of 2.2 W/cm2. In contrast, ultrasound had no effect on the enzymatic oxidation of cholesterol (2.5 g/L) by cholesterol oxidase. A high loading of cholesterol (5.0 g/L) in sonicated microbial systems had, however, an adverse effect. The ultrasound enhancement is discussed in terms of an increased dissolution rate of the sustrate crystals and more importantly, in terms of the uniquely ultrasound‐induced enhancement of mass transfer inside and outside a cell.
Influence of immobilization procedure and salt environment on functional stability of chloroplast membranes: Experimental data and numerical analysis
Authors: Thomasset Brigitte , Thomas Daniel , Lortie Robert
Published: December 2004

The interactions between chloroplast membranes and their microenvironment within artificial matrices (albumin‐glutaraldehyde matrix, polyurethane foam) where investigated. Particularly, the influence of a high‐ionic‐strength medium (0.75 M potassium citrate) on the stability of the photosynthetic ferricyanide reduction by immobilized thylakoids has been studied. A method of data analysis based on a nonlinear identification method combined with the numerical integration of the equation of the transient state of the continuous stirred tank reactor (CSTR) is proposed to estimate the actual degradation of the photosynthetic electron transfer. A statistical analysis achieved on the parameter values has allowed a quantitative assessment of the global behavior of immobilized chloroplast membranes. From the mathernatical analysis of the experimental data, we demonstrate that citrate used in the reaction media prevents the photoinactivation of the electron transfer chain whatever the nature of the matrix or the type of the reactor. The use of an albumin‐glutaraldehyde matrix or an open reactor during experiments also has allowed a better stabilization of the photosystems under operational conditions.
Bacterial removal of pyrite from concentrated coal slurries
Authors: Andrews G. , Darroch M. , Hansson T.
Published: December 2004

To be economically feasible, bacterial coal desulfurization must be done at a high rate in concentrated coal slurries. The rate may be restricted by gas transfer limitation (O2 or CO2), inhibition of the Thiobacilli, or ceil death by mechanical abrasion. Experiments designed to differentiate between these limitations show that CO2 transport is limiting in shake flasks for slurries of more than 20% (wt/wt) of a coal containing 1% pyritic sulfur. Air sparging removed this limitation in slurries of up to 50%, leaving inhibition as the main limitation. Heterotrophic bacteria that establish themselves in a recycled cell culture increase process rates not only by removing organic inhibitors but also by increasing the O2–CO2 ration required by the biomass to the value that is supplied naturally from air.
Protein enrichment of sweet potato residue with amylolytic yeasts by solid‐state fermentation
Authors: Yang Shang‐Shyng
Published: December 2004

Starchy agricultural wastes were inoculated with amylolytic yeasts for protein enrichment by solid‐state fermentation. The moisture content of substrate was 65–69%, and water activity was equivalent to 0.98–0.99. The optimum conditions for protein enrichment were initial moisture content 65%, initial pH 4.5, a 1:1 mixture of ammonium sulfate and urea was incrementally added to the ferment with 1% added at zero time, 1% added at 24 h, and 0.5% added at 48 h, and incubation with amylolytic yeasts (1.0 × 1010/100 g substrate) at 30°C for 2–3 days. The final product contained 16.11–20.82% protein.
A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH
Authors: Miller W. M. , Blanch H. W. , Wilke C. R.
Published: December 2004

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale‐up procedures. An SP2/0‐derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model1at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth‐associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1–7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.
Oxygen transfer properties of a bioreactor for use within a nuclear magnetic resonance spectrometer
Authors: Drury David D. , Dale Bruce E. , Gillies Robert J.
Published: December 2004

Nuclear magnetic resonance (NMR) spectroscopic analysis of whole cells is an important emerging technique for noninvasive and nondestructive monitoring of cell physiology. However, this technique requires extremely high cell densities. Attempts to maintain densities above the carrying capacity of a maintenance system result in the demise of the entire culture. To define conditions for maintaining mammalian cells at high densities for NMR studies, we have designed a bioreactor to operate under defined, oxygen‐limited conditions within an NMR spectrometer. The bioreactor utilizes hollow fibers to deliver nutrients and remove wastes from an agitated cell suspension. The mass transfer properties of the fibers with respect to oxygen were determined. Ehrlich Ascites Tumor (EAT) cells were supplied with glutamine as the respiratory carbon source. The maximum viable cell density supported by a given oxygen concentration in the fluid flowing through the fiber lumen was predicted and then confirmed experimentally on the bench and in the spectrometer.
Physical mechanisms of cell damage in microcarrier cell culture bioreactors
Authors: Cherry Robert S. , Papoutsakis Eleftherios Terry
Published: December 2004

The negative effects of excessive agitation on tissue cells in microcarrier culture have often been ascribed to “shear.” Analysis of the fluid mechanics occurring suggests that there are actually three potential damage mechanisms: collisions of a cell‐covered microcarrier with other beads, collisions with parts of the reactor (primarily the impeller), and interaction with turbulent eddies the size of the microcarrier beads. Review of the available quantitative information on agitation effects in cell cultures does not establish which mechanism is predominant; the range of experimental variables reported emphasizes power input over the other reactor and impeller parameters. The bead–bead collision model is tentatively supported by the available data, but the other mechanisms may still be significant in some systems. The formation of bead aggregates by cellular bridging provides a parallel means of damaging cells. Breaking of these bridges by any of the three means identified earlier can cause cell destruction and/or the net transfer of cells to formerly bare beads. High concentrations of bridges are favored by lower agitation rates, presumably because the bridges are not as quickly destroyed after formation.
Serum‐free media in hybridoma culture and monoclonal antibody production
Authors: Glassy Mark C. , Tharakan John P. , Chau Pao C.
Published: December 2004

The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum‐free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum‐free formulations and selection rules for the serum‐free ingredients. In general, serum‐free media which are “lipid rich” can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum‐free media, irrespective of the lipid content.
Reductive dissolution of fe(III) oxides by Pseudomonas sp. 200
Authors: Arnold Robert G. , DiChristina Thomas J. , Hoffmann Michael R.
Published: December 2004

The kinetics and mechanism of reductive dissolution of Fe(III) oxides were examined in pure, batch cultures of Pseudomonassp. 200. Primary factors controlling hematite dissolution kinetics were mineral surface area (or concentration of high‐energy surface sites), ligand concentration, and cell number. In the presence of nitrilotriacetic acid (NTA), saturation kinetics were apparent in the relationship governing reductive dissolution of hematite. A kinetic expression was developed in which overall iron‐reduction rate is functionally related to the concentrations of both NTA and Fe(III).

Addition of NTA resulted in a 20‐fold increase in the microbial rate of mineral (reductive) dissolution. Mechanisms in which NTA served as a bridging ligand, shuttling respiratory electrons from the membrane‐bound microbial electron transport chain to the metal center of the iron oxide, or accelerated the departure of Fe(II) centers (bound to ligand) from the oxide surface following reduction have been postulated. Experimental results indicated that cell–mineral contact was essential for reductive dissolution of goethite.
Cellulase immobilization on Fe3O4 and characterization
Authors: Garcia Albert , Oh Sangha , Engler Cady R.
Published: December 2004

A successful procedure for attaching cellulase to 45 μm iron oxide particles with a high‐molecular‐weight (1000) ligand has been developed. Mass and activity balances were calculated for immobilized enzymes with different loadings. The highest specific activity of the immobilized enzyme was 5.9 mmol glucose/g bound protein/h. The efficacy of retaining enzymatic activity was 128%. The optimum pH was 5.5 compared to 4.0 of the free enzyme. The half‐life of the IMC was extended to 272 h compared to 0.77 h of the free enzyme.
Peptide synthesis with immobilized carboxypeptidase Y
Authors: Cramer Steven M. , Horváth Csaba
Published: December 2004

Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 μm microparticulate amino‐silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino‐silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed‐bed reactors for peptide synthesis. Packed‐bed reactors (40 × 4.6 mm, 60 × 4.6 mm) were used to catalyze the synthesis of 170 mg N‐benzoyl‐L‐arginyl‐L‐methioninamide, 380 mg N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucinamide, and 200 mg N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucyl‐L‐phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L‐methionyl‐L‐leucyl‐L‐phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N‐benzoyl‐L‐arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucyl‐L‐phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.
Hydrolysis of lactose in skim milk by immobilized β‐galactosidase in a spiral flow reactor
Authors: Bakken Andrew P. , Hill Charles G. , Amundson Clyde H.
Published: December 2004

β‐galactosidase from Aspergillus Oryzae immobilized in a spiral flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Residence time distribution measurements were used to assess the amount of longitudinal dispersion occurring as a consequence of the spiral flow pattern and the semiporous nature of the polymeric material used to construct the spiral. It was possible to model the flow conditions as tubular flow with a Peclet number that was a linear function of the reactor space time. Nonlinear regression methods were used to determine the kinetic parameters of three proposed enzymatic rate expressions. The best fit of the data was obtained using a rate expression containing separate terms for competitive inhibition of the reaction by both the a and β anomers of galactose. This kinetic model also incorporates the kinetics of the mutarotation between these forms. At 30°C and a space time of 7 minutes, 80% of the lactose present in skim milk can be converted to glucose and galactose.
In vivo nuclear magnetic resonance analysis of immobilization effects on glucose metabolism of yeast Saccharomyces cerevisiae
Authors: Galazzo Jorge L. , Bailey James E.
Published: December 2004

Fermentation rates and intracellular compositions have been determined for alginate‐entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus‐31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose‐1,6 diphosphate, glucose‐6‐phosphate, and 3‐phosphoglycerate and in internal pH. Carbon‐13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.
Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture
Authors: Ebina Y. , Ekida M. , Hashimoto H.
Published: December 2004

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four‐electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H+ ions produced by some accumulated acidic substances, and the impedance thus increased.
Regulation of mammalian cell growth by autocrine growth factors: Analysis of consequences for inoculum cell density effects
Authors: Lauffenburger D. , Cozens C.
Published: December 2004

Effects of inoculum cell density on mammalian cell growth in culture have been observed in a variety of experimental systems. Although these effects have been attributed generally to medium conditioning by the cells, there has previously been no quantitative theory proposed for this phenomenon based on developments in molecular and cell biology. In this article, we offer such a theory founded on the regulatory action of autocrine growth factors. A particularly relevant example of these is platelet‐ derived growth factor (PDGF), which is produced by fibroblastic cells in response to stimulation by transforming growth factor β (TGFβ), a common serum constituent, and provides a mitogenic signal for the same cells. A simple mathematical model for the production, diffusive transport, and binding of autocrine growth factors to cell surface receptors, coupled to a model for the dependence of cell proliferation on growth factor receptor binding allows prediction of initial cell population growth rate as a function of inoculum cell density. We focus on situations involving anchorage‐dependent cell growth, in which the cells are attached to a surface. A number of clear results are obtained, most notably the following: 1) for cells cultured on spherical microcarrier bead surfaces, the inoculum cell density needed to produce a given growth rate is linearly proportional to the bead radius; and 2) all other factors being equal, the inoculum cell density on a unit surface area basis needed to produce a given growth rate is greater for spherical microcarrier surfaces than for flat culture dish surfaces. These two results are consistent with the experimental observations of Hu and coworkers1,2 for fibroblast growth in minimal medium plus serum. The model also allows elucidation of the influence of other system parameters, both biological and physical, on initial cell proliferation rate and the inoculum cell density dependence.
Reactive extraction of penicillin G from mycel‐containing broth in a countercurrent extraction decanter
Authors: Likidis Z. , Schlichting E. , Bischoff L. , Schügerl K.
Published: December 2004

Penicillin was recovered from mycel‐containing fermentation broth by direct reactive extraction into a counter‐current extraction decanter, Type CA 226–290 of the Westfalia Separator Co., at room temperature via steady state operation. Penicillin concentrations in the feed varied from 3 to 41 g L−1, Amberlite LA‐2 carrier concentrations from 7 to 20 g L−1and/or DITDA carrier concentrations from 7.2 to 84 g L−1, the LA‐2‐to‐penicillin mole concentration ratio from 4 to 6.4, and/or the DITDA‐to‐penicillin mole concentration ratio was maintained at 2. The throughputs of the fermentation broth (520 to 880 L h−1) of the solvent phase (200 to 860 L h−1) and the over all throughput (800 to 1750 L h−1) were high. Extraction degrees of 72 to 96% were achieved between pH 4.6 and 5.1. Without carriers in the same pH range, extraction degrees of only 17 to 19% were attained. By reducing the pH to 2.3 and in the absence of carriers, the degree of extraction was increased to 61%. However, during the extraction, 6.5% of the penicillin decomposed. At these high throughputs, the steady state was attained within 1 to 4 min. Through the mechanical stress, the length of the hyphae was reduced and the protein content of the broth was increased by 50 to 100%. However, this protein content had no appreciable influence on the phase separation.
Media and environmental effects on phenolics production from tobacco cell cultures
Authors: Schmidt Andrew J. , Lee James M. , An Gynheung
Published: December 2004

The effects of various factors in culture medium on the phenolics production from cultured tobacco cells (free and immobilized) were studied. It was found that removing the growth hormone from the medium increased the productivities of phenolics for both free and immobilized cultures. Low initial sucrose concentration in the medium restricted growth and resulted in high cellular productivities of the phenolics for freely suspended cells, but not for the immobilized cells. Addition of 1.4% DMSO to standard culture medium greatly increased phenolics productivities without affecting cell viability in both free and immobilized cell cultures. Continuous operation of a packed‐column reactor of the immobilized cells was achieved for 500 hours. Aeration was accomplished by diffusing pure oxygen through silicone tubing placed inside the reactor. It was found that prolonged cell viability was contingent upon initially operating the reactor with total recycle for several days, and then introducing fresh feed while maintaining a high recycle rate. Immobilized cells packed in a continuous column reactor achieved productivities more than twice that achieved in any batch run.
Analysis of a framework using material balances in metabolic pathways to elucidate cellular metabolism
Authors: Niranjan S. C. , San Ka‐Yiu
Published: December 2004

A framework using material balances in metabolic pathways to study cellular metabolism is examined and the results are discussed. Rate measurements on extracellular compounds alone were found to be not always sufficient to validate proposed unique intracellular mechanisms. The conditions to delineate among candidate mechanisms based solely on extracellular measurements are established. The number of half reactions comprising the reaction network is found to be an important parameter.
Biotransformation of benzaldehyde by Saccharomyces cerevisiae: Characterization of the fermentation and toxicity effects of substrates and products
Authors: Long A. , Ward O. P.
Published: December 2004

Although higher initial rates of phenylacetyl carbinol formation were observed in fermentations containing a high starting benzaldehyde level, a massive reduction in yeast viability was observed resulting in early cessation of production formation. Pulse feeding to maintain lower benzaldehyde concentrations resulted in a lower initial reaction rate, but prolonged yeast viability and the biotransformation. This resulted in higher overall product tilers. As benzaldehyde concentration was increased, yeast growth rate was reduced (0.5 g/L), inhibited (1–2 g/L), or cell viability reduced (3 g/L). Benzaldehyde appeared to alter the cell permeability barrier to substrates and products. Reductions in yeast biomass levels and especially protein and lipid content were observed during the biotransformation. The effects of benzaldehyde and reaction products on yeast pyruvate decarboxylase and alcohol dehydrogenase stability were determined. Homogenized yeast cells produced similar phenylacetyl carbinol levels to whole yeast only if supplemented with thiamine pyrophosphate and magnesium.
Fundamental studies of biomolecule partitioning in aqueous two‐phase systems
Authors: Diamond Alan D. , Hsu James T.
Published: December 2004

Phase diagram data at 4°C was determined for the aqueous two‐phase systems composed of polyethylene glycol, dextran, and water. The Flory–Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two‐phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH2 group on the c‐terminal amino acid residue and by utilizing a set of low‐molecular‐weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH2 group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low‐molecular‐weight proteins gave a linear relationship with the tie line compositions of a phase diagram.
Shear degradation as a probe of microalgal exopolymer structure and rheological properties
Authors: Ramus J. , Kenney B. E.
Published: December 2004

The bulk theological properties of exopolymers produced by three species of microalgae are destroyed by shear stress. The properties are drag reduction in capillary pressure flow and low shear rate viscosity. As such, shear stress constitutes an experimental probe into the macromolecular structure which effects bulk Theological properties. Native and sheared exopolymer solutions were subjected to analysis by electrophoresis, size exclusion chromatography, hydrolysis, dialysis, and reducing end‐group analysis. The evidence indicates that shearing did not break the glycoside backbone of these exopolymers, rather shearing disrupted subtle interactions between copolymers. The interactions necessary for bulk rheological properties are likely at the quaternary level of macromolecular organization, specifically weak aggregations.
Stability testing of ligninase and Mn‐peroxidase from Phanerochaete chrysosporium
Authors: Aitken Michael D. , Irvine Robert L.
Published: December 2004

The white‐rot fungus Phanerochaete chrysosporium produces extracellular peroxidases (ligninase and Mn‐peroxidase) believed to be involved in lignin degradation. These extracellular enzymes have also been implicated in the degradation of recalcitrant pollutants by the organism. Commercial application of ligninase has been proposed both for biomechanical pulping of wood and for wastewater treatment. In vitro stability of lignin degrading enzymes will be an important factor in determining both the economic and technical feasibility of application for industrial uses, and also will be critical in optimizing commercial production of the enzymes. The effects of a number of variables on in vitro stability of ligninase and Mn‐peroxidase are presented in this paper. Thermal stability of ligninase was found to improve by increasing pH and by increasing enzyme concentration. For a fixed pH and enzyme concentration, ligninase stability was greatly enhanced in the presence of its substrate veratryl alcohol (3,4‐dimethoxybenzyl alcohol). Ligninase also was found to be inactivated by hydrogen peroxide in a second‐order process that is proposed to involve the formation of the unreactive peroxidase intermediate Compound III. Mn‐peroxidase was less susceptible to inactivation by peroxide, which corresponds to observations by others that Compound III of Mn‐peroxidase forms less readily than Compound III of ligninase.