bluuebunny/crossref_metadata_2025 huggingface dataset:
On‐line rheological measurements and control in fungal fermentations
Authors: Olsvik E. S. , Kristiansen B.
Published: December 2004

A system for on‐line rheological measurements and control in filamentous fermentations is presented. The output signals from the control unit can be used in terms of process control. Diluting the broth in a growth controlled feed pattern was found to influence the viscosity of the broth and lead to process improvements. Just diluting the fermentation broth to keep the viscosity below a preset value was seen to give only temporary process improvements. The higher the viscosity, the less effective the viscosity controlled dilutions. The failure to get full control over the viscosity by the dilution techniques used is caused by the large number of factors influencing the rheological properties of an Aspergillus niger culture. The factors shown in this work to influence the rheological properties of the fermentation broth were the biomass concentration, the specific growth rate, mixing qualities (impeller speed and working volume), and the dissolved oxygen concentration. © 1992 John Wiley & Sons, Inc.
Effect of temperature on hybridoma cell cycle and MAb production
Authors: Bloemkolk Jan‐Willem , Gray Murray R. , Merchant Fahar , Mosmann Timothy R.
Published: December 2004

The kinetics of growth and antibody formation of an anti–interleukin‐2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34°C to 39°C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37°C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G1‐phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G1‐phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. © 1992 John Wiley & Sons, Inc.
How to predict product yield in kinetically controlled enzymatic peptide synthesis
Authors: Gololobov Mikhail Yu. , Kozlova Elena V. , Borisov Ivan L. , Schellenberger Ute , Schellenberger Volker , Jakubke Hans‐Dieter
Published: December 2004

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters α and β for papain‐catalyzed synthesis of Mal‐L‐Phe‐L‐Ala‐LLeuNH2 from Mal‐L‐Phe‐L‐AlaOMe and L‐LeuNH2. The experimental results demonstrate that this theory adequately describes the real process. © 1992 John Wiley & Sons, Inc.
Urea sensor with single poly(propylene) membrane
Authors: Wang Y. J. , Chen C. H. , Hsiue G. H. , Yu B. C.
Published: December 2004

Urease was immobilized on the plasma‐aminated surface of a hyfrophobic poly(propylene) (PP) membrane. This membrane, with urease matrix on one side while maintaining its original hydrophobic property on the other, was used to construct the urea sensor. The new urea sensors had response sensitivities ranged from 19 mV/decade to 30 mV/decade depending on the conditions of the plasma reaction. The enzyme electrode using single membrane gave a shorter response time as compared to the corresponding conventional electrode employing two seperate PP membranes. The sensitivity of the enzyme electrode increased with increasing buffer pH and reached a maximal level (40 mV/decade) at pH 7.6. The response sensitivity of the electrode was not affected by the change of buffer strength. Deamination of the plasma‐modified hydrophobic PP membrane did not occur in aqueous environment judging from the stability of the urea electrode up to 12 days of operation. © 1992 John Wiley & Sons. Inc.
Efficient recovery of recombinant proteins using membrane‐based immunoaffinity chromatography (MIC)
Authors: Nachman Michele , Azad Abdul R. M. , Bailon Pascal
Published: December 2004

A systematic approach to the design and development of membrane‐based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon‐α2a is used as a model system to determine the operational parameters in membrane‐based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen‐antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon‐α2a, interleukin‐2, and interleukin‐2 receptor, have been purified efficiently employing membrane‐based immunoaffinity chromatography. Overall, membrane‐based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial‐scale production of recombinant proteins. © 1992 John Wiley & Sons, Inc.
Enzymatic resolution of racemic amines in a continuous reactor in organic solvents
Authors: Gutman Arie L. , Meyer Elazar , Kalerin Evgeny , Polyak Felix , Sterling Jeffrey
Published: December 2004

An enzymatic process has been developed for the continuous production of the pharmaceutically important intermediate (R)‐1‐aminoindan and of the chiral resolving agent (R)‐1‐(1‐naphthyl)ethylamine. The process consists of the subtilisin catalyzed stereoselective aminolysis of the racemic primary amine with an active ester in organic solvent. The competing nonenzymatic reaction has been suppressed by appropriate choice of solvent and reactant's concentration and by minimizing the time of contact between the amine and the active ester. Subtilisin was immobilized on glass beads and the reaction carried out in a continuous‐flow column bioreactor. By using a 450‐mL column bioreactor containing 5.7 g of subtilisin immobilized on 570 g of glass beads, 1.6 kg of racemic 1‐(1‐naphthyl)ethylamine was resolved after 320 h of continuous operation with only a slight loss of the enzymatic activity. During the whole process, the optical purity of the chiral amine eluting from the column was higher than 90%. A facile procedure was developed for separating the unreacted (R)‐amine from the (S)‐amide and for the recycling of the solvent 3‐methyl‐3‐pentanol and the active ester 2,2,2‐trifluoroethyl butyrate. © 1992 John Wiley & Sons, Inc.
Selective separation of microorganisms by lectins: Yeast and concanavalin A as a model system
Authors: Stratford Malcolm , Bond Chris J.
Published: December 2004

Specific aggregation and separation of microorganisms was investigated using yeasts and concanavalin A as a model system. Cells of Saccharomyces cerevisiae were specifically aggregated and so separated from those of Schizosaccharomyces pombe. Optimum aggregation with over 99% of cells aggregated was achieved by adjustment to pH value and applied agitation. Dimeric lectin structure caused a far higher degree of aggregation than did tetrameric. Degree of aggregation was also strongly influenced by the ratio of lectin/cell concentrations, optimum aggregation occurring in the middle range of ratios. A high ratio of lectin to cells inhibited aggregation, occupation of most of the available receptors preventing intercellular bonding by divalent lectins. Detachment and reuse of concanavalin A was demonstrated using switching from moderate to low pH value. Potential uses for species‐specific‐separation of microorganisms are discussed. © 1992 John Wiley & Sons, Inc.
Estimating cell concentration in the presence of suspended solids: A light scatter technique
Authors: Kennedy M. J. , Thakur M. S. , Wang D. I. C. , Stephanopoulos Gregory N.
Published: December 2004

A novel sensor was developed, based on light scatter, to estimate the cell concentration in the presence of suspended solids. The light scatter properties of cells in the presence of suspended solids were investigated. Two crucial observations were made: first, that the light scatter from cells is essentially a linear function of cell concentration and, second, that invariant regions are present in the light scatter spectrum of cell/solid substrate mixtures. Invariant regions are wavelength intervals of the light scatter spectrum in which the light scatter reading is independent of solid substrate concentration and only a function of cell concentration. The occurrence of invariant regions is the key behavior which allowed the quantification of cell concentration in the presence of suspended solids.

An algorithm was developed for the estimation, from light scatter data, of cell concentration in the presence of solid substrate. The light scatter approach was validated by comparing cell concentrations estimated by this technique to those obtained from DNA and carbon dioxide evolution rate measurements during a series of fermentations. The model system used was Bacillus subtilis var sakainensis ATCC 21394 growing on fishmeal as the sole nitrogen source.

A model was developed based on the interactions of scatter and absorbance. This model reflects the hypothesis that invariant regions are caused by changes in the absorbance of the solid substrate as a function of wavelength. © 1992 John Wiley & Sons, Inc.
Damaging agitation intensities increase DNA synthesis rate and alter cell‐cycle phase distributions of CHO cells
Authors: Lakhotia Sanjay , Bauer Kenneth D. , Papoutsakis Eleftherios T.
Published: December 2004

The effects of fluid‐mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2‐L bioreactors has been examined. A two‐color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high‐agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S‐phase cell of the high‐agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high‐agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250‐rpm culture had a higher fraction of S‐phase and a lower fraction of G1‐phase cells than the 235‐rpm culture. The DNA sunthesis rate per viable S‐phase cell of the 250‐rpm culture was greater than of the 235‐rpm culture. © 1992 John Wiley & Sons, Inc.
Development of a single‐pass ceramic matrix bioreactor for large‐scale mammalian cell culture
Authors: Applegate Mark A. , Stephanopoulos Gregory
Published: December 2004

A single‐pass, plug‐flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via sllicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side‐by‐side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single‐pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale‐up. © 1992 John Wiley & Sons, Inc.
Viability of Candida shehatae in D‐xylose fermentations with added ethanol
Authors: Kastner James R. , Ahmad Maqsood , Jones W. Jack , Roberts R. S.
Published: December 2004

Ethanol was added at concentrations of 25 and 50 g/L to active cultures of Canida shehatae under oxygen‐limited (fermentative) conditions. Added ethanol completely inhibited grwoth and fermentation of D‐xylose by C. shehatae. Cultures with added ethanol rapidly declined in cell viability as measured by plate counts and methylene blue staining. The rate of decline in cell viability was dependent on the amount of added ethanol. Over the course of the fermentation, cell viability, as measured by plate counts, was significantly lower in all experiments (with or without ethanol addition) compared with the viability measurements by methylene blue staining. Thus, data from the plate counts provided a more sensitive measure of the toxic effects of added ethanol and long‐term anaerobiosis on C. shehatae growth/fermentation. Mean cell volume and total cell volume declined in fermentations with added ethanol. © 1992 John Wiley & Sons, Inc.
Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105
Authors: Robas N. , Zouheiry H. , Branlant G. , Branlant C.
Published: December 2004

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. © 1993 John Wiley & Sons, Inc.
Intensification of mass transfer in aqueous two‐phase systems
Authors: Save Sanjiv V. , Pangarkar Vishwas G. , Kumar S. Vasanth
Published: December 2004

A novel technique which intensifies conventional aqueous two‐phase extraction by conversion of dispersed phase into colloidal gas aphrons (CGAs) has been developed for extraction of an enzyme. In the present work, amyloglucosidase (1,4‐α‐D‐glucan glucohydrolase) was extracted using a polyethylene glycol‐sodium sulfate‐water system. The lighter phase, i.e., polyethylene glycol (PEG) rich phase, was converted into CGAs which were then dispersed into a salt rich phase. The effect of type of surfactant and its concentration, dispersed phase velocity, phase composition, and type of sparger on the dispersed phase mass transfer coefficient was investigated. The results suggests 9–16 times higher values of mass transfer coefficient compared to spray column. The multiorifice sparger at concentrations of 0.33 g/L of cetyl trimethyl ammonium chloride yielded best results. © 1993 John Wiley & Sons, Inc.
Cell–cell adhesion and aggregation: Influence on the growth behavior of CHO cells
Authors: Renner W. A. , Jordan M. , Eppenberger H. M. , Leist C.
Published: December 2004

The influence of cell‐cell adhesion on the growth behavior of Chinese hamster ovary (CHO) cells in suspension culture was investigated. CHO cells form aggregates under suboptimal growth conditions. Clusters are formed around decaying and dead cells. The deoxyribonucleic acid (DNA) released from these cells was found to mediate the cells was found to mediate the cell‐cell adhesion. Cluster formation dramatically influenced the growth behavior of the cells. First, cells within aggregates showed a strongly reduced specific proliferation rate, and second, shear forces exerted on large aggregates caused a considerable higher specific death rate than those exerted on single cells. These factors led to a reduction of the specific growth rate up to 50%. This decrease could be avoided by addition of DNase 1 to the medium. It is shown that the separate determination of the specific proliferation and death rates is not feasible with state‐of‐the‐art methods. To achieve a more profound and precise description of the growth pattern of animal cells, we propose an extended Monod model and describe the relevant methods. © 1993 John Wiley & Sons, Inc.
Evaluation of a hepatocyte‐entrapment hollow fiber bioreactor: A potential bioartificial liver
Authors: Nyberg Scott L. , Shatford Russel A. , Peshwa Madhusudan V. , White James G. , Cerra Frank B. , Hu Wei‐Shou
Published: December 2004

We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte‐entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort‐term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation. © 1993 John Wiley & Sons, Inc.
Immobilization of lipase for effective interesterification of fats and oils in organic solvent
Authors: Cho Sang‐Woo , Rhee Joon Shick
Published: December 2004

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8‐fold that of the original activity of free lipase when the spacer was 7‐aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half‐life of 97 days. © 1993 John Wiley & Sons, Inc.
Reverse micelles in protein separation: The use of silica for the back‐transfer process
Authors: Leser Martin E. , Mrkoci Kristina , Luisi Pier Luigi
Published: December 2004

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein‐loaded reverse micellar solution with a water phase containing an appropriate salt (back‐transfer). In this article we describe an alternative “back extraction” procedure which is based on the addition of silica to the protein‐containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2‐ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60–80 of the total protein (α‐chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80−100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short‐chain lecithin was used. It is shown that the recovery of a α‐chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. © 1993 John Wiley & Sons, Inc.
Fuzzy control of ethanol concentration its application to maximum glutathione production in yeast fed‐batch culture
Authors: Alfafara Catalino G. , Miura Keigo , Shimizu Hiroshi , Shioya Suteaki , Suga Ken‐ichi , Suzuki Kazuyuki
Published: December 2004

A fuzzy logic controller (FLC) for the control of ethanol concentration was developed and utilized to realize the maximum production of glutathione (GSH) in yeast fedbatch culture. A conventional fuzzy controller, which uses the control error and its rate of change in the premise part of the linguistic rules, worked well when the initial error of ethanol concentration was small. However, when the initial error was large, controller overreaction resulted in an overshoot.

An improved fuzzy controller was obtained to avoid controller overreaction by diagnostic determination of “glucose emergency states” (i.e., glucose accumulation or deficiency), and then appropriate emergency control action was obtained by the use of weight coefficients and modification of linguistic rules to decrease the overreaction of the controller when the fermentation was in the emergency state. The improved fuzzy controller was able to control a constant ethanol concentration under conditions of large initial error.

The improved fuzzy control system was used in the GSH production phase of the optimal operation to indirectly control the specific growth rate μ to its critical value μc. In the GSH production phase of the fed‐batch culture, the optimal solution was to control μ to μc in order to maintain a maximum specific GSH production rate. The value of μc also coincided with the critical specific growth rate at which no ethanol formation occurs. Therefore, the control of μ to μc could be done indirectly by maintaining a constant ethanol concentration, that is, zero net ethanol formation, through proper manipulation of the glucose feed rate. Maximum production of GSH was realized using the developed FLC; maximum production was a consequence of the substrate feeding strategy and cysteine addition, and the FLC was a simple way to realize the strategy. © 1993 John Wiley & Sons, Inc.
Continuous measurement of microbial heat production in laboratory fermentors
Authors: van Kleeff B. H. A. , Kuenen J. G. , Heijnen J. J.
Published: December 2004

The possibility of continuously measuring the heat produced by microorganisms in an ordinary laboratory fermentor was studies. An inventory of the heat flows influencing the temperature of the culture was made. The magnitude and standard deviation in these heat flows were studied from theoretical and practical viewpoints. Calibration procedures were tested, and a model describing the heat flows in steady state and during dynamic conditions was made. Microbial heat production could be calculated accurately with the help of this model, appropriate temperature measurements, and equipment properties measured during the calibration procedures. It was found that the measurement of heat production could be done with an accuracy similar to that in the O2 uptake measurement. © 1993 John Wiley & Sons, Inc.
Dynamics of transcriptional and translational processes in hepatocytes cultured in a collagen sandwich
Authors: Dunn James C. Y. , Tompkins Ronald G. , Yarmush Martin L.
Published: December 2004

A mathematical model which simulates the dynamic behavior of the hepatocytes cultured in a collagen sandwich is presented. Using several independently determined experimental parameters (e.g., albumin gene nuclear runoff activity, the level of albumin mRNA, and the albumin secretion rate), we have used this model to calculate the in vivo albumin gene transcriptional rate (0.27 molecules per second per hepatocyte), the half‐life of albumin mRNA (3.3 days) in cultured hepatocytes, and the albumin polypeptide elongation rate (10 amino acids per second). In addition, the characteristic time constants for the transient increases in transcription (5 days) and in translation (10 days) were also obtained. These bestfit parameters were used to predict the rate of albumin secretion in rescued hepatocytes. © 1993 John Wiley & Sons, Inc.
Factors affecting the performance of crossflow filtration of yeast cell suspension
Authors: Tanaka Takaaki , Kamimura Ryoji , Itoh Kazutaka , Nakanishi Kazuhiro , Matsuno Ryuichi
Published: December 2004

Factors affecting the performance of crossflow filtration were investigated with a thin‐channel module and yeast cells. In crossflow filtration of Saccharomyces cerevisiae cells cultivated with YPD medium (Yeast extract, polypeptone, and dextrose) and suspended in saline, a steady state was attained within several minutes when the cell concentration was low and the circulation flow rate was high. The steady‐state flux and the change in flux during the initial unsteady state were explained well by conventional filtration theory, with the amount of cake deposited and the mean specific resistance to the cake measured in a dead‐end filtration apparatus used in calculation. When the circulation flow rate was lower than a critical value, a part of the channel of the crossflow filtration module was plugged with cell cake, and thus the steady‐state flux was low. In crossflow filtration of suspensions of commercially available baker's yeast, the flux gradually decreased, and the flux after 8 h of filtration was lower than the value calculated by filtration theory. Fine particles contaminating the baker's yeast was responsible for the decrease. A similar phenomenon was responsible for the decrease. A similar phenomenon was observed in crossflow filtration of a broth of S. cerevisiae cells cultivated in molasses medium, which also contains such particles, had no effect of the permeation flux during crossflow filtration. © 1993 John Wiley & Sons, Inc.
Effect of oxygen fluctuations on recombinant Escherichia coli fermentation
Authors: Namdev Pradyumna K. , Irwin Nancy , Thompson B. G. , Gray Murray R.
Published: December 2004

Escherichia coli DH5α, carrying the pUC19 plasmid for the lacZ fragment of β‐galactosidase and ampicillin resistance, was grown in a batch fermentor under conditions of fluctuating oxygen supply. A Monte Carlo method was used to control the on/off supply of air to simulate circulation of cells in a large fermentor. Rapid changes in oxygen supply reduced the rates of oxygen uptake the carbon dioxide release and prolonged the active second growth phase in batch culture, compared to growth with continuous aeration. Amplification of the plasmid was observed during the stationary phase when air supplied continuously, but not during the Monte Carlo experiments. © 1993 John Wiley & Sons, Inc.
A Simple genetically structured model of trp repressor–operator interactions
Authors: Koh Boon Tong , Yap Miranda G. S.
Published: December 2004

A genetically structured mathematical model of the trp operon based on known molecular interactions of aporepressor, corepressor, and inducer is proposed. The model simulates, both qualitatively and quantitatively, the influence of these regulatory species on the extent of repression and expression of cloned gene products. It shows that at low aporepressor concentration, full repression is not possible even with high tryptophan levels, resulting in leaky expression. Calculations based on the model enabled predictions of optimum levels of aporepressor and tryptophan for effective repression and, concurrently, the β‐indoleacrylic acid concentrations required for induction for both low and high plasmid copy number clones. Using the model we attempted to provide explanations for seemingly anomalous and sometimes contradictory observations by researchers when working with the trp promoter. © 1993 John Wiley & Sons, Inc.
A Simple morphologically structured model describing the growth of filamentous microorganisms
Authors: Nielsen Jens
Published: December 2004

Based on the reported mechanisms for filamentous growth, a simple morphologically structured growth model is set up. The model may describe the growth of filamentous microorganisms both on a solid medium and in a submerged culture. For description of a submerged culture the model is combined with a simple population model, which is derived from a balance for the distribution function for the hyphal elements. The model is compared with experimental data for three species of filamentous microorganisms: Geotrichum candidum, Streptomyces hygroscopicus, and Penicillium chrysogenum. © 1993 John Wiley & Sons, Inc.
Diffusivity of Cu2+ in calcium alginate gel beads
Authors: Chen Dong , Lewandowski Zbigniew , Roe Frank , Surapaneni Prasad
Published: December 2004

A linear absorption model (LAM) is used to describe the process of metal binding to spherically shaped biopolymers particles. The LAM was solved using a numerical algorithm which calculates diffusivities of metal ion in biopolymer gels. It assumes attainment of rapid metal‐biopolymer binding equilibrium accompanied by rate limiting diffusion of the metal ions through the gel. The model was tested using batch experiments in which copper (Cu2+) binding with calcium alginate beads was investigated. Biopolymer density in the beads was varied between 2% and 5%. The diffusion coefficient of Cu2+ calculated from the LAM ranged from 1.19 × 10−9 to 1.48 × 10−9 m2 s−1 (average 1.31 ± 0.21 × 10−9 m2 s−1), independent of biopolymer density. The LAM has theoretical advantages over the shrinking core model (shell progressive model). The latter calculated an unreasonable exponential increase in the diffusion coefficient as density of alginate polymer in the bead increased. © 1993 John Wiley & Sons, Inc.
Recombinant trypsin production in high cell density fed‐batch cultures in Escherichia coli
Authors: Yee L. , Blanch H. W.
Published: December 2004

Fed‐batch techniques were employed to obtain high cell density cultures (92–100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth‐inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease. © 1993 Wiley & Sons, Inc.
Population and biomass kinetics in fed‐batch cultures of Daucus carota L. somatic embryos
Authors: Huang Li‐Chung , Chi Chung‐Ming , Vits Hugo , Staba E. John , Cooke Todd J. , Hu Wei‐Shou
Published: December 2004

The population dynamics of developing somatic embryos of carrot (Daucur carota L.) was investigated in batch and fed‐batch cultures using modified Murashige and Skoog medium. These substrate limitations coincided not only with stoppage of biomass increase, but also with the increase in total concentration of embryos as well as the advancement of the embryo into a more mature stage. Both glucose and ammonium were depleted from the culture. Restoring either glucose, or ammonium and nitrate, as to approximately initial concentrations in fed‐batch experiments, did not result in a significant increase of the total normal embryo concentration. On the other hand, medium replacement led to increase in biomass concentration, total embryo number, and improved embryo maturity. The addition of a mixture of glucose, ammonium, and nitrate to the spent medium resulted in variable increases in biomass and embryo number, but always less than those resulting from media replacement. Although the total number of embryos was higher after medium replacement, the fraction of embryos reaching torpedo stage was still only 50%. The need for a better means of population characterization for further kinetic studies is discussed. © 1993 Wiley & Sons, Inc.
Stability of bilirubin oxidase in organic solvent media: A comparative study on two low‐water systems
Authors: Skrika‐Alexopoulos Eleftheria , Muir Jacqueline , Freedman Robert B
Published: December 2004

The storage stability of bilirubin oxidase was studied in water‐in‐oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a “fast” phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady‐state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the Km of the enzyme for bilirubin. Stability was also studied in a liquid/solid two‐phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the “fast” phase rate observed in the water‐in‐oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the “slow” phase observed in water‐in‐oil microemulsions. © 1993 Wiley & Sons, Inc.
A novel membrane bioreactor for detoxifying industrial wastewater: I. Biodegradation of phenol in a synthetically concocted wastewater
Authors: Livingston Andrew Guy
Published: December 2004

A novel process has been used to biodegrade phenol present in an acidic (1 M HCI) and salty (5% w/w NaCl) synthetically bioreactor, in which the phenol present in the wastewater is separated from the inorganic components by means of a silicone rubber membrane. Transfer of the phenol from the wastewater and into a biological growth medium allows biodegradation to proceed under controlled conditions which are unaffected by the hostile inorganic composition of the wastewater. At a wastewater flow rate of 18 mL h−1 (contact time 6 h), 98.5% of the phenol present in the wastewater at an inlet concentration of 1000 mg was degraded; at a contact time of 1.9 h, 65% of the phenol was degraded. Phenol degradation was accompanied by growth of a biofilm on the membrane tubes and by conversion of approximately 80% of the carbon entering the system to CO2 carbon. Analysis of the transport of phenol across the membrane revealed that the major resistance to mass transfer arose in the diffusion of phenol across the silicone rubber membrane. A mathematical model was used to describe the transfer of phenol across the membrane and the subsequent diffusion and reaction of phenol in the biofilm attached to the membrane tube. This analysis showed that (a) the attached biofilm significantly lowers the mass transfer driving force for phenol across the membrane, and (b) oxygen concentration limits the phenol degradation rate in the biofilm. These conclusions from the model are consistent with the experimental results. © 1993 Wiley & Sons, Inc.
Continuous, high level production and excretion of a plasmid‐encoded protein by Escherichia coli in a two‐stage chemostat
Authors: Fu Jeffrey , Wilson David B. , Shuler Michael L.
Published: December 2004

The stable continuous overproduction of a plasmidencoded protein, β‐lactamase, for at least 50 days by Escherichia coli K‐12, RB791(pKN), with release into the culture medium has been demonstrated in two‐stage chemostats. The second‐stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of β‐lactamase could not be sustained with this strain in a single‐stage chemostat because of cell death and selection for lac−1 cells. β‐Lactamase production in the second stage was sensitive to the second‐stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high β‐lactamase levels. The best operating conditions found at 20°C were a first‐stage dilution rate of 0.12 h−1, a second‐stage dilution rate of 0.03 h−1, and equal glucose feed supplied to each stage. Enzymatically active β‐lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg β‐lactamase/L that was 50% pure at an OD600 < 6. © 1993 Wiley & Sons, Inc.
Affinity thermoprecipitatin: Contribution of the efficiency of ligand–protein interaction and access of the ligand
Authors: Galaev I. Yu. , Mattiasson B.
Published: December 2004

Conjugates to two thermoprecipitating polymers, poly(N‐vinyl caprolactam) and poly(N‐isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu‐iminodiacetic acid, and p‐aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100‐1000‐fold decrease in enzyme‐affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10−7‐10−8 M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10−9‐10−11 M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand‐polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand‐polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. © 1993 Wiley & Sons, Inc.
In situ fermentation monitoring with recombinant firefly luciferase
Authors: Lasko Daniel R. , Wang Daniel I. C.
Published: December 2004

A novel method is described for the on‐line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 106 cells/mL, or an OD600 of 0.004, are easily detectable and concentrations greater than 1010 cells/mL are well within range. The data are collected by a computer, so adaptation to on‐line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. © 1993 John Wiley & Sons, Inc.
Gluco‐oligosaccharide synthesis by free and immobilized β‐glucosidase
Authors: Ravet C. , Thomas D. , Legoy M. D.
Published: December 2004

Gluco‐oligosaccharides were synthesized through the enzymatic condensation of D‐glucose at high concentration using a commercial almond β‐glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (aw) in the presence of different glucose concentrations. The yield and the composition of the gluco‐oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di‐, tri‐, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL · mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. © 1993 John Wiley & Sons, Inc.
Neural network modeling of batch cell growth pattern
Authors: Syu Mei‐Jywan , Tsao George T.
Published: December 2004

The capability of neural networks in modeling batch cell growth by providing initial conditions only is tested in this study. The neural network tested is of the back‐propagation‐type including a newly discovered saturation‐type transfer function. The simulation and prediction results of this neural network modeling will be demonstrated. © 1993 John Wiley & Sons, Inc.
Transport of sugars via two anomer‐specific sites on mannose–phosphotransferase system in Lactococcus cremoris: In vivo study of mechanism, kinetics, and adaptation
Authors: Benthin Stig , Nielsen Jens , Villadsen John
Published: December 2004

Glucose uptake in Lactococcus lactis subsp. cremoris FD1 occurs via the mannose phosphotransferase system (Man–PTS), which is quite unspecific and allows transport of many different sugars and sugar analogues. It was previously shown (Benthin, S., Nielsen, J., Villadsen, J. Biotechnol. Bioeng. 40:137–146, 1992) that the kinetics of in vivo glucose uptake in a glucose‐limited chemostat culture is best described by assuming that the glucose transport system has two anomer‐specific sites with a relative uptake rate of 36% through the α‐site. In the present study, the existence of anomer‐specific sites on Man–PTS is shown by experiments where α‐glucose, β‐glucose, mannose, and 2‐deoxyglucose are added to glucose‐limited chemostat cultures. A quantitative description of the competitive uptake of the involved sugars at the two sites is given. In a mannose‐limited chemostat culture, the relative glucose flux via the α‐site is 50%, corresponding to a change toward the equilibrium composition of mannose (68%). Furthermore, when the feed to a mannose‐limited chemostat culture is changed to glucose, the rate of change of relative glucose flux through the α‐site corresponds to constitutive synthesis of Man–PTS with 36% α‐site stoichiometry in new cells. When N‐acetylglucosamine (73% α‐anomer at equilibrium) is the limiting substrate, the relative glucose flux through the α‐site is also 48% to 50%. With a feed of α‐glucose generated enzymatically from nonmetabolizable sucrose the relative glucose flux through the α‐site can be as high as 78%. Finally, growth in the presence of nonmetabolizable α‐methylglucoside leads to formation of cells with a relative glucose flux through the α‐site of 29% to 30%. The adaptation of the flux distribution between the α‐ and β‐site is tentatively explained by the hypothesis that two integral membrane proteins of Man–PTS are involved in this process. © 1993 John Wiley & Sons, Inc.
Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis
Authors: Goel Akshay , Ferrance Jerome , Jeong Jinwook , Ataai Mohammed M.
Published: December 2004

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose‐limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by‐products, CO2 evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide‐adenine dinucleotide phosphate (NADPH) used in biosynthesis. © 1993 John Wiley & Sons, Inc.