bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of AcPheLeuNH2 by α‐chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system
Authors: Serralheiro M. L. M. , Cabral J. M. S.
Published: December 2004

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the α‐chymotrypsin‐mediated AcPheLeuNH2 synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the α‐chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. © 1994 John Wiley & Sons, Inc.
Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism
Authors: Sharfstein Susan T. , Tucker Sean N. , Mancuso Anthony , Blanch Harvey W. , Clark Douglas S.
Published: December 2004

Carbon‐13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon‐13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of 13C label from the TCA cycle. These results illustrate that 13NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. © 1994 John Wiley & Sons, Inc.
Solvation of CBZ–amino acid nitrophenyl esters in organic media and the kinetics of their transesterification by subtilisin
Authors: Reimann Antje , Robb Donald A. , Halling Peter J.
Published: December 2004

Subtilisin Carlsberg adsorbed on silica particles has been used to catalyze the transesterification of CBZ–Ala–ONp and CBZ–Leu–ONp with 1–butanol in organic systems preequilibrated to water activity of 0.93. Initial reaction rates are conveniently followed by extraction of the released nitrophenol into an alkaline aqueous phase. Kinetic parameters were determined for varied ester concentrations in toluene, isopropyl ether, and hexane. The effect of solvent on substrate solvation was determined by solubility measurements. Much of the observed effect of solvent on Vm/Km may be accounted for by solvation differences. The residual effect of solvent on Km, after discounting solvation differences, is completely opposite to the apparent trend. © 1994 John Wiley & Sons, Inc.
Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p‐xylene mixture
Authors: Lee Jang‐Young , Roh Jae‐Rang , Kim Hak‐Sung
Published: December 2004

For the complete biodegradation of a mixture of benzene, toluene, and p―xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate―cis‐glycol dehydrogenase in the tol pathway was found to attack benzene―cis―glycol, toluene―cis―glycol, and p―xylene―cis―glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis―glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis―glycol compounds by the action of toluate―cis―glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p‐xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. © 1994 John Wiley & Sons, Inc.
A novel method to prepare size‐regulated spheroids composed of human dermal fibroblasts
Authors: Yamazaki Manabu , Tsuchida Michiko , Kobayashi Ken‐yu , Takezawa Toshiaki , Mori Yuichi
Published: December 2004

A simple method to prepare size‐regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly‐N‐isopropyl‐acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37°C, then when lowering the temperature of culture medium the cells attached to it detach as a self‐supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30°C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV‐irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 μg/cm2 (collagen ratio, 4.5%), UV‐irradiation energy of 2000 J/m2 was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV‐irradiation, it was possible to obtain cell‐adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 μm in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 μm in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size‐regulated spheroids for different applications; for example, in toxicology tests. © 1994 John Wiley & Sons, Inc.
Affinity cross‐flow filtration: Purification of IgG with a novel protein a affinity matrix prepared from two‐dimensional protein crystals
Authors: Weiner Christian , SáRa Margit , Dasgupta Gautam , Sleytr Uwe B.
Published: December 2004

In this article, we describe the use of 1‐ to 2‐μm sized affinity microparticles for the isolation and purification of IgG from artificial IgG‐human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross‐flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111‐69, in which the peptidoglycan‐containing layer was completely covered with a hexagonally ordered S‐layer lattice. After crosslinking the S‐layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S‐layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S‐layer protein release under cross‐flow conditions between pH 2 to 12. The IgG‐binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. © 1994 John Wiley & Sons, Inc.
Application of the transition time of metabolic system as a criterion for optimization of metabolic processes
Authors: Torres Néstor V.
Published: December 2004

Transition time of metabolic systems in introduced as a suitable optimization criterion for biotechnological processes in which it is desirable to reduce the lag time and minimize the mass contained within the system. Lag time is the time needed for the system to attain the steady state. Results obtained from the sensitivity analysis of this steady state response are presented within the metabolic control analysis and applied to 3 case studies. In all of them the information provided by the transition time control profile allows the implementation of a strategy for biotechnological manipulations aimed at the improvement of the process. © 1994 John Wiley & Sons, Inc.
Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations
Authors: Hiller G. W. , Clark D. S. , Blanch H. W.
Published: December 2004

The effects of step‐change increase in the concentrations of amino acids and vitamins on the metabolism, growth, and antibody productivity of a murine hybridoma cell line grown in continuous culture on serum‐free medium are presented. Additions of the amino acids cysteine with methionine, tryptophan, and isoleucine with valine and vitamin B12 (as cyanocobalamin) resulted in significant increases in viable cell concentrations. Additions of aspartate with asparagine, and threonine with vitamin B1 (as thiamine hydrochloride) resulted in significant increases in final antibody concentrations. Substantial decrease in the fraction of amino acid nitrogen excreted as ammonia occurred upon supplementation with three times the normal concentrations of branched chain amino acids. Decreases in the fraction of amino acid nitrogen converted to ammonia were paralleled by increases in the fraction converted to alanine. © 1994 John Wiley & Sons, Inc.
Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A
Authors: Wilson S.‐A. , Daniel R. M. , Peek K.
Published: December 2004

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac‐Phe‐OEt and Bz‐Ala‐OMe. Tur‐NH2, Trp‐NH2, Leu‐pNA, and Val‐pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz‐ala‐Tyr‐NH2 was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz–ala‐OMe, 25 mM Tyr‐NH2, 70°C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz‐Ala‐OMe and 615 mM Tyr‐NH2 at 40°C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; ‐βNA and ‐pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. © 1994 John Wiley & Sons, Inc.
An on‐line sampling system for fermentation monitoring Using membrane inlet mass spectrometry (MIMS): Application to phenoxyacetic acid monitoring in penicillin fermentation
Authors: Hansen Ken Flemming , Lauritsen Frants R. , Degn Hans
Published: December 2004

A sampling system for on‐line monitoring of organic compounds of low volatility in complex fermentation media uses membrane inlet mass spectrometry (MIMS). A Syringe pump draws a continuous flow of microfiltered broth from the reactor and circulates it after acidification through a membrane inlet, in which a membrane is the only interface between the sample and the high vacuum of a mass spectrometer. All operations run automatically, i.e., sampling, acidification measurement, and calibration. The on‐stream acidification enables MIMS monitoring of carboxylic acids, as they must be undissociated in order to pass the hydrophobic membrane. The performance of the monitoring system was tested by measurements of standard solutions of phenoxyacetic acid (POAA, the sie chain precursor of penicillin‐V) as well as on POAA during 200 h penicillin‐V fermentation. During the entire fermentation POAA was monitored n low millimolar concentrations with high accuracy and fast response to step changes in POAA concentration. Tandem mass spectrometry (MS/MS) allowed direct identification of peaks in the mass spectrum of the broth that were not accounted for by POAA. These peaks were identified as SO2 and SCO. © 1994 John Wiley & Sons, Inc.
Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus
Authors: Schlatmann J. E. , Moreno P. R. H. , Vinke J. L. , ten Hoopen H. J. G. , Verpoorte R. , Heijnen J. J.
Published: December 2004

Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. © 1994 John Wiley & Sons, Inc.
Transport of bacteria in porous media: I. An experimental investigation
Authors: Sarkar A. K. , Georgiou George , Sharma Mukul M.
Published: December 2004

The convective transport of concentrated suspension of bacteria in porous media is of interest for several processes such as microbial enhanced oil recovery and in situ bioremediation. The parameters which affect the transport of the bacterium Bacillus licheniformis JF‐2, a candidate microorganism for microbial enhanced oil recovery, were investigated experimentally in sandpacks. Bacteria retention and permeability reduction occurred primarily in the first few centimeters upon entering the porous medium. In downstream sections of the sandpack, the permeability reduction was low, even in cases in which high cell concentrations (108 cfu/mL) were detected in the effluent. The effect of (i) addition of a dispersant, (ii) linear velocity of injection, (iii) cell concentration, (iv) salinity (v) temperature, and (vi) the presence of a residual oleic phase were determined experimentally. A lower reduction in permeability and a higher effluent bacterial concentration were obtained in the presence of dispersant, high injection velocities, low salinities, and at a higher temperature. Macroscopic measurements at different linear velocities and in the presence or absence of dispersants suggest that the formation of reversible microaggregates and multiparticle hydrodynamic exclusion may be the primary mechanisms for bacterial retention and permeability reduction. © 1994 John Wiley & Sons, Inc.
Symmetric enzyme distribution in asymmetric UF polysulfone membranes
Authors: Gille Michael , Staude Eberhard
Published: December 2004

Invertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane‐impermeable macromolecules as well as a membrane‐permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. © 1994 John Wiley & Sons, Inc.
Surfactant‐induced breakthrough effects during the operation of two‐phase biocatalytic membrane reactors
Authors: Vaidya A. M. , Halling P. J. , Bell G.
Published: December 2004

Surface‐active components, both reactants and products, are frequently encountered in two‐phase, aqueous‐organic, biocatalytic reactions, When such reaction are carried out in a membrane reactor, employing a membrane selectively wetted by one of the two reactants, changes in the content of these surfactants– as a consequence of the progress of the reaction–can lead to wetting transitions at the two membrane‐liquid interfaces as a result of adsorption of the tenside. This can lead to a decrease in the pressure required to cause the, initially, nonwetting phase to break through the membrane. Such effects render difficult the operation of two‐phase membrane bioreactors. Hence, it is necessary to make a careful selection of the membrane material and type by considering factors such as UF versus MF and low MWCO versus high MWCO to enable the reactor to be operated without breakthrough, but without significantly compromising the reaction rates that can be maintained.

The phenomena leading to breakthrough effects are discussed in this paper, and experimental results for the hydrolysis of ethyl laurate by lipase from Candida rugosa in a batch flat sheet membrane reactor are presented with the reactor operated with a variety of membranes. An experimental result showing the decrease in the pressure required to cause breakthrough of the organic phase (for the system ethyl laurate‐lauric acid–water) as the content of the highly surface‐active lauric acid in the organic phase is increased is also presented for an asymmetric, hydrophilic meta‐aramid ultrafiltration membrane. © 1994 John Wiley & Sons, Inc.
Closed‐loop control of fed‐batch cultures of recombinant Escherichia coli using on‐line HPLC
Authors: Turner Claire , Gregory Malcolm E. , Thornhill Nina F.
Published: December 2004

This article describes a fully automated system for the on‐line monitoring and closed‐loop control of a fed‐batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed‐batch fermentations. The system had two components. The first components, on‐line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on‐line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. © 1994 John Wiley & Sons, Inc.
Oxygen requirements and mass transfer in hairy‐root culture
Authors: Yu Shaoxiong , Doran Pauline M.
Published: December 2004

Oxygen mass transfer in clumps of Atropa belladonna hairy roots was investigated as a function of root density and external flow conditions. Convection was the dominant mechanism for mass transfer into root clumps 3.5 to 5.0 cm in diameter; Peclet numbers inside the clumps ranged from 1.4 × 103 to 7.1 × 104 for external superficial flow velocities between 0.4 and 1.4 cm s−1. Local dissolved‐oxygen levels and rates of oxygen uptake were measured in aflow chamber and in bubble column and stirred bioreactors. When air was used as oxygen source, intraclump dissolved‐oxygen tensions ranged from90% to 100% air saturation at high external flow velocity andlow root density, to less than 20% air saturation in dense root clumps. Specific oxygen‐uptake rate declined with increasing root density. When external boundary layers around individual roots were eliminated byforcing liquid through the clumps at superficial velocities between 0.2 and1.0 cm s−1, internal dissolved‐oxygen tension was maintained at 95% to 100% air saturation and rate of oxygen uptake at 1.6 × 10−6 g g−1 s−1 dry weight. Liquid culture of single A. belladonna hairy roots was used to investigate the effect of dissolved‐oxygen tensionon root growth and morphology. Total root length and number of root tips increased exponentially at oxygen tensions between 70% and 100%air saturation. Specific growth rate increased with oxygen tension up to 100% air saturation; this result demonstrates that hairy roots aeratedwithout oxygen supplementation are likely to be oxygenlimited. No growth occurred at 50% air saturation. Growth of hairy roots proceeded with an average length per tip of about 1 cm; this value was essentially independent of dissolved‐oxygen tension between 70% and 100% air saturation. © 1994 John Wiley & Sons, Inc.
Improved taxol yield by aromatic carboxylic acid and amino acid feeding to cell cultures of taxus cuspidata
Authors: Fett‐Neto Arthur G. , Melanson Stewart J. , Nicholson Sherwin A. , Pennington Jason J. , DiCosmo Frank
Published: December 2004

Cell culture of Taxus cuspidata represents an alternative to whole plant extraction as a source of taxol and related taxanes. Feeding phenylalanine to callus cultures was previously shown to result in increased taxol yields, probably due to the involvement of this amino acid as a precursor for the N‐benzoylphenylisoserine side chain of taxol. Inthis study, we have examined the effect of various concentrations of phenylalanine, benzoic acid, N‐benzoylglycine, serine, glycine, alanine, and 3‐amino‐3‐phenyl‐propionic acid on taxol accumulation in 2‐year‐old cell suspensions of Taxus cuspidata, cell line FCL1F, and in developing callus cultures of T. cuspidata. All compounds tested were included in media at stationary phase (suspensions) or after the period of fastest growth (calli). Alanine and 3‐amino‐3‐phenyl‐propionicacid were tested only in callus cultures and did not affect taxol accumulation. Significant increases or trends toward increases in taxol accumulationin callus and suspensions were observed in the presence of phenylalanine, benzoic acid, N‐benzoylglycine, serine, and glycine. The greatest increases in taxol accumulation were observed in the presence of various concentrations of phenylalanine (1 mM for callus; 0.05, 0.1, and 0.2 mM for suspensions) and benzoic acid (0.2 and 1 mM for callus and 0.05, 0.1, and 0.2 mM for suspensions). Increases in taxol yields of cell suspensions in the presence of the most effective precursors brought taxol amounts at stationary phase from 2 μg · g−1 to approximately 10 μg . g−1 of the extracted dry weight. The results are discussed in termsof possible implications to taxol biosynthesis and in terms of practical applications to large‐scale cell culture systems for the production ofthis drug. © 1994 John Wiley & Sons, Inc.
Use of respiratory quotient as a control parameter for optimum oxygen supply and scale‐up of 2,3‐butanediol production under microaerobic conditions
Authors: Zeng A.‐P. , Byun T.‐G. , Posten C. , Deckwer W.‐D.
Published: December 2004

The respiratory quotient (RQ) was found to be a suitable control parameter for optimum oxygen supply for the production of 2,3‐butanediol + acetoin under microaerobic conditions. In laboratory scale continuous cultures optimum production of 2,3‐butanediol + acetoin was obtained at an RQ value between 4.0 to 4.5. This agreed well with optimum RQ value (4.0) stoichiometrically derived from the bioreactions involved. In fed‐batch cultures product concentrations as high as 102.9 g/L (96.0 g/L butanediol + 6.9 g/L acetoin) can be achieved within 32 h cultivation with an RQ control algorithm for oxygen supply. Under similar conditions only 85.7 g/L product (77.6 g/L butanediol + 8.1 g/L acetoin) was obtained with control of constant oxygen supply rate throughout the cultivation.

In pilot scale batch cultures under identical oxygen supply rate the achievable RQ value was found to be strongly influenced by the reactor type and scale. The initial oxygen supply rate influenced the achievable RQ as well. However, in all the reactors studied the specific product formation rate of cells in the exponential growth phase was only a function of RQ. The same optimum RQ value as found in continuous cultures was obtained. It was thus concluded that RQ can be used as a control parameter for optimum production of 2,3‐butanediol + acetoin in both laboratory and pilot plant scale reactors. © 1994 John Wiley & Sons, Inc.
Enhancement of oxygen transfer in highly viscous non‐newtonian fermentation broths
Authors: Kawase Yoshinori , Tsujimura Masanari
Published: December 2004

The influence of short draft tubes covered by perforated plates on gas‐liquid mass transfer was examined in external‐loop airlift bioreactors. The volumetric mass transfer coefficients in a model external‐loop airlift bioreactor were measured with water and non‐Newtonian media. It was found that introduction of draft tubes covered with perforated plates in the riser significantly improved the mass transfer rate, particularly in higher viscous non‐Newtonian fermentation media. The enhancement of mass transfer rate might be due mainly to an increase in bubble coalescence and redispersion. © 1994 John Wiley & Sons, Inc.
Use of plant material for the decontamination of water polluted with phenols
Authors: Dec Jerzy , Bollag Jean‐Marc
Published: December 2004

Plant materials were found useful in the decontamination water polluted with phenolic contained in the plant tissue. The enzymes mediated oxidative coupling of the pollutants, followed by precipitation of the formed polymers from the aqueous phase. An industrial wastewater contaminated with 2,4‐dichlorophenol (up to 850 ppm) and other chlorinated phenols was successfully treated using minced horseradish, potato, or white radish (amended with H2O2). Horseradish‐mediated removal of 2,4‐dichlorophenol from model solutions was comparable with that achieved using purified horseradish peroxidase. In addition, horseradish could be reused up to 30 times. Due to the apparent ease of application, the use of plat material may present a breakthrough in the enzyme treatment of contaminated water. © 1994 John Wiley & Sons, Inc.
High‐density algal photobioreactors using light‐emitting diodes
Authors: Lee Choul‐Gyun , Palsson Bernhard Ø.
Published: December 2004

Lack of high‐density algal photobioreactors (PBR) has been a limitation in exploiting the biotechnological potential of algae. Recent developments of highly efficient light‐emitting diodes (LED using gallium aluminum arsenide chips) have made the development of a small LED‐based PBR possible. We have calculated theoretical values of gas mass transfer requirements and light‐intensity requirement to support high‐density algal cultures for the 680 nm monochromatic red light from LED as a light source. A prototype PBR has been designed based on these calculations. A cell concentration of more than 2 × 109 cells/mL (more than 6.6% v%sol;v), cell doubling times as low as 12 h, and an oxygen production rate as high as 10 mmol oxygen/L culture/h were achieved using on‐line ultrafiltration to periodically provide fresh medium. © 1994 John Wiley & Sons, Inc.
Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents
Authors: Orsat Bernard , Drtina Gray J. , Williams Michael G. , Klibanov Alexander M.
Published: December 2004

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. © 1994 John Wiley & Sons, Inc.
Multirate state and parameter estimation in an antibiotic fermentation with delayed measurements
Authors: Gudi R. D. , Shah S. L. , Gray M. R.
Published: December 2004

This article discusses issues related to estimation and monitoring of fermentation processes that exhibit endogenous metabolism and time‐varying maintenance activity. Such culture‐related activities hamper the use of traditional, software sensor‐based algorithms, such as the extended kalman filter (EKF). In the approach presented here, the individual effects of the endogenous decay and the true maintenance processes have been lumped to represent a modified maintenance coefficient,mc. Model equations that relate measurable process outputs, such as the carbon dioxide evolution rate (CER) and biomass, to the observable process parameters (such as net specific growth rate and the modified maintenance coefficient) are proposed. These model equations are used in an estimator that can formally accommodate delayed, infrequent measurements of the culture states (such as the biomass) as well as frequent, culture‐related secondary measurements (such as the CER). The resulting multirate software sensor‐based estimation strategy is used to monitor biomass profiles as well as profiles of critical fermentation parameters, such as the specific growth for a fed‐batch fermentation ofStreptomyces clavuligerus. © 1994 John Wiley & Sons, Inc.
Characterization of a prototype industrial on‐line analyzer for bicarbonate/carbonate monitoring
Authors: Guwy Alan J. , Hawkes Dennis L. , Hawkes Freda R. , Rozzi Alberto G.
Published: December 2004

In many biological reactors bicarbonate is the major species determining pH buffering capacity, or alkalinity. In anaerobic digesters bicarbonate levels should be within 10 to 50 mM for stable operation. Bicarbonate alkalinity in wastewater treatment processes in routinely measured off‐line titrimetrically. Recently we have described the principle of a novel on‐line method of measuring bicarbonate alkalinity. In the prototype device described here, a continuous stream (15 cm3 min−1) of the substrate to be monitored was saturated with gaseous CO2, acidified by the addition of excess acid, and the rate of carbon dioxide evolution, proportional to the concentration of bicarbonate/carbonate in the liquid flow, continuously measured by a sensitive gas meter. The instrument was robust and its response was satisfactory for wastewater treatment process control applications, with linearity in the range 5 to 50 mM HCO3−, a response time in the order of 30 min, and accuracy of the order of 7% in the concentration range 5 to 50 mM sodium bicarbonate. The device was not affected by interference from volatile fatty acids, does not make use of pH probes which in many wastes are subject to fouling, and may form the basis of a digester control strategy. © 1994 John Wiley & Sons, Inc.
Effects of diffusion limitation on immobilized nitrifying microorganisms at low temperatures
Authors: Wijffels R. H. , Hunik J. H. , Leenen E. J. T. M. , Günther A. , de Castro J. M. Obón , Tramper J. , Englund G. , Bakketun Å.
Published: December 2004

Activation energies of suspended and immobilized nitrifying bacteria were determined and compared to determine if diffusion limitation results in decreased sensitivity for temperature. The activation energy for the respiration activity of suspended Nitrosomonas europaea and Nitrobacter agilis was found to be 86.4 and 58.4 kJ mol−1, respectively. The activation energy for oxygen diffusion in the support material, κ‐carrageenan, determined from the effect of temperature on the effective diffusion coefficient (D), was 17.2 kJ mol−1. Consequently, the apparent actvation energy of diffusion limited cells should be lower. It was indeed shown that due to the effect of diffusion limitation and to temperature effects on the Monod constant Ks, the immobilized‐cell activity was less sensitive to temperature. The apparent activation energy for immobilized Ns. europaea was between 28.6 and 94.2 kJ mol−1 and for immobilized Nb. agilis between 1.4 and 72.9 kJ mol−1, depending on the oxygen concentration and temperature. © 1995 John Wiley & Sons, Inc.
Rheologies and morphologies of three actinomycetes in submerged culture
Authors: Warren S. J. , Keshavarz‐Moore E. , Shamlou P. Ayazi , Lilly M. D. , Thomas C. R. , Dixon K.
Published: December 2004

The broth rheologies and morphologies of three actinomycetes (Saccharopolyspora erythraea, Actinomadura roseorufa, and Streptomyces rimosus) in submerged culture have been examined. The rheology of all the broths became pseudoplastic as soon as significant growth occurred with the power law index, n, falling to 0.20 to 0.25. The consistency index, K, rose with biomass concentration although in some instances it fell later in the fermentation. The mean main hyphal lengths of all cultures were in the range, 15 to 25 μm, and did not alter greatly even when large changes in K were occurring. © 1995 John Wiley & Sons, Inc.
Real‐time fuzzy‐knowledge‐based control of Baker's yeast production
Authors: Siimes Terhi , Linko Pekka , von Numers Camilla , Nakajima Mikio , Endo Isao
Published: December 2004

A real‐time fuzzy‐knowledge‐based system for fault diagnosis and control of bioprocesses was constructed using the object‐oriented programming environment Small‐talk/V Mac. The basic system was implemented in a Macintosh Quadra 900 computer and built to function connected on line to the process computer. Fuzzy logic was employed in handling uncertainties both in the knowledge and in measurements. The fuzzy sets defined for the process variables could be changed on‐line according to process dynamics. Process knowledge was implemented in a graphical two‐level hierachical knowledge base. In on‐line process control the system first recognizes the current process phase on the basis of top‐level rules in the knowledge‐base. Then, according to the results of process diagnosis based on measurement data, the appropriate control strategy is subsequently inferred making use of the lower level rules describing the process during the phase in question. © 1995 John Wiley & Sons, Inc.
Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p‐xylene mixture
Authors: Lee Jang‐Young , Jung Kwan‐Hye , Kim Hak‐Sung
Published: December 2004

A hybrid metabolic pathway through which benzene, toluene, and p‐xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146–1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad‐host‐range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt‐2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p‐xylene by P. putida TB103 were increased by about 9.3‐, 3.7‐, and 1.4‐fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. © 1995 John Wiley & Sons, Inc.
Evaluation and applications of optical cell density probes in mammalian cell bioreactors
Authors: Wu P. , Ozturk S. S. , Blackie J. D. , Thrift J. C. , Figueroa C. , Naveh D.
Published: December 2004

On‐line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 × 106 cells/mL to 20 × 106 cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of “step perfusion adjustments” based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. © 1995 John Wiley & Sons, Inc.
Quantitative analysis of biofilm thickness variability
Authors: Murga Ricardo , Stewart Philip S. , Daly Don
Published: December 2004

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 μm) and as thick as 1000 μm. Week‐old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 μm mean thickness) or K. pneumoniae (100 μm mean thickness) were thinner than the binary population biofilm (400 μm mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. © 1995 John Wiley & Sons, Inc.
Mass balancing in kinetic resolution: Calculating yield and enantiomeric excess using chiral balance
Authors: Straathof A. J. J. , Rakels J. L. L. , Heijnen J. J.
Published: December 2004

When kinetic resolution is applied for the production of enantiomerically pure compounds, process options may be used which involve more than one chiral substrate and one chiral product, such as sequential or parallel enzymatic kinetic resolutions or hydrolysis of diastereomers. Although the relation between the yields (y) of the chiral compounds is straightforward in these cases, the relation between their enantiomeric excess (ee) values is not. Combining mass balances into a so‐called chiral balance (Σ y · eeR = 0) provides the relation between enantiomeric excess values in a useful manner. This chiral balance easily shows which nonmeasured enantiomeric excess values and yields can be calculated from measured values. The chiral balance is only valid when configurations at chiral centers are conserved. © 1995 John Wiley & Sons, Inc.
Reduction of nitrate and nitrite in a cyclically operated continuous biological reactor
Authors: Wang J.‐H. , Baltzis B. C. , Lewandowski G. A.
Published: December 2004

Biological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two‐dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain of Pseudomonas denitrificans [American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. © 1995 John Wiley & Sons, Inc.
Mathematical model of self‐cycling fermentation
Authors: Wincure B. M. , Cooper D. G. , Rey A.
Published: December 2004

This article presents a mathematical model for biomass, limiting substrate, and dissolved oxygen concentrations during stable operation of self‐cycling fermentation (SCF). Laboratory experiments using the bacterium Acinetobacter calcoaceticus RAG‐1 and ethanol as the limiting substrate were performed to validate the model. A computer simulation developed from the model successfully matched experimental SCF intracycle trends and end‐of‐cycle results and, most importantly, settled into an unimposed periodicity characteristic of stable SCF operation. © 1995 John Wiley & Sons, Inc.
Modeling of an industrial alcohol fermentation and simuiation of the plant by a process simulator
Authors: Pascal F. , Dagot C. , Pingaud H. , Corriou J. P. , Pons M. N. , Engasser J. M.
Published: December 2004

The aim of the present study was the development of a general simulation module for fermentation within the framework of existing chemical process simulators. This module has been applied to an industrial plant which produces ethanol from beet molasses and fresh beet juice by Saccharomyces cerevisiae. An unstructured mechanistic model has been developed with kinetic laws that are based on a chemically defined reaction scheme which satisfies stoichiometric constraints. This model can be applied to different culture conditions and takes into account secondary byproducts such as higher alcohols. These byproducts are of prime importance and need to be correctly estimated because a sequence of distillation columns follow the fermentor in the plant. Important measurement campaigns have been performed on the plant to validate the model. Plant operation has been successfully simulated using the same kinetic model for both continuous and fed‐batch modes of production. © 1995 John Wiley & Sons, Inc.
Altered physicochemical characteristics of polyethylene glycol linked beet stem oxalate oxidase
Authors: Varalakshmi P. , Lathika K. M. , Raghavan K. G. , Singh B. B.
Published: December 2004

Oxalate oxidase (EC 1.2.3.4), obtained from the beet stem, was covalently linked to polyethylene glycol (PEG). Compared with native enzyme, the modified oxalate oxidase exhibited decreased electrophoretic mobility, increased storage stability, higher thermal stability, and resistance to heavy metal inactivation and proteolytic digestion. The chemical modification of oxalate oxidase with PEG also brought about a marked shift in its optimal pH, from pH 4.5 to 6.5, without altering its Michaelis constant (Km) significantly. These acquired properties of the immobilized oxalate oxidase render it suitable for possible applications in clinical, nutritional, and medical fields. © 1995 John Wiley & Sons, Inc.