bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic effects of simultaneous inhibition by substrate and product
Authors: Van Den Heuvel J. C. , Beeftink H. H.
Published: December 2004

The starting point for the present investigations was the finding that increasing influent concentrations from 10 to 380 mmol/L glucose decreased the attainable growth rate of an acidogenic population in continuous culture from 0.52 to 0.05 h−1 To account for this phenomenon, a new kinetic model is developed that combines substrate and product inhibition. Both effects are connected through the product yield, giving rise to a complex dependency of the growth rate on the substrate concentration. As a main feature, the maximum attainable growth rate decreases almost hyperbolically above some optimal substrate concentration in the influent. Furthermore, under certain conditions the kinetic model predicts the existence of three steady states: a high‐conversion and a low‐conversion state that are both stable and a metastable intermediate state. The latter states from the multiple‐steady‐state region are to be avoided, and eventual transitions to these states may have important consequences for the stability and the operation of such reaction systems. Substrate as well as product inhibition is reported for Propionibacterium freundenreichii and recently could be demonstrated for the above‐mentioned acidogenic population. The proposed model allows optimization of anaerobic wastewater treatment processes and is applicable also to other fermentations.
Immobilization of E. coli β‐galactosidase and its derivatives by polyacrylamide gel
Authors: Khare S. K. , Gupta M. N.
Published: December 2004

We have recently prepared some crosslinked derivatives of Escherichia coli β‐galactosidase by treating the enzyme with bisimidoesters. In this article, we report the results obtained when the native and these crosslinked derivatives are entrapped in polyacrylamide gel lattice. It was found that use of combination of three protective agents, viz., bovine serum albumin, cysteine, and lactose, during immobilization gave an increased yield of 190% in the case of DMA crosslinked preparation. In the case of native enzyme, the Km, pH optimum, and temperature optimum were found to remain unchanged on immobilization. The DMA crosslinked preparation entrapped in polyacrylamide in the presence of BSA, lactose, and cysteine was found to be a significantly better catalyst and hydrolyzed 47% milk lactose as compared to 31% hydrolysis by entrapped native enzyme in 6 h.
Action of Bacillus subtilis α‐amylase on native wheat starch
Authors: Colonna Paul , Buléon Alain , Lemarié Fabienne
Published: December 2004

Native starch granules from wheat have been subjected to enzymatic depolymerization with an α‐amylase from Bacillus subtilis. Crystallites made from short‐chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x‐ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by α‐amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of α‐amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.
A structured mechanistic model of the kinetics of enzymatic lysis and disruption of yeast cells
Authors: Hunter J. B. , Asenjo J. A.
Published: December 2004

A structured, mechanistic model has been built for the kinetics of yeast cell lysis by microbial cell lytic enzymes, based on an understanding of the two‐layer yeast cell wall structure and the properties of yeast‐lytic enzyme systems. The model predicts the release of protein, peptides and carbohydrates from four cell structures: the outer and inner wall layers, the cytosol and organelles or proteins present in particles; it also predicts organelle or particle lysis or solubilization and the breakdown of released proteins to peptides. Applications of the model to design and optimization of selective product release are discussed.
Defined bacterial culture development for methane generation from lactose
Authors: Yang Shang‐Tian , Tang I‐Ching , Okos Martin R.
Published: December 2004

The defined microbial cultures for methane generation from lactose were investigated. A mixed culture consisting of homolactic (Streptococcus lactis), homoacetic (Clostridium formicoaceticum), and acetate‐utilizing methanogenic (Methanococcus mazei) bacteria was used to convert lactose and whey permeate to methane at mesophilic temperatures (35–37°C) and a pH around 7.0. Lactose was first converted to lactic acid by S. lactis, then to acetic acid by C. formicoaceticum, and finally to methane and CO2 by M. mazei. About 5.3 mol methane were obtained from each mole of lactose consumed, and the conversion of acetate to methane was the rate‐limiting step for this mixed‐culture fermentation.
Cybernetic modeling of bacterial cultures at low growth rates: Mixed‐substrate systems
Authors: Turner Brian G. , Ramkrishna D. , Jansen Norman B.
Published: December 2004

A cybernetic model to predict the low‐growth‐rate behavior of bacteria in mixed‐substrate environment is presented. Using only growth and maintenance parameters from single‐substrate experiments, the model accurately predicts the simultaneous substrate utilization and maintenance energy effects in constant fed‐batch cultures of Klebsiella oxytoca. The robustness of the model was examined more rigorously by perturbing glucose‐limited fed‐batch cultures with additions of arabinose, xylose, and fructose. In all cases, reasonable agreement of the model prediction with the experimental data was observed.
Fluorescence sensing of fermentation parameters using fiber optics
Authors: Junker B. H. , Wang D. I. C. , Hatton T. A.
Published: December 2004

A method has been developed for measuring fermentation parameters such as dissolved oxygen, pH, and cell density that differs from traditional techniques that require electrodes and off‐line samples. Fluorescent indicators, each sensitive to a single variable, are dissolved directly into a fermentation broth. A fiber‐optic probe fluorimeter measures the fluorescence intensities that can then be correlated with parameter values. In addition, an integrated scatter scanning technique can be used to monitor cell density in situ. Results have been obtained using simulated baker's yeast broth and during actual baker's yeast fermentations.
Foam separation of microbial cells
Authors: Parthasarathy S. , Das T. R. , Kumar R. , Gopalakrishnan K. S.
Published: December 2004

Batch foam separation has been employed to separate Saccharomyces carlsbergensis cells from their broth without the use of any external surface‐active agent. A model has been developed to predict the foamate cell concentration as well as the variation of cell concentration in the bulk liquid in the foam column as a function of time. The model assumes a linear equilibrium relation between the cell concentrations at the interface and the bulk. The foam has interface as well as interstitial liquid. The interface is assumed to be in equilibrium with the interstitial liquid, which in turn is assumed to have the same concentration as the bulk. The interfacial area is calculated by assuming the foam bubbles to be pentagonal dodecahedral in shape. The model has been found to explain the results of foam separation of cells quite well, particularly with respect to the effect of bubble size and aeration rate.
Recirculating bioreactor–separator system for simultaneous biotransformation and recovery of product: Immobilized L‐aspartate β‐decarboxylase reactor system
Authors: Takamatsu Satoru , Ryu Dewey D. Y.
Published: December 2004

A new combined bioreactor‐separator system was designed and its operational feasibility demonstrated in order to develop a bioprocess that enables us to handle simultaneous biotransformation and recovery of product by crystallization. Enzymatic conversion of L‐aspartate to L‐alanine by L‐aspartate β‐decarboxylase from Pseudomonas dacunhae (ATCC 21192) was used as a model system for this study to demonstrate the principles involved in the bioprocess design. Immobilized cells of P. dacunhae containing the enzyme were fluidized in a tapered column type of bioreactor and a filter‐crystallizer combination was used as a separator unit in our experimental system.

It was found that almost a theoretical yield was achieved, and the process control for both the bioreactor operation and separation was relatively easy. The Production systems, namely, the recirculating bioreactor separator combination system and the conventional batch reactor system, were analyzed and compared based on the results obtained form this study, and it was found that a significant cost reduction, by about 20%, can be achieved when the recirculating bioreactor–separator combination system was employed. Based on these findings, it is anticipated that the conceptual design of the bioreactor–separator combination system evaluated in this study has some potential for industrial application.
Design and mathematical description of differential contactors used in extractive fermentations
Authors: Roffler Stephen R. , Wilke Charles R. , Blanch Harvey W.
Published: December 2004

The performance of differential contactors for use in extractive fermentation is complicated by the effects of product formation in the contactor. When product formation is significant, approximate analytical solutions are presented for the performance of the contactor for two limiting cases: high and low substrate concentrations. When products are formed at a constant rate, there is a minimum raffinate solute concentration that can be obtained, in contrast to the behavior of a column in the absence of product formation. General equations describing the behavior of the system for product formation with backmixing in both phases are presented. The case of a stripping factor not equal to unity is considered.
Aerobic fungal cell immobilization in a dual hollow‐fiber bioreactor: Continuous production of a citric acid
Authors: Chung Bong Hyun , Chang Ho Nam
Published: December 2004

Aspergillus niger B60 was immobilized in a dual hollow‐fiber bioreactor (DHFBR) to produce citric acid continuously. The fungi proliferated well in the interstitial region formed by a parallel arrangement of three microporous polypropylene hollow fibers contained within a silicone tube. Long‐term operation with nitrogen‐enriched medium was not possible due to expansion of the silicone tubes by continual cell growth. The fungal growth could be controlled by supplying a nitrogen‐deficient medium at the production stage. With pure oxygen aeration and nitrogen‐deficient medium, volumetric productivity reached 1.62 g/L h at a residence time of 4.02 h, which corresponded to a 27‐fold increase over that of shake‐flask fermentation. When the residence time was increased to 20.1 h, citric acid at a concentration of 26 g/L was continuously produced, with a yield of 80‐90% and a volumetric productivity of 1.3 g/L h. This represents a significant improvement in final concentration, yield, and the volumetric productivity over the equivalent values of the corresponding batch fermentation, which were 18 g/L, 40%, and 0.06 g/L h, respectively.
A constant‐depth laboratory model film fermentor
Authors: Peters A. C. , Wimpenny J. W. T.
Published: December 2004

A laboratory model constant‐depth film fermentor was developed. Film grew on the surface of polytetrafluoroethylene (PTFE) plugs and was limited to a predetermined depth by mechanically removing excess film. Six film‐forming organisms were isolated from river water and used to assess the operating characteristics of the fermentor. Film accumulation was logarithmic, and a steady state was maintained. Electron micrographs show early film development. The fermentor enables film to be grown on any substratum and allows discrete, reproducible, and representative samples to be taken.
Effects of charged groups in haptens on adsorption equilibrium of hapten antibody
Authors: Sada Eizo , Katoh Shigeo , Miyoshi Hirotoshi , Yamanaka Kuniko , Kondo Akihiko
Published: December 2004

Antisera against charged (p‐azobenzoate and p‐azoben zenesulfonate) and uncharged (dinitrophenyl) haptenic groups were produced in rabbits, and the equilibrium characteristics of hapten‐antibody were measured by use of immunoadsorbents. The antibody to the uncharged hapten formed a stable binding with the hapten to the changes in ionic strength and pH. On the other hand, the antibodies to the charged haptens showed affinities sensitive to the changes in pH and ionic strength. Therefore, the effect of the pKa of ionizable haptens on the pH dependence of the hapten‐antibody binding was studied by comparing the interactions between a series of para‐substituted benzoic acids and the anti‐p‐azobenzoate antibody. The pH dependence of the interactions was strongly affected by the pKa of ionizable groups in haptens. Furthermore, the equilibrium characteristics of anti‐p‐aminobenzoyl dipeptides were compared. The characteristics of interactions were affected by the features of amino acid residues.
A new immobilization technique of whole cells and enzymes with colloidal silica and alginate
Authors: Fukushima Yaichi , Okamura Katsutoshi , Imai Kazutaka , Motai Hiroshi
Published: December 2004

A mixed gel composed of colloidal silica and alginate (As gel) was prepared for the immobilization of enzymes or microorganisms. The physical strength of AS gel increased with the amount of colloidal silica. The ethanol production rate of Saccharomyces cerevisiae (IFO 0224) immobilized in AS gel was higher than in alginate gel (Al gel) in the early phase of growth. At a concentration of glucose of more than 10%, the ethanol production of immobilized yeast in AS gel was higher than in Al gel. Any difference was not recognized in the diffusion coefficient of glucose between AS and Al gels. The AS gel had an ability to retain proteins such as bovine serum albumin and γ‐globulin. The alkaline protease and β‐galactosidase in AS gel continued their function for a long time, but those immobilized in Al gel did not. Immobilized β‐galactosidase in AS gel had a higher thermal stability than in Al gel or free enzymes.
Membrane reactor coupled with electrophoresis for enzymatic production of aspartic acid
Authors: Lee C. K. , Hong J.
Published: December 2004

Aspartic acid production by aspartase reaction on ammonium fumarate was carried out in a membrane reactor coupled with electrophoresis. A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. An electric field was applied across the membrane to preferentially remove the product aspartate from the reactor into the permeate stream. The charged molecule, aspartate, is much smaller than the molecular‐weight cutoff of the membrane (104) so that the ions would move freely through pores of the membrane. The concentration of aspartate in the permeate stream is determined by the electromigration velocity of the ions and the permeation rate of solvent (water) through the membrane. The permeation rate of solvent could be controlled by the applied pressure, and the migration velocity of the ions could be controlled by the electric field strength applied. The equilibrium conversion of ammonium fumarate to the aspartate was 70%. In the presence of electric field, the aspartase activity was not disturbed. Also, it is shown that the aspartate concentration in the permeate stream was 20% higher than that in the reaction solution with the permeate flow rate of 0.7 mL/min. The steady‐state conversion was 60%. Instead of aspartate, aspartic acid can be recovered directly from the permeate stream by controlling the circulation of buffer electrolyte in the anode compartment.
Polyelectrolyte precipitation of proteins: I. The effect of reactor conditions
Authors: Fisher Rod R. , Glatz Charles E.
Published: December 2004

Lysozyme was recovered from egg white by continuous precipitation with polyacrylic acid (molecular weight of 4 × 106). Precipitator residence time and shear rate had significant effects on the size distribution of the precipitate, but no clear effects on the compositions. Precipitate mean size increased with higher shear, indicating growth phenomena predominating over breakage. Also, an enhancement of growth rate at small sizes was noted. The Camp number successfully characterized the interaction of shear rate and residence time on the particle size.
Adsorption of Trichoderma reesei cellulase on cellulose: Experimental data and their analysis by different equations
Authors: Steiner W. , Sattler W. , Esterbauer H.
Published: December 2004

The adsorption of cellulase from Trichoderma reesei MCG 77 on Avicel was measured at varying cellulase (2–8 g/L) and Avicel (10–200 g/L) concentrations at pH 4.8 and 50°C. Different mathematical equations were derived for the evaluation of the experimental data. The fraction of cellulase protein that can maximally be adsorbed is 0.96, and 1 g Avicel can bind maximally 0.092 g cellulase protein. The Michaelis constant for the adsorption equilibrium [cellulase] + [Avicel] ⇌ [cellulase Avicel] complex is between 2.0 and 2.3 · 10−5 mol/L. This value is based on the assumption that cellulase has an average molecular weight of 48.000. The apparent molecular weight of Avicel, i.e., that amount in grams that can bind 1 mol cellulase, is 520,000. Under maximum binding the enzyme covers on Avicel a surface of 42 m2/g, and the occupied volume is 0.186 cm3/g Avicel.
Effects of microcarrier concentration in animal cell culture
Authors: Croughan Matthew Shane , Hamel Jean‐François P. , Wang Daniel I. C.
Published: December 2004

Results are presented which show how the microcarrier concentration affects the hydrodynamic environment in animal cell bioreactors. At low levels of agitation, no physical effects of microcarrier concentration were found. However, cell growth was strongly influenced by cell concentration. At high levels of agitation, a strong detrimental effect of microcarrier concentration was found. A new mechanism of hydrodynamic damage was identified which is second order in microcarrier concentration. The identification of this mechanism adds to the fundamental understanding of hydrodynamic phenomena in microcarrier bioreactors.
Shear stress induced stimulation of mammalian cell metabolism
Authors: Frangos J. A. , McIntire L. V. , Eskin S. G.
Published: December 2004

A flow apparatus has been developed for the study of the metabolic response of anchorage‐dependent cells to a wide range of steady and pulsatile shear stresses under well‐controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/cm2, and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. This study demonstrates that shear stress in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.
Enzymatic transesterifications of carbonates in water‐restricted environments
Authors: Abramowicz Daniel A. , Keese Charles R.
Published: December 2004

We investigated the ability of several hydrolases to catalyze reactions with an abiotic water‐insoluble substrate, carbonic acid diphenyl ester, also known as diphenyl carbonate (DPC). In single‐phase water/organic systems, turnover numbers (TN) of greater than 2 × 104 min−1have been achieved for the hydrolysis of DPC. The Km values for the hydrolytic reaction were measured to be 200μM and 330μM for Candida cylindracea lipase and Porcine liver esterase, respectively. In addition to hydrolysis, we observed transesterification of carbonates with a wide variety of alcohol and phenol species. Transesterifications of DPC with bifunctional alcohols resulted in the synthesis of polycarbonates. We investigated the stability and transesterification activity of these enzymes in several water‐restricted environments to limit competing hydrolysis reactions. We find that, with the removal of water, hydrolysis is reduced more than four orders of magnitude while transesterification is diminished only 10‐fold (turnover numbers of 600 min−1 in water‐miscible systems to 60 min−1 in water‐restricted environments with pure Candida lipase). Stability of the Candida lipase in these water‐restricted environments (half‐life of longer than 3 days) is much greater than in water/organic single phase systems (5 h in 20% methanol). In addition, the Candida lipase displayed enantiomeric selectivity in transesterifications of DPC with racemic 2‐butanol (greater than 80% ee).
Energy requirements for the size reduction of poplar and aspen wood
Authors: Holtzapple M. T. , Humphrey A. E. , Taylor J. D.
Published: December 2004

The energy requirements associated with conventional mechanical size reduction of poplar and aspen wood are compared to a new method of size reduction employing a wood planer. Although the planer requires about 2.3 times less energy to achieve the same size reduction as conventional methods, large‐scale equipment to implement this approach does not currently exist. Explosive depressurization was also compared to conventional mechanical size reduction. The conventional mechanical methods require roughly 70% more energy to achieve the same size reduction as explosive depressurization. Thus, explosive depressurization appears to be the preferred method and has the added benefit of altering the chemical structure of the wood to enhance the enzymatic hydrolysis of the cellulose fraction.
Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells
Authors: Mulchandani Ashok , Luong John H. T. , LeDuy Anh
Published: December 2004

Immobilization of Aureobasidium pullulans by adsorption on solid supports and entrapment in open pore polyurethane foam were attempted. By adsorption, the highest cell loading of 0.012–0.018 g dry wt/cm2 support was obtained in pH 2.0 medium. Under this acidic condition, the net surface charges (zeta potentials) of both the cells and supports were close to zero and no pullulan was synthesized. Cationic coatings of Cytodex and polyethylenimine were not efficient in enhancing the binding strength between the cells and the supports. Surface immobilized cells and polyurethane foam entrapped cells exhibited a similar fermentation characteristics resulting in ca. 18 g/L pullulan and ca. 5 g/L leaked cells. However, cells entrapped in the polyurethane foam were more shear resistant. The immobilized cells thus could be repeatedly used for pullulan biosynthesis.
Immobilized enzymes in reverse micelles: Studies with gel‐entrapped trypsin and α‐chymotrypsin in AOT reverse micelles
Authors: Fadnavis Nitin W. , Luisi Pier Luigi
Published: December 2004

Trypsin and α‐chymotrypsin were immobilized by gelentrapment in polyacrylamide cross‐linked with N,N1‐methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30‐40% activity after 1 week. The enzymes obey Michaelis‐Menten kinetics in the investigated concentration range with Km values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nα‐benzoyl‐L‐arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., ZAlaPheLeuNH2) using water‐insoluble substrate has been performed with gelentrapped α‐chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.
Disruption of native and recombinant Escherichia coli in a high‐pressure homogenizer
Authors: Sauer Thomas , Robinson Campbell W. , Glick Bernard R.
Published: December 2004

The disruption of native and recombinant strains of Escherichia coli was studied using a high‐pressure homogenizer (Microfluidizer). The cells were grown in both batch and continuous fermentations. Cell suspensions ranging from 4 to 175 g dry wt/L were investigated at disruption pressures ranging from 30–95 MPa and at up to five passes. For both types of cells, the fraction of cells disrupted was dependent on the growth rate and concentration of the cells, the disruption pressure, and the number of passes through the disrupter. A model is presented that correlates the fractional disruption with these operating variables. The recombinant strain disrupted more readily than the native strain; 95 to 98% disruption of the former was achieved in two to three passes at a pressure of 95 MPa.
Dynamic pressure method for kla measurement in large‐scale bioreactors
Authors: Linek V. , Beneš P. , Vacek V.
Published: December 2004

A dynamic method is proposed for kla measurement in aerated and agitated reactors, in which a change in the total pressure in the reactor by approximately 20% leads to a simultaneous change in the oxygen concentration in all the bubbles in the dispersion. This procedure suppresses the influence of nonideal mixing of the gas phase on the kla value. Other dynamic methods so far used do not possess this advantage. They are based on a step change in oxygen concentration in the entering gas, where the interfacial nitrogen transport and the finite rate of the concentration change propagation into the individual bubbles in the dispersion can cause an error in the reported kla values of more than hundreds of percent. The reliability of the pressure method is tested by comparison both with the standard dynamic method, in which pure oxygen is absorbed in a liquid from which all other gas components were previously removed, and with the steady‐state sulphite method. The signal of the oxygen probe used in the experiments must be independent of the pressure. A test for this in dependence is described. The pressure method is also suitable for large‐scale reactors since the necessary pressure changes are sufficiently small and, morever, air can be used.
A new method for the adaptive determination of optimum pH and temperature
Authors: Harmon Jeff. L , Lyberatos Gerasimos , Svoronos Spyros A.
Published: December 2004

An adaptive control algorithm for the on‐line determination of optimal temperature or pH for biomass production in a continuous fermentor is presented. The algorithm requires no prior information and uses a dynamic Hammerstein model to identify parameters and to estimate an optimal steady‐state control value. A check of the estimated performance measure second derivative is included to ensure that the target extremum is an optimum. The process is driven towards this optimum with a variable step size that depends on the quality of the on‐line identified model. Numerical simulations are performed on a dynamic chemostat model that incorporates a metabolic time delay. The algorithm successfully finds the optimum temperature or pH values and maintains the reactor at the optimum steady state.
Formal modeling of approximate relations in biochemical systems
Authors: Mavrovouniotis Michael L. , Stephanopoulos George , Stephanopoulos Gregory
Published: December 2004

Aiming to satisfy the need for the formalization of semiquantitative reasoning in the analysis of biochemical systems, the O[M] formalism for reasoning with orders of magnitude and approximate relations has been developed. It is based on seven primitive relations among quantities and compound relations which are formed as implicit disjunctions of primitives. O[M] can perform inferences by using formal approximate relations, algebraic equations, inequalities, if–then rules, assumptions, and goals. The applications discussed include Michaelis–Menten kinetics, different modes of inhibition of an enzymatic reaction, analysis of fluxes in biochemical networks, and identification of rate‐limiting steps of biochemical pathways. In these applications, O[M] provides a medium for acquisition and formalization of previously informal concepts, analysis of systems at the order‐of‐magnitude level of detail, and automation of commonsense reasoning.
Economic evaluation of preconcentration in production of ethanol from dilute sugar solutions
Authors: Zacchi G. , Axelsson A.
Published: December 2004

The economic feasibility of preconcentrating dilute sugar solutions prior to fermentation is investigated. Two methods, evaporation and reverse osmosis, are compared. A computer program to determine the optimal preconcentration conditions for glucose solutions of 1.5–16 wt% has been developed. It was used to compute the fractional cost for labor and maintenance, preconcentration, fermentation, and distillation. Preconcentration with evaporation resulted in a higher total cost, compared with no preconcentration, for all cases studied, although a six‐effect unit was used. Reverse osmosis was found to be economically feasible for preconcentration to about 5–10 wt % depending on the concentration of the feed.
Bioactive polymers. 56: Urease immobilization on carboxymethylcellulose
Authors: Dumitriu Severian , Popa Marcel , Artenie Vlad , Dan Florin
Published: December 2004

This article reports on the reaction of urease immobilization through its covalent bonding on carboxymethylcellulose. The reaction is activated by dicyclohexylcarbodiimide. The coupling reaction is influenced by the enzyme–support and activator–support ratios, as well as by duration. Starting from a rotating, composed experimental program of the second order, the function correlating the activity of the immobilized enzyme with the reaction parameters is established. Immobilized urease exhibits thermal stability higher than that of free enzyme, regarding both pH and the inhibiting action of some metal ions or organic substances. The stability over time of the immobilized urease is high, its enzymatic activity being maintained at over 85% of the initial value three months after synthesis.
Improved method for preparing high maltose conversion syrups
Authors: Saha Badal C. , Zeikus J. Gregory
Published: December 2004

An improved method is presented for producing high maltose conversion syrups from liquefied and raw starch. It comprises saccharifying the starch at higher temperatures than presently used with environmentally compatible thermostable β‐amylase and other thermostable enzymes.
Theoretical analysis of G6P production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow‐fiber reactor
Authors: Ishikawa Haruo , Tanaka Takenori , Takase Susumu , Hikita Haruo
Published: December 2004

The performance of an ultrafiltration hollow‐fiber reactor, in which the enzymatic synthesis of glucose 6‐phosphate from glucose and cofactor ATP and the enzymatic regeneration of ATP from ADP and acetyl phosphate are performed simultaneously, was analyzed theoretically. A simple analytical model in which the liquid flowing in the fiber tubes is assumed to be plug flow, and the radial concentration gradients in the tube and shell sides are both neglected, could simulate the reactor performance with satisfactory accuracy. The simulation elucidated the effects of the reactor configurations and various operational conditions on glucose conversion, ATP recycle number, and space–time yield. If the fiber tubes, through which the permeability of the relevant components such as substrates is high, were packed as much as possible in the reactor, good reactor performance could be expected. Furthermore, with a sufficiently high enzyme concentration, low ATP concentration in the feed solution, and appropriate space velocity, good space–time yield with high glucose conversion and with very high ATP recycle number is theoretically possible.
Continuous production of 3‐fluoro‐L‐alanine with alanine dehydrogenase
Authors: Ohshima Toshihisa , Wandrey Christian , Conrad Dietrich
Published: December 2004

Alanine dehydrogenase catalyzed the conversion of 3‐fluoropyruvate into 3‐fluoro‐L‐alanine in the presence of NADH and ammonia. The optimum pH of the reaction was 7.8. The Km values of the enzyme for 3‐fluoropyruvate, polyethylene glycol‐bound NADH, and ammonia were 2.94, 0.56, and 105mM, respectively. 3‐Fluoro‐L‐alanine was selectively and continuously produced from 3‐fluoropyruvate and ammonium formate in an enzyme membrane reactor by the multienzyme reaction system of alanine dehydrogenase and formate dehydrogenase with a simultaneous coenzyme regeneration. The average conversion and the space–time yield were 73% and 75 g/L day, respectively, with operation of the reactor for 4 days. Alanine dehydrogenase and formate dehydrogenase consumed were 11, 370 and 22, 950 units/kg 3‐fluoro‐L‐alanine, respectively. The cycle number was 3150 mol/mol NAD.
Mathematical model for microbial oxidation of pure lead sulfide by Thiobacillus ferrooxidans
Authors: Kargi Fikret
Published: December 2004

A shrinking‐core mathematical model describing bioleaching of lead sulfide is developed considering the deposition of insoluble bio‐oxidation products on metal sulfide particle surfaces. Variations in particle size are considered as it affects diffusion limitations.
Esterification‐coupled extraction of organic acids: Partition enhancement and underlying reaction and distribution equilibria
Authors: Aires‐Barros M. R. , Cabral J. M. S. , Willson R. C. , Hamel J.‐F. P. , Cooney C. L.
Published: December 2004

A novel means of extracting carboxylic acids from aqueous solutions is described which involves the use of a lipolytic enzyme to convert the acid to a more hydrophobic ester. A water‐immiscible long‐chain alcohol serves both as a reactant in esterifying the acid, and as a solvent for extraction of the ester. Radiochemical tracer studies of the underlying reaction and extraction equilibria established that a low equilibrium concentration of ester (0.04–0.5mM) in the presence of water is counterbalanced by the high distribution coefficient of the ester (220–3380, wt. basis). The net result is a substantial (4‐ to 15‐fold) increase in the apparent distribution coefficient of the acid; 80%–95% of the extracted acid is in the esterified form. The method is applicable to a variety of alcohols and acids.
Integral kinetics of α‐galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose
Authors: Porter Jill E. , Herrmann Klaus M. , Ladisch Michael R.
Published: December 2004

α‐Galactosidase from soybean (Glycine max) was purified by a five‐step procedure. The enzyme's natural substrates, raffinose and stachyose, have Km's of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a Ki of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first α‐(1,6)‐bond in the tetrasaccharide is the rate‐limiting step. Since the stachyose hydrolysis yields raffinose, soybean α‐galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.
Morphological measurements on filamentous microorganisms by fully automatic image analysis
Authors: Packer H. L. , Thomas C. R.
Published: December 2004

Characterization of mycelial morphology is important for physiological and engineering studies of filamentous fermentations, and in the design and operation of such fermentations. Image analysis has been developed as a method for this characterization, and has been shown to be faster and generally more accurate than previous methods. A fully automatic system has been developed, in which speed is gained, but with loss of accuracy in some cases. The method has been tested on Streptomyces clavuligerus and Penicillium chrysogenum P1 batch fermentations. It has also been tested on a fed‐batch Penicillium chrysogenum P2 fermentation, in which the medium contained solid ingredients. Fully automatic image analysis for morphological characterization of filamentous microorganisms is an important development which will make practical many engineering and physiological studies of such fermentations that have so far not been completely satisfactory.
Pellet formation and cellular aggregation in Streptomyces tendae
Authors: Vecht‐Lifshitz S. E. , Magdassi S. , Braun S.
Published: December 2004

In submerged cultures, Streptomyces tendae tended to form fluffy spherical pellets of the noncoagulative type. An increase in the average pellet size could be attained by decreasing any of the following: shear rate, pH, temperature, or inoculum size. Conditions leading to oxygen limitation tended to reduce the average pellet size and induced pulpy growth, whereas oxygen sufficiency seemed to induce pellet formation. Factors inducing pellet formation simultaneously increased cell wall hydrophobicity. It is therefore proposed that the main forces inducing cellular aggregation in S. tendae are hydrophobic interactions of cell walls, and these interactions are controlled by availability of dissolved oxygen.
Effects of stirring and sparging on cultured hybridoma cells
Authors: Gardner A. R. , Gainer J. L. , Kirwan D. J.
Published: December 2004

The response of hybridoma cells to fluid shear caused by stirring and sparging has been investigated in a 2‐L turbine‐agitated bioreactor. Viable cell count, lactate dehydrogenase (LDH) release, and antibody secretion were measured over the course of batch culture experiments under varied conditions of stirring and gas sparging. The effectiveness of Pluronic F68 as a protective agent in sparged cultures was also studied. Growth was found to be unaffected by stirring of the culture under surface aerated conditions, but gas sparging had a significant detrimental effect on growth and antibody production. The effect of sparging was reduced when cultures were supplemented with Pluronic at a level of 0.4% (w/v). Experimental data were analyzed through formulation of models for LDH release and antibody production. Rates of cell lysis could be estimated by correlating extracellular LDH levels through the model for LDH release. The lysis rate estimated for sparged conditions was sufficiently large to approximately account for the observed decrease in the specific growth rate of the culture. The presence of Pluronic apparently interfered with the LDH release mechanism, so precise estimation of lysis rates under these conditions was not possible. Sparging was found not to have a detrimental effect on antibody production in cultures without Pluronic added. Specific antibody production rates in cultures supplemented with Pluronic were about 25% higher than in sparged cultures without Pluronic added.
Oxygen and carbon dioxide mass transfer and the aerobic, autotrophic cultivation of moderate and extreme thermophiles: A case study related to the microbial desulfurization of coal
Authors: Boogerd F. C. , Bos P. , Kuenen J. G. , Heijnen J. J. , van der Lans R. G. J. M.
Published: December 2004

Mass transfers of O2, CO2, and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high‐pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, kLa measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H2SO4 in water) phase as a function of the temperature has been studied in a laboratory‐scale fermentor. At 15, 30, 45, and 70°C, (kLa)o values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (kLa)o was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (kLa)o was plotted against the temperature. By multiplying the equilibrium concentration of O2 in water with (kLa)o maximal, O2 transfer capacities were calculated. It appeared that in finite of a decreased solubility of O2 at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70°C. However, in an air‐mixed fermentor the transfer capacity of O2 decreased slowly but steadily.

Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO2 transfer capacity, calculated as the product of the equilibrium CO2 concentration times (kLa)c, decreased slowly as the temperature increased over the range 15–70°C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO2 limitation is more likely to occur than O2 limitation.
Formation of CC bonds by mandelonitrile lyase in organic solvents
Authors: Wehtje Ernst , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

Mandelonitrile lyase (EC 4.1.2.10) catalyzes the formation of D‐mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production of D‐mandelonitrile. In order to obtain optically pure D‐mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture of D, L‐mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity of D‐mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in product yield.
Kinetics of CO2 hydration in fermentors: pH and pressure effects
Authors: Yegneswaran P. K. , Gray M. R. , Thompson B. G.
Published: December 2004

Fluctuations in pH and head‐space pressure in a fermentor introduce temporary changes in off‐gas CO2 concentrations. These changes are quantified using a simple model based on kinetics of CO2 hydration and gas–liquid mass transfer. The model is verified experimentally. An eigenvalue analysis of the model indicates that mass transfer is the parameter which controls the dynamics of CO2 equilibration.
A comparison of mathematical model predictions to experimental measurements for growth and recombinant protein production in induced cultures of Escherichia coli
Authors: Betenbaugh Michael J. , Dhurjati Prasad
Published: December 2004

Recombinant cell growth and protein synthesis by a recombinant Escherichia coli under various inducing conditions are compared to the predictions of a mathematical model. The mathematical model used was a combination of two literature models: (1) an empirical kinetic model for recombinant growth and product formation and (2) a genetically structured model of the lac promoter‐operator on a multicopy plasmid. The experimental system utilized was recombinant E. coli CSH22 bearing the temperature‐sensitive plasmid pVH106/172, which codes for the synthesis of β‐galactosidase and the other lac operon genes under the control of a lac promoter. Mathematical model predictions for recombinant β‐galactosidase yield and specific growth rate were compared with fermentation measurements of these same quantities for conditions of chemical induction with cyclic AMP and IPTG, copy number amplification (by shifting culture temperature), and combined chemical induction and copy number amplification. The model successfully predicted experimental product yields for most cases of chemical induction even though the product yields varied from 0.34 × 103 to 1500 × 103 units/g cell mass. The kinetic model also correctly predicted a decline in the specific growth rate with increasing levels of plasmid and recombinant protein. The model was less successful at predicting product amplification at high copy numbers. A comparison of model predictions and experimental results was also used to investigate some of the assumptions used in constructing the mathematical models.
Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: I. Modeling and simulation
Authors: Vos Henk J. , Groen Dick J. , Potters Jacques J. M. , Luyben Karel Ch. A. M.
Published: December 2004

For the application of immobilized enzymes, fixed bed reactors are used almost exclusively. Fixed bed reactors have specific disadvantages, especially for processes with a deactivating catalyst. Therefore, we have studied a novel reactor type with continuous transport of the immobilized biocatalyst. Flow of biocatalyst is countercurrent to the substrate solution. Because of a stagewise reactor design, back‐mixing of biocatalyst is very limited and transport is nearly plug flow. The reactor operates at a constant flow rate and conversion, due to constant holdup of catalytic activity. The reactor performance is compared with a configuration of fixed bed reactors. For reactions in the first‐order regime, enzyme requirements in this new reactor are slightly less than for fixed bed processes. The multistage fluidized bed appears to be an attractive reactor design to use biocatalyst to a low residual activity. However, nonuniformity of the particles might affect plug flow transport of the biocatalyst. A laboratory scale reactor and experiments are described in Part II1 of this series. Hydrodynamic design aspects of a multistage fluidized bed are discussed in more detail in Part III.2
Mechanisms for biopolymer accumulation in immobilized Acinetobacter calcoaceticus system
Authors: Wang Sy‐Dar , Wang Daniel I. C.
Published: December 2004

The gram‐positive bacteria, Acinetobacter calcoaceticus, is capable of accumulating biopolymer in the carrier matrix of an immobilized cell system. Several possible mechanisms for the biopolymer accumulation are evaluated. It appears that direct solid surface polymer adsorption and polymer diffusion limitation within the pore space are minor factors in biopolymer accumulation. Calculations demonstrate that the cell bound polymer to dry cell weight ratio is much higher for immobilized cells than for free cells. The higher cell‐bound polymer to dry‐cell‐weight ratio for immobilized cells as well as the accumulation of the immobilized cells in the Celite matrix are believed to be the main factors for biopolymer accumulation in the Carrier matrix. Further studies reveal that the cell‐bound polymer to dry‐cell‐weight ratio is strongly affected by shear forces. At zero shear stress, such as would be present in the carrier matrix, cell bound polymer to dry cell weight ratio can be as high as 1.6. As the shear stress increases, this ratio decreases. When shear stress increases above 5 dyn/cm2, a level equivalent to the shear experienced by free cells in a stirred tank fermentation, cell‐bound polymer decreases to less than 20% of dry cell weight. A macroscopic model is developed to describe the effect of shear stress on the cell‐bound polymer to dry‐cell‐weight ratio.