bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix method for determining steps most rate‐limiting to metabolic fluxes in biotechnological processes
Authors: Westerhoff Hans V. , Kell Douglas B.
Published: December 2004

The metabolic control theory developed by Kacser, Burns, Heinrich, and Rapoport is briefly outlined, extended, and transformed so as optimally to address some biotechnological questions. The extensions include (i) a new theorem that relates the control of metabolite concentrations by enzyme activities to flux ratios at branches in metabolic pathways; (ii) a new theorem that does the same for the control of the distribution of the flux over two branches; (iii) a method that expresses these controls into properties (the so‐called elasticity coefficients) of the enzymes in the pathway; and (iv) a theorem that relates the effects of changes in metabolite concentrations on reaction rates to the effects of changes in enzyme properties on the same rates. Matrix equations relating the flux control and concentration control coefficients to the elasticity coefficients of enzymes in simple linear and branched pathways incorporating feedback are given, together with their general solutions and a numerical example. These equations allow one to develop rigorous criteria by which to decide the optimal strategy for the improvement of a microbial process. We show how this could be used in deciding which property of which enzyme should be changed in order to obtain the maximal concentration of a metabolite or the maximal metabolic flux.
Forced‐flow electrophoresis of proteins and viruses
Authors: Mullon C. , Mason N. , Sparks R.
Published: December 2004

This article describes a model for forced‐flow electrophoresis (FFE), considering the desired species fraction removal, other fraction removals, and outlet concentrations of all species in the system. The model predicts the necessary inlet flow rate of the retentate chamber and the rate of filtration and the voltage gradient and also provides an appropriate heat balance permitting consideration of possible heat denaturation of the species. The equipment consists of two membranes and a filter, the electric field being imposed by means of external electrodes, and two fractions are obtainable. The main discriminating factor is not the pore sizes of the filter but the relative solute ionization, which depends on the pH and the ionic strength of the buffer solution. Serum proteins (albumin, γ‐globulin) and bacteriophages (M13, MS2, ϕX174) have been used to characterize the separation process.
A comparison of some methods of estimating volumetric mass‐transfer coefficients in an external‐loop airlift fermenter
Authors: Kamp F. , Wase D. A. J. , McManamey W. J. , Mendoza O. , Thayanithy K.
Published: December 2004

Volumetric mass‐transfer coefficients were measured in an 11‐L external‐loop airlift fermenter with deionized water, a fermentation medium, and during a fermentation. Both a Mackareth oxygen electrode and a novel rapid‐response probe were used. When the conventional step‐change dynamic method was used for water, the long, nonlinear response time of the Mackareth electrode made correction of its readings difficult; this problem did not occur when the rapid‐response probe was used. A comparison was made with a method of mass‐transfer coefficient determination which does not involve any assumptions about the gas residence time distribution. However, this method requires that the liquid phase is well‐mixed and this requirement was not met in the airlift fermenter. Comparison of the present results with other KL a determinations for airlift fermenters showed that KL a in water depends on the active gas holdup, the value of KL a/ϵ at 20°C being ca. 0.37 s−1. Although higher gas holdups were obtained with the fermentation medium than for water, the values of KL a/ϵ were lower, ca. 0.22 s−1 at 20°C.
Co‐operative binding interactions in affinity chromatography: Theoretical considerations
Authors: Hubble John
Published: December 2004

A theoretical relationship has been developed to allow the effect of free ligand concentration on the capacity of an affinity Chromatography matrix to be determined where the protein adsorbed shows co‐operative binding. Computer simulations using literature values for association constants show that under optimal conditions resin capacity can be increased significantly in the presence of a small but finite concentration of free ligand. The model also allows prediction of the soluble ligand concentration required for biospecific elution. The results obtained suggest the possibility of a new elution technique, “reverse biospecific elution,” that reduces the amount of free ligand required to effect elution.
Immobilized heparinase: In vitro reactor model
Authors: Bernstein Howard , Yang Victor C. , Langer Robert
Published: December 2004

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 ± 0.048 (N = 10). The immobilized enzyme has a Km of 0.15 ± 0.03 mg/mL and an activation energy of 10.3 ± 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 × 106 daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.
Stratified ecology techniques in the startup of an anaerobic downflow fixed film percolating reactor
Authors: Canovas‐Diaz M. , Howell J. A.
Published: December 2004

A two‐column pilot scale downflow percolating fixed film (DPFF) anaerobic reactor treating deproteinized cheese whey (DPW) was used in this study. The system was operated at one‐third of the maximum working volume in an attempt to avoid mass transfer limitations of the gas products (since two‐thirds of the immobilized microbial culture was unsubmerged), as well as to minimize the effects of localized high volatile fatty acids (VFA) levels and low pH on its methanogenic activity. A new method to shorten the start‐up period was used by growing methanogenic and acetogenic (H2‐producers) bacteria on a synthetic medium until reaching a stable methane production rate and then switching the reactor over to DPW and allowing acidogenic growth. The start‐up period was reduced to 35 days as a result of this operational mode. The reactor was able to handle 8 kg COD/ m3·d after 35 days of operation.
Catabolite repression of amylase synthesis in yeast
Authors: Pasari Ajay B. , Korus Roger A. , Williams Ronald A. , Heimsch Richard C.
Published: December 2004

Amylase synthesis by the yeasts Saccharomycopsis fibuligera and Schwanniomyces castellii and alluvius is repressed by glucose. Steady state continuous culture data for amylase activity, E, biomass concentration, X, and reducing sugar concentration, S, were fitted to the three‐parameter catabolite repression model

and biomass productivity, DX, and amylase productivity, DE, were determined for S. castellii and S. alluvius.
A mathematical model for prediction of plasmid copy number and genetic stability in Escherichia coli
Authors: Ataai M. M. , Shuler M. L.
Published: December 2004

The design of bioreactors for genetically modified bacterial cultures would benefit from predictive models. Of particular importance is the interaction of the external environment, cell physiology, and control of plasmid copy number. We have recently developed a model based on the molecular mechanisms for control of replication of Co1E1 type plasmids. The inclusion of the plasmid model into a single‐cell E. coli model allows the explicit prediction of the interaction of cell physiology and plasmid‐encoded functions. The model predictions of the copy number of plasmids with the Co1E1 origin of replication carrying a variety of regulatory mutations is very close to that observed experimentally.

All of the model parameters for plasmid replication control can be obtained independently and no adjustable parameters are needed for the plasmid model. In this article we discuss the model's use in predicting the effect of operating conditions on production of a protein from a plasmid encoded gene and the stability of the recombinant cells in a continuous culture.
Dynamics of a biological fixed film for phenol degradation in a fluidized‐bed bioreactor
Authors: Worden Robert Mark , Donaldson Terrence L.
Published: December 2004

Experimental and modeling studies were conducted to analyze the dynamic response behavior of a phenol‐oxidizing fixed film using a differential, fluidized‐bed bioreactor in a recycle loop with a well‐mixed reservoir. With the bioreactor at steady state, a pulse of phenol was added to perturb the system, and the phenol concentration was monitored continuously until steady state was again achieved.

The experimental dynamics were compared with a dynamic mathematical model based on diffusion and reaction within the biofilm, liquid mixing, and biofilm growth. Constant‐pH experiments could be adequately described using an unstructured, double‐Monod kinetic expression with substrate inhibition by phenol.

However, in dynamic experiments without pH control, the pH of the liquid phase dropped, and damped oscillations were observed in the phenol concentration and reaction rate trajectories. Oscillatory solutions could not be induced in the model, even when product inhibition was included, and a linear stability analysis did not reveal tendencies toward instability. The cause of the experimental oscillations remains unknown.
Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media
Authors: de Boer E. , Plat H. , Tromp M. G. M. , Wever R. , Franssen M. C. R. , van der Plas H. C. , Meijer E. M. , Schoemaker H. E.
Published: December 2004

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3′,5,5′‐tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper‐oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1‐propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.
Characteristics of immobilized yeast reactors producing ethanol from glucose
Authors: Anselme M. J. , Tedder D. W.
Published: December 2004

The evolutionary performance of up flow reactors are affected by the cell immobilization matrix and the matrix particle size distribution. Higher productivities are obtained using a low‐density brick with a particle size of ca. 400–1400 μm. A medium condition favoring growth quickly leads to large biomass gradients within the reactors and, eventually, reductions in average productivities due to bed plugging. These systems can be accurately modelled using Monod kinetics when dispersion and the biomass gradient are considered. The productivity was apparently not controlled by substrate diffusion in these cases.
Potential enhancement of photosynthetic energy conversion in algal mass culture
Authors: Sukenik A. , Falkowski P. G. , Bennett J.
Published: December 2004

Actual laboratory data obtained from steady‐state Dunaliella tertiolecta cultures grown under a wide range of photon flux densities were used in a simple model to calculate daily production in a conventional algal mass culture system. In spite of large physiological and biochemical variations between low‐light‐ (LL) and high light‐ (HL) adapted cultures, the overall calculated daily productivity is almost identical for both strains grown at optimal conditions. When production of fine biochemicals is considered, however, a hypothetical HL strain, which cannot shade adapt, is advantageous. Based on biochemical and biophysical analysis of D. tertiolecta responses to growth irradiance levels, specific targets are defined for genetic manipulation to enhance productivity in algal mass culture systems. The targets identified are (1) amplification of the carboxylation enzyme ribulose‐1,5‐bisphosphate carboxylase‐oxygenase relative to the electron transport complexes, which should increase photosynthetic capacity at light saturation, and (2) enlargement of the light‐harvesting complexes by varying their pigment composition in order to increase light harvesting at low photon flux densities.
Formation of oligosaccharides during enzymatic lactose hydrolysis and their importance in a whey hydrolysis process: Part II: Experimental
Authors: Prenosil J. E. , Stuker E. , Bourne J. R.
Published: December 2004

Enzymatic lactose hydrolysis using two yeast and two fungal lactases that are of current technical interest was studied. The enzymes were compared regarding their oligosaccharide production. Parameters influencing oligosaccharide formation, together with the effect of immobilization were examined and conditions minimizing oligosaccharide content in the hydrolysis product were proposed. Enzymatic whey hydrolysis was also considered. A possibility of enzymatic lactose recombination from its hydrolysis products was shown.
Recovery of penicillin by reactive extraction in centrifugal extractors
Authors: Likidis Z. , Schügerl K.
Published: December 2004

Penicillin V and/or G were extracted from fermentation broth by Amberlite LA‐2 in n‐butylacetate at pH 5 in a laboratory centrifugal extractor, Type SA 01 of Westfalia, up to an overall phase throughput rate of 30 L/h, in a bench‐scale four‐stage extraction system, consisting of Type TA 1 of Westfalia, up to an overall phase throughput rate of 110 L/h, and in a pilot‐plant three‐stage extraction unit, consisting of Type TA 7 extractors of Westfalia, up to a phase throughput rate of 990 L/h with very high degrees of extraction (up to 99%). The reextraction from the ion‐pair complex, which contained organic solvent, was performed by phosphate, borate, or carbonate buffer at pH 7.5–8.5 in all three extractor systems with degrees of extraction up to 98%, but at considerably lower overall phase throughput rates than those of the extraction, since no satisfactory phase separation is possible at higher throughput rates.
Alum flocculation and bioflocculation of activated sludge for vacuum filtration
Authors: Novak Richard A.
Published: December 2004

This study investigated the relationship between sludge loading rate, COD‐to‐nitrogen ratio of influent waste, and maximum difference in specific resistance as a result of chemical conditioning (ΔZ). It also related ΔZ to sludge carbohydrate content, protein content, and surface charge. This research also explored the necessity of chemical conditioning when an activated sludge exhibits excellent bioflocculation characteristics.
Bioflocculation as a microbial response to substrate limitations
Authors: Logan Bruce E. , Hunt James R.
Published: December 2004

Previous theories of nutrient supply to microbial floes assumed that transport within the flocs was by molecular diffusion, and they predict that overall nutrient uptake is reduced in floes compared to dispersed cells. Calculations, supported by recent advances in understanding fluid flow through suspended aggregates, however, have shown that substantial fluid flow may occur through highly permeable bacterial floes. Since bioflocculation of microorganisms in bioreactors is known to occur under conditions of low substrate availability, the rate of substrate uptake is assumed to be mass transfer limited. The hydrodynamic environment of a cell then determines cellular uptake rates. Through development of a relative uptake factor, the overall uptake by cells in flocs in sheared fluids and floes attached to bubbles are compared with the uptake by an identical quantity of dispersed cells. Bioflocculation is found to increase the rate of substrate transport to cells in permeable floes compared to dispersed cells, particularly for large‐molecular‐weight substrates and when bubbles are present.
Kinetics and regulation of β‐lactamase formation in methylotrophic bacterium
Authors: Samuelov Nissim S.
Published: December 2004

The regulation and kinetics of formation of an inducible, cell‐bound oxacillin‐hydrolyzing β‐lactamase (M‐OXA) by a methanol‐grown Pseudomonas strain were investigated in batch, chemostat, and two‐stage continuous cultures. The extent to which enzyme production occurs declines at increased growth rates, and the rate of M‐OXA β‐lactamase production follows a kinetic pattern that is partially growth independent and partially inversely growth linked. Growth and enzyme formation are regulated differently by medium constituents. The initial steps of M‐OXA β‐lactamase synthesis takes place during the exponential growth phase, and active enzyme is produced by induced nonproliferating cells, probably through a turnover mechanism.
Mathematical modeling for mixed culture growth of two bacterial populations with opposite substrate preferences
Authors: Kim Sun U. , Kim Daniel C. , Dhurjati Prasad
Published: December 2004

An unstructured mathematical model is proposed for mixed culture growth of two different bacterial species that exhibit “opposite” substrate preferences in response to the “same” environmental conditions. The model incorporates enzymatic control mechanisms such as induction, repression, and inhibition in the microorganisms as manifested in their preferential utilization of substrates and microbial interactions such as amensalism and competition. The model predicts cell mass, substrate concentrations, dissolved oxygen tension, as well as key enzyme levels. The predictions of the model are compared with experimental data for pure culture growth and for mixed culture growth on two substrates, glucose and citrate, in a batch reactor.
High‐Density culture of mouse–human hybridoma in serum‐free defined medium
Authors: Takazawa Yoshiharu , Tokashiki Michiyuki , Murakami Hiroki , Yamada Koji , Omura Hirohisa
Published: December 2004

Mouse–human hybridoma 4H11 cells producing anti‐Pseudomonas sp. monoclonal antibody (IgA) grew in a serum‐free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high‐density culture in a serum‐free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC‐43 in a culture vessel equipped with hollow‐fiber modules for medium exchange. Total cell density reached 1.1 × 107 cells/mL (viable cell density was 7.6 × 106 cells/mL), and the IgA productivity was around 20 μg/106 cells/day in the serum‐free medium, which corresponded to the levels in serum‐supplemented medium.
Fed‐batch fermentation for the production of α‐amylase by Bacillus amyloliquefaciens
Authors: Yoo Young J. , Cadman Theodore W. , Hong Juan , Hatch Randolph T.
Published: December 2004

Fed‐batch cultures were performed to maximize the α‐amylase activity in a bioreactor. Kinetic equations containing a catabolite repression effect were used to model the enzyme formation from Bacillus amyloliquefaciens. Fed‐batch culture experiments were performed using maltose to implement the optimal feeding strategy. Optimal fed‐batch culture based on sequential parameter estimation was performed successfully using off‐line analysis while the fermentation was in progress. The enzyme activity from the fed‐batch culture employing maltose was higher than that of the batch culture by 60%. Enzyme production using starch showed similar trends to those obtained using maltose.
Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi
Authors: Gervats P. , Molin P. , Grajek W. , Bensoussan M.
Published: December 2004

The water activity (aw) of substrates has been related to the mycelial growth and the sporogenesis of two molds. In the absence of other limiting factors, optimal aw values were determined for growth and sporogenesis as 0.99 and 0.98, respectively, for Trichoderma viride TS and 0.97 and 0.96 for Penicillium roqueforti. In all cases, the accuracy of the optimal value would justify the regulation of this parameter. A model was proposed which establishes a relationship between the mycelial growth and the water activity value of the substrate.
Development of ultraporous fired bricks as support for yeast cell immobilization
Authors: Opara C. C. , Mann J.
Published: December 2004

Ultraporous fired bricks (porosities from 56 to 72%) were developed from materials locally available in Nigeria. The grog particle size was used to modulate the porosity of the bricks. The porous bricks produced were then employed as supports for the immobilization of a yeast strain isolated from a local alcoholic beverage, palm wine. The influence of a brick's porosity and particle size on the cell‐loading capacity and cell growth inside the fired brick support were studied. The study revealed that a brick's porosity varied linearly with the mean particle size of the grog, increasing from a porosity of 56% at a particle size of 0.805 mm to 72% at a particle size of 0.075 mm. Cell saturation of the surface area available within the support matrix was completed within four hours of contact between the cell and the adsorbing surface especially for the most porous samples. Cell growth was therefore not observed in such cases; however, the less porous samples supported some cell growth upon incubation. Cell holdup was also observed to increase exponentially when either the porosity was increased or the particle size was decreased. The influence of particle size, however, became insignificant at very high porosities.
A new pilot reactor for solid‐state fermentation: Application to the protein enrichment of sugar beet pulp
Authors: Durand A. , Chereau D.
Published: December 2004

A new pilot reactor for solid‐state fermentation has been used for single‐cell protein production on raw sugar beet pulp with a mutant, Trichoderma viride T.S. This pilot plant, having a maximum working capacity of one ton (ca. 200 kg dry matter) can be scaled up to the production plant level. During the process, the protein content increases from 9 to 20–21% (on the basis of dry matter) in 48 h. A material and heat balance is presented in relation with temperature and moisture level regulation during the process.
Photo‐osmosis through liquid membrane bilayers generated by bacteriorhodopsin
Authors: Srivastava R. C. , Tandon A.
Published: December 2004

Liquid membrane bilayers, generated by bacteriorhodopsin on a supporting membrane, exhibit photo osmosis. The phenomenon has been shown to be a consequence of light‐induced electrical potential differences which develop across the liquid membrane bilayer due to the light‐driven proton pumping action of bacteriorhodopsin. The variations of photo osmotic velocity with wavelength, intensity of light, and proton acceptor concentrations has been studied.
The effect of wood‐derived inhibitors on 2, 3‐butanediol production by Klebsiella pneumoniae
Authors: Nishikawa Nora K. , Sutcliffe Roger , Saddler John N.
Published: December 2004

Four potential inhibitors, hydroxymethylfurfural, furfural, syringaldehyde and vanillin, were assayed over a range of concentrations and times to simulate the amounts that might be expected to accumulate when the hydrolyzed water‐soluble fraction from steam‐treated aspenwood was added in a fed‐batch mode to K. pneumoniae. Combinations of the individual materials resulted in a cumulative inhibitory effect. Prolonged incubation could alleviate some of the inhibition.
Problematics and stability of on‐line pH measurements in anaerobic environments: The jellied combined electrode
Authors: Monzambe Kuma M. , Naveau Henry P. , Nyns Edmond‐Jacques , Bogaert Nicolas , Bühler Hans
Published: December 2004

Discrepancies of one pH unit and more have been observed after a few days, between continuous on‐line in situ pH measurements and instant off‐line pH measurements during anaerobic digestion of an agroindustrial wastewater. Concomitantly, the electrical resistance across the porous diaphragm of the on‐line electrode increased, and a black clogging developed on its diaphragm. Measurements of the relative liquid junction potential in KCl or Na2S solutions excluded that high concentrations of ions such as, K+, Na+, Cl−, HS−, or S2− were the major cause of the drifts in pH values. It has been possible to limit the rapid increase of the liquid junction potential and the black clogging formation on the porous diaphragm either by acidification and/or by overpressurization of the electrode‐filling liquid. Continuous on‐line in situ pH values consistent with instant off‐line pH values over long periods of time have been obtained with a newly designed pH sensor in which a special jellied electrode filling replaced the porous diaphragm.
Reversible immobilization of soybean β‐amylase on phenylboronate–agarose
Authors: Viera Francisco Batista , Barragan Beatriz Brena , Busto Beatriz Luna
Published: December 2004

Soybean β‐amylase (EC 3.2.1.2) wap immobilized on phenylboronate‐agarose by strong interactive binding. The insoluble derivative was active and more stable to temperature changes than the free enzyme. The absence of enzyme leakage even in the presence of substrate was demonstrated. Changes in pH over a wide range (4.0–8.0) did not affect the stability of the complex. The support could be recovered by sorbitol elution, which demonstrated the reversibility of the binding. Since the enzyme was not retained on phenylagarose under similar conditions, we rejected hydrophobic interactions as a cause of the strong binding of the enzyme to phenylboronate‐agarose. We suggest that the bonding of the enzyme to the phenylboronate ligand occurs by a charge transfer mechanism between the trigonal boronate and the side chain nitrogenated groups. It was concluded that phenylboronate‐agarose has good properties as a support, which recommends its use for the preparation of immobilized enzymes.
Influence of growth substrate on production of cellulase enzymes by Trichoderma harzianum E58
Authors: Mes‐Hartree M. , Hogan C. M. , Saddler J. N.
Published: December 2004

Cellulase production by Trichoderma harzianum E58 grown on lactose and various cellulosic substrates such as Solka Floe, Avicel, and steamed aspenwood was investigated. The culture filtrates of T. harzianum E58 obtained after growth on these substrates were assayed for their cellulase activities and overall hydrolytic activities. The severity of the steaming conditions used for the aspenwood had a pronounced effect on the cellulolytic activity of the produced culture filtrates. Those substrates that were more readily hydrolyzed by the cellulase complex were the poorest substrates for inducing an active cellulase complex. Substrates such as acid‐impregnated aspenwood and lactose induced a less hydrolytic efficient cellulase complex than more recalcitrant substrates such as microcrystalline cellulose.
Determination of intrinsic parameters for immobilization reactions of catalase and amyloglucosidase in porous glass supports
Authors: Hossain Md. M. , Do D. D.
Published: December 2004

Experiments have been carried out for immobilizing enzyme‐catalase and amyloglucosidase in controlled‐pore glass particles of two different pore sizes. The experimental results have been analyzed, initial‐stage analysis for the rate parameters of immobilization reactions and long‐time analysis for determining the evolution of the immobilization process. These investigations suggest that the overall process of immobilization is controlled by the restricted diffusion of enzymes into the pores of the support. As a result, immobilized enzyme (IME) can penetrate only up to a certain distance into the support. The penetration depth of IME for the support–enzyme system mentioned have been evaluated from the experimental bulk enzyme concentration data in a batch recirculation reactor.
Glucoamylase immobilized on acid‐hydrolyzed starch graft polyacrylonitrile
Authors: Slininger P. J. , Fanta G. F. , Abbott T. P.
Published: December 2004

A continuous stirred reactor fed maltose as substrate was used to show that acid hydrolyzed starch‐g‐polyacry‐lonitrile and other polysaccharide graft copolymers can bind and retain significant quantities of active glucoamy‐lase. Glucose productivities up to 2.7 g/g carrier/h were observed with the immobilized glucoamylase, and half‐lives up to 1800 h were indicative of activity longevity. Factors influencing the immobilized enzyme activity and first‐order decay rate included temperature, pH, and carrier composition. In all cases, maltose was converted quantitatively to glucose with no evidence of reversion product formation.
Performance of recombinant fermentation and evaluation of gene expression efficiency for gene product in two‐stage continuous culture system
Authors: Lee Sun Bok , Ryu Dewey D. Y. , Seigel Robert , Park Sung Hoon
Published: December 2004

In order to develop a general methodology for evaluation of the gene expression efficiency for gene product, theoretical and experimental studies were undertaken using a recombinant Escherichia coli K12ΔH1Δtrp/ pPLc23trpA1 as a “gene–host cell” model system in a two‐stage continuous‐culture system. For this, a genetically structured kinetic model proposed earlier for biosynthesis of gene product in batch cultivation was extended to the two‐stage continuous‐culture system. A partial list of key parameters of the model includes the rate of plasmid segregation, specific growth rate of recombinant cell, plasmid content, rates of transcription and translation, and other parameters related to product biosynthesis. The dynamics of heterogeneous cell population containing plasmid‐harboring and plasmid‐free cells were also studied. Theoretical analysis of cell population dynamics shows that the recombinant cells could be maintained stably for a prolonged time in a two‐stage continuous‐culture system. Fermentation performance of the recombinant E. Coli cells in a two‐stage continuous bioreactor system was examined experimentally, and the gene expression efficiency of a cloned gene product was determined based on the genetically structured kinetic model proposed. Based on our experimental results, the gene expression efficiency of the model gene–host cell system was found to be about twofold more efficient (i. e., 41.8 mg TrpA protein/mg plasmid DNA) as compared to the average rate of protein biosynthesis by E. coli cells. The performance of two‐stage recombinant fermentation was also simulated using a mathematical model developed. General trends obtained from the model simulation agree reasonably well with the currently available experimental data, although further refinements need to be made. The methodology illustrated in this article could be used for evaluation of the gene expression efficiency of other genetically engineered recombinants once such recombinants with certain gene–host cell systems are constructed.
Adaptive optimization of a continuous culture with the use of a bilevel forgetting factor
Authors: Chang Y. K. , Pyun Y. R. , Lim H. C.
Published: December 2004

An adaptive optimization algorithm using a dynamic identification scheme with a bilevel forgetting factor (BFF) has been developed. The simulation results show superiority of this method to other methods when applied to maximize the cellular productivity of a continuous culture of baker's yeast, Saccharomyces cerievisiae. Within the limited ranges of tuning parameters tested the BFF algorithm is found to be superior in terms of initial optimization speed and accuracy and reoptimization speed and accuracy when there is an external change and long term stability (removal of “blowing up” phenomena). Algorithms tested include those based on a constant forgetting factor, an adaptive variable forgetting factor (VFF) and moving window (MW) identification.
Anaerobic acidogenesis of a complex wastewater: I. The influence of operational parameters on reactor performance
Authors: Dinopoulou Georgia , Rudd Thomasine , Lester John N.
Published: December 2004

The influence of operational, parameters, such as hydraulic retention time, organic loading rate, influent substrate concentration, pH, and temperature, on the performance of the first phase of anaerobic digestion has been investigated. A complex substrate based on beef extract was used, and six series of experimental runs were conducted, each one showing the effect of one operational variable. The predominant fermentation products were always acetic and propionic acid, independent of the values of the operational parameters. For initial COD concentrations and hydraulic retention times above the critical values identified as 3 g/L and 6 h, respectively, the degree of acidification achieved was between 30 and 60%. The degree of acidification was found to increase with the hydraulic retention time and decrease with the influent substrate concentration and organic loading rate, while the opposite held true for the rate of product formation. Furthermore, it has been demonstrated that acidification is primarily determined by the hydraulic retention time and the rate of product formation by the influent substrate concentration. The concentration of the acetic acid produced was found to depend on the operational parameters. However, the concentration of propionic acid produced depended only on the substrate availability with a consistent proportion of 8% initial COD converted to it. The optimum pH and temperature were 7 and 40°C, respectively. The percentage of acetic acid as a proportion of the total volatile fatty acids produced was found to increase with increasing pH and temperature, while the percentage of propionic acid seemed to decrease accordingly. Finally the effect of the temperature on the rate of acidification followed an Arrhenius type equation with an activation energy equal to 4739 cal/mol.
The barley protein degradation: Mechanism of protein solubilization during barley mashing with neutral proteinase
Authors: Marković Ivan , Topolovec Velimir , Marić Vladimir , Johanides Vera
Published: December 2004

Kinetics of protein solubilization during barley mashing with neutral proteinase were studied. By plotting the kinetics data in Foster–Niemann coordinates for barley concentration range of 10–30% linear relationships with high correlation coefficients (r ⩾ 0. 999) were obtained. The slopes of straight lines were very close to corresponding reciprocal initial insoluble nitrogen concentrations. Barley proteolytic inhibitors affected the ordinate intercept; by their addition the values of ordinate intercept decreased. The data suggest that the modified Foster–Niemann equation can be proposed to interpret kinetics of insoluble barley protein degradation. The proteolytic activity decay was studied as well. The enzyme decay was faster in buffer solution than during barley mashing, but in both cases first‐order kinetics can be applied. A mathematical model describing protein solubilization and enzyme decay kinetics were developed. The results of computer simulation were in good agreement with experimental data.
Continuous regeneration of ATP in enzyme membrane reactor for enzymatic syntheses
Authors: Berke Wolfgang , Schüz Hans‐Jürgen , Wandrey Christian , Morr Michael , Denda Gudrun , Kula Maria‐Regina
Published: December 2004

This work shows that the enzyme membrane reactor offers the opportunity to carry out the enzymatic regeneration of ATP providing continuous operation with high performance. In this system, the coenzyme is immobilized on a water‐soluble polymer. These high‐molecular weight derivates are entrapped within an ultrafiltration membrane together with the enzymes for production of regeneration. Several polymer derivatives of ATP have been prepared for this immobilization technique. Coenzymatic activity of these derivatives was studied with several enzymes for both ATP‐requiring and ATP‐regenerating reactions. PEG‐N6‐aminohexyl‐ATP was selected as the appropriate coenzyme for operating the enzyme membrane reactor. Acetate kinase was the only enzyme providing enough activity for regeneration. Production of glucose‐6‐phosphate is cited as the first example. The kinetics of acetate kinase and hexokinase were examined and used to choose the operating conditions of the process. The process operated continuously for more than 1 month. With a mean conversion of 80%, the space–time yield amounted to 348 g glucose‐6‐phosphate/L d. The cycle number of ATP was estimated as 20, 000 mol/mol. With the continuous production of γ‐glutamylcysteine and NADP, the feasibility of the system was proven.
Controlled fed‐batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen
Authors: Hsieh Jih‐Han , Shih Kun‐Yu , Kung Hsiang‐Fu , Shiang Ming , Lee Lan‐Ying , Meng Meng‐Hsiao , Chang Ching‐Chuan , Lin Hui‐Min , Shih Shu‐Ching , Lee Shu‐Ying , Chow Teh‐Yuan , Feng Teng‐Yung , Kuo Tsong‐Teh , Choo Kong‐Bung
Published: December 2004

We have performed controlled fed‐batch fermentation experiments to compare the production level of hepatitis B surface antigen (HBsAg) by recombinant yeast Saccharomyces cerevisiae strains (YNN27/pYBH‐1, YNN27/ p2μ‐S11, YNN27/pDCB‐S2) containing plasmid vector with alcohol dehydrogenase (ADH1), acid phosphatase (PHO5), and glyceraldehyde‐3‐phosphate dehydrogenase (GPD) promoter, respectively. Yeast cell concentrations of 15‐35 g dry cell weight/L were obtained. By limiting phosphorous concentration, HBsAg expression level for the YNN27/p2μ‐S11 strain with inducible PHO5 promoter reached 0.2–0.3 mg/L. By controlling nutrient addition rate and dissolved oxygen concentration, HBsAg concentrations of 3‐10 mg/L were achieved in 60–70 h fermentation using the YNN27/pDCB‐S2 strain with the constitutive GPD promoter.
Algae flocculation in reservoir water
Authors: Sridhar P. , Namasivayam C. , Prabhakaran G.
Published: December 2004

Removal of algae in the reservoir water was studied by electroflocculation using a bipolar cell with aluminum electrodes and flocculation by treatment with commercial alum. Comparison of both the methods is discussed.
Fermentation of single and mixed substrates by the parent and an acid‐tolerant, mutant strain of Clostridium Thermoaceticum
Authors: Brumm Phillip J.
Published: December 2004

Growth of the parent and acid‐tolerant mutant strains of Clostridiurn thermoaceticum was examined on a variety of substrates and mixtures of substrates. Nondiauxic growth was noted for both strains on combinations of carbohydrates, organic acids, or a carbohydrate and an organic acid. The mutant strain was able to grow on DL‐lactate as sole energy source. The parent strain would not grow on lactate as sole energy source but consumed lactate when presented with a second fermentable substrate. Neither strain would grow on formate as sole energy source, but both consumed formate when presented with a second fermentable substrate.
Mathematical modeling of affinity ultrafiltration process
Authors: Luong J. H. T. , Male K. B. , Nguyen A. L. , Mulchandani A.
Published: December 2004

An affinity ultrafiltration process has been developed by exploiting affinity binding in conjunction with cross‐flow filtration. The process was proven to possess high resolution, high recovery yield, and ease of scale‐up. The process could purify trypsin from a trypsin‐chymotrypsin mixture batchwise or continuously. Essential for applying this concept was the synthesis of a water‐soluble high‐molecular‐weight polymer bearing m‐aminobenzamidine, a strong and specific trypsin in hibitor. A mathematical model was also developed to describe the dynamic behavior of the newly developed purification process. The model was able to predict the profiles of enzyme concentrations in the process with high accuracy.
Correlation for volumetric mass transfer coefficient in mechanically agitated aerated vessel for oxygen absorption in aqueous electrolyte solutions
Authors: Linek V. , Beneš P. , Holeček O.
Published: December 2004

Empirical equations used so for for computing kla in mechanically agitated aerated vessels agree well with the measured dependence of Kla on the power input of the stirrer and the superficial velocity of the gas. However, they do not fit the dependence of Kla on the physical properties of the liquid batch. From the data published so far it can be deduced that the equations suitable for the description of the dependence of kmola on the liquid physical properties must include some independent variable, which characterizes the rate of coalescence of the bubbles. About 400 experimental values of Kla were compared with those calculated from different relations. This comparison shows that the rate of coalescence for the aqueous electrolyte solutions is well described on the basis of the Gibbs theory of adsorption of the solute in the surface layer of the solution.
The hydrolysis of triglycerides by immobilized lipase in a hydrophiiic membrane reactor
Authors: Pronk W. , Kerkhof P. J. A. M. , Van Helden C. , Riet K. van'T
Published: December 2004

In the present article a method is described to immobilize lipase from Candida rugosa on a hollow fiber membrane, and the use of such a system for the hydrolysis of lipids is reported. The membranes were ENKA hydro philic Cuprophan®‐type hollow fibers, having a large specific surface area. The immobilized lipase exhibited a high stability: the half‐life time was 43 days at a temperature of 30°C. Furthermore, it is proved that kinetic studies can be carried out with this system, operated in a batch or continuous mode. The relation between conversion rate and degree of hydrolysis was determined. On this basis, a dynamic model of the process was developed that describes the relation between reaction conditions and the conversion rate.
Biochemical switching device: Monocyclic enzyme system
Authors: Okamoto Masahiro , Sakai Toshio , Hayashi Katsuya
Published: December 2004

The switching characteristics of a monocyclic enzyme system, in which two enzymes share substrates or co‐factors in a cyclic manner, such as, → X1 + B + E1 ⇄ A + E1 + X2 →, → X3 + A + E2 ⇄ B + E2 + X4 → (E1, E2 are enzymes, X1, X3 are substrates, X2, X4 are products, A, B are cofactors), were demonstrated using computer simulations. The detailed mathematical models of biochemically possible cyclic enzyme systems were built up and the effects of rate constants and the effects of initial concentrations of enzymes and cofactors on switching characteristics were discussed. The cyclic enzyme system could function as a switching circuit when the initial concentrations of enzymes or cofactors are over a certain threshold value. Based on the present results, we further discuss the dynamic characteristics of a biochemical reactor system (bioreactor) involving this cyclic enzyme system as a switching controller.
The influence of bond chemistry on immobilized enzyme systems for ex vivo use
Authors: Comfort Ann R. , Mullon Claudy J.‐P. , Langer Robert
Published: December 2004

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50–80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25–30%) with tresyl chloride activated agarose and oxirane activated acrylic.

The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme‐linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme‐support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross‐linking with glutaraldehyde also improves the enzyme‐support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.
Use of polyelectrolyte complex‐stabilized calcium alginate gel for entrapment of β‐amylase
Authors: Kokufuta Etsuo , Shimizu Noboru , Tanaka Hideo , Nakamura Isei
Published: December 2004

Calcium alginate gel stabilized with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate (KPVS) and trimethylammonium glycol chitosan iodide (TGCI) was used for the immobilization of β‐amylase. The immobilization was made by gelling aqueous droplets of enzyme solution including both sodium alginate and KPVS in a CaCl2 solution containing TGCI. The activity of the enzyme entrapped into the stabilized gel beads was evaluated by studying the batch reaction kinetics of enzyme‐catalyzed hydrolysis of maltotetraose. Repeated kinetic measurements, totaling 18, were carried out at fixed time intervals. After each measurement the beads were stirred for 1 day in a freshly prepared 10 mM NaCl solution at 3°C. It was found that the immobilized system remained stable without leading to a serious loss of the activity or to a large leakage of the enzyme from the support. This was explained as being due to a PEC‐crosslinked contracted network structure of the stabilized gel matrix.