bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth yield, maintenance requirements, and lipid formation in the oleaginous yeast Apiotrichum curvatum
Authors: Ykema Adrie , Bakels Ronald H. A. , Verwoert Ira I. G. S. , Smit Henk , van Verseveld Henk W.
Published: December 2004

For guiding and improving the efficiency of the production of lipid, complete insight into the flow of carbon and energy during growth and product formation is necessary. Therefore, data have been collected to determine various important growth parameters for the oleaginous yeast Apiotrichum curvatum. Chemostat experiments at specific growth rates, ranging from 0.05 to 0.15 h−1 and recycling experiments with 100% biomass retention, with growth rates decreasing from 0.10 to 0.004 h−1, demonstrated that maintenance requirements of A. curvatum are very low, compared to maintenance requirements described for other yeasts as Saccharomyces cerevisiae and Candida parapsilosis.

It also appeared that growth and lipid production are proportional to substrate consumption when specific growth rates are higher than approximately 0.02 h−1, but that lipid production stops at growth rates below this value. The practical consequences of these data are that fed batch cultures, which are often applied in fermentation industry, can only be useful with lipid producing yeasts when the growth rate in the process is carefully monitored to ensure specific growth rates higher than 0.02 h−1. Dilution of the culture, partial recycling and/or a continuously increasing nutrient feed are solutions for the expected problems at low growth rates.
Optimization of Escherichia coli growth by controlled addition of glucose
Authors: Robbins J. W. , Taylor K. B.
Published: December 2004

During aerobic growth of Escherichia coli (recombinant K‐12 and strain B) on protein hydrolysate (L‐broth) and a carbon source (glucose), acetic acid is produced via glucose metabolism until the late log phase. At this point, the culture pH starts to increase and the growth rate decreases. In cultures without further glucose supplementation, these changes are associated with the accumulation of ammonia, the utilization of acetic acid, the depletion of amino acids, and the complete depletion of glucose. We hypothesize that, after depletion of the glucose, the bacteria catabolize amino acids for energy and carbon and give off the nitrogen as ammonia. Also contributing to the overall increase in pH is the depletion of the acetic acid produced earlier as it is metabolized upon exhaustion of glucose. However, there is a lag time of about 1 hour after the initial pH increase before the sustained accumulation of ammonia begins. This lag indicates that an unidentified factor, in addition to the increase in ammonia, contributes to the increase in pH. Advantage was taken of the turnaround from acid production to base production as reflected in the culture pH to implement the addition of glucose. In growth experiments during which the pH was controlled in the basic direction by glucose addition, the observed decrease in growth rate was significantly postponed and the pH change in the basic direction was reversed as a result of acid production by the cells from the newly added glucose. Furthermore, coll densities of twice that obtained without glucose feeding were demonstrated. Based on the media cost per unit cell density, the data indicate a 31% cost savings.
Immobilized enzyme cellulose hollow fibers: III. Physical properties and in vitro biocompatibility
Authors: Comfort Ann R. , Berkowitz Sharon , Albert Elizabeth , Langer Robert
Published: December 2004

The initial testing of the safety of a cellulose–heparinase hollow fiber device was assessed with respect to physical properties and in vitro biocompatibility. The material cleared urea and creatinine without passing albumin, even at high flow rates. The clearance of urea and creatinine by cellulose–heparinase was equal or slightly reduced in comparision to the cellulose device. The cellulose–neparinase device tolerance to now rates was also unchanged. In addition, scanning electron microscopy of the lumen established the uniformity of the material. The analysis of clearance rates and the scanning electron micrographs show there to be no damage to the cellulose membrane after tresyl chloride activation and heparinase immobilization. The investigation of biocompatibility in an in vitro test system with whole human blood indicated that there were no significant changes in the biocompatibility of cellulose with bound heparinase. There was no change in the level of red blood cells, white blood cells, or platelets over the course of in vitro whole blood perfusion through cellulose or cellulose–heparinase hollow fiber devices. Low levels of plasma hemoglobin and complement activation were observed with cellulose and cellulose–heparinase devices. Thus, the cellulose hollow fibers can be functionalized without any changes in in vitro performance.
Isolation of urokinase by affinity ultrafiltration
Authors: Male K. B. , Nguyen A. L. , Luong J. H. T.
Published: December 2004

A water‐soluble, ligand‐bound polymer has been synthesized for the purpose of isolation of urokinase, an important plasminogen activator. The affinity polymer was formed by copolymerizing N‐acryloyl‐m‐aminobenza‐midine and acrylamide in the absence of oxygen. An affinity ultrafiltration process was then developed for isolating urokinase from an artificial solution containing peroxidase and urokinase and from a crude urine source. The process yields were determined to be 86% and 49%, respectively. The recovered urokinase exhibited a specfic activity close to that of the highest commercial grade. This article also presents a new technique for assaying urokinase by coupling plasminogen with L‐benzoyl arginine‐p‐nitroanilide (L‐BAPNA), an inexpensive chromogenic substrate.
Mathematical modeling of the effects of ammonium ion on the intracellular pH of hybridoma cells
Authors: McQueen Anne , Bailey James E.
Published: December 2004

A mathematical model for the effects of NH4Cl addition to the growth medium on hybridoma cell intracellular pH (pHi) is presented which includes a detailed description of the properties of the Na+/H+ exchanger. The model is used to calculate the steady‐state value of pHi as a function of the extracellular NH4Cl concentration, employing parameter values taken from the literature except for the cell permeability to NH4+ This parameter value, estimated to be 3.5 × 10−7 cm/s, is obtained by fitting simulation results to experimental data obtained previously for the steady‐state value of pHi in the presence of 10mM NH4Cl. The model simulates well the major features of previously observed experimental pHi changes following NH4Cl addition: the transient characteristics, dependence on the NH4Cl concentration, and lack of dependence on the external pH value. The effect of parameter variations on model results is also investigated in additional simulations. The general modeling framework employed here should be useful for other types of mammalian cells.
Invertase‐catalyzed reactions in alcoholic solutions
Authors: Selisko Barbara , Ulbrich Renate , Schellenberger Alfred , Müller Ute
Published: December 2004

The formation of alkyl β‐D‐fructofuranosides by invertase from sucrose in aqueous solutions of methanol, ethanol, or n‐propanol is studied for the dependence on alcohol and invertase concentrations as well as on reaction time. The yield of alkyl β‐D‐fructosides is shown to be controlled by three competitive reactions: the alcoholysis of sucrose, the hydrolysis of sucrose, and the hydrolysis of alkyl β‐D‐fructosides. Both the conversion rate of sucrose and the fraction of alkyl β‐D‐fructosides in the product mixture are dependent on the chain length of the alcohols. They decrease in the sequence methanol > ethanol > n‐propanol. Alkyl β‐D‐fructosides are also formed by invertase starting from alcoholic solutions of fructose.
Modeling of immobilized cell reactor for propionic acid fermentation
Authors: Crespo J. P. S. G. , Almeida J. S. , Moura M. J. , Carrondo M. J. T.
Published: December 2004

A mathematical model of steady state reactor behavior was developed, combining the tanks‐in‐series model with fermentation kinetic description. The model was tested with experimental data‐from an immobilized cell reactor for propionic acid production operated under different residence times. In order to determine the effects of liquid flow rate and gas evolution on the degree of mixing, residence time distribution studies (RTDs) were performed during fermentation. The tanks‐in‐series model represented adequately the hydrodynamic behavior of the reactor. Association of an intrinsic kinetic model obtained from chemostat studies with the tanks‐in‐series model allows a “chemostat equivalent” cell concentration profile and a substrate consumption profile to be obtained. The advantage of having a two‐level pH adjustment within the reactor is also discussed.
Enhancing penicillin fermentations by increased oxygen solubility through the addition of n‐hexadecane
Authors: Ho Chester S. , Ju Lu‐Kwang , Baddour Raymond F.
Published: December 2004

n‐Hexadecane was added to fermentation media to increase the medium oxygen solubilities, thus enhancing oxygen transfer rates in penicillin fermentations. For shake flask fermentations, cells were found to grow faster in the flasks with n‐hexadecane than those without. The addition of n‐hexadecane to penicillin fermentations was shown to significantly increase cell growth and penicillin production and reduce formation of mycelial pellets. The result was attributed to the enhancement of oxygen transfer in mycelial fermentations due to the higher oxygen solubilities of fermentation media achieved by adding n‐hexadecane.
High‐level recombinant protein production in bioreactors using the baculovirus–insect cell expression system
Authors: Caron Antoine W. , Archambault Jean , Massie Bernard
Published: December 2004

In order to develop an efficient process for large‐scale production of recombinant protein, various factors were studied which affect the productivity of Sf‐9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac‐BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10‐fold when host cell density at the time of infection increased from 2 × 106 to 3 × 106 cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4–6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum‐free medium. Fine‐tuning of infection parameters in a 4‐L surface‐aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.
Parametric analysis of the errors associated with the Michaelis–Menten equation
Authors: Brown Russell F. , Holtzapple Mark T.
Published: December 2004

Based on the well‐known mechanism describing Michaelis–Menten kinetics, three rate expressions may be developed: the exact solution (Model 1), a rate equation resulting from the pseudo‐steady‐state assumption (Model 2), and Model 2 with the additional assumption that the amount of free substrate is approximately equal to the total amount of substrate (Model 3). Although Model 1 is the most precise, it must be integrated numerically and it requires three experimentally determined parameters. Models 2 and 3, however, are simpler and require only two parameters. Using dimensionless forms of the three models, we have evaluated the errors in the two simplified models relative to the exact solution using a wide range of parameter values. The choice of model for reactor design depends on the initial substrate to enzyme ratio (α0), and on the ratio of the Michaelis–Menten constant to the enzyme concentration (σ). Based on a 2% model error criteria, when α0 > 15 or σ ≥ 100, Model 3 is adequate; if 5 < α0 < 15, or if σ ≥ 10, then Model 2 may be used; and if α0 < 5 and σ < 10, then the exact solution (Model 1) is required.
A simple model to describe transient differences between cell number and biomass growth rates of Escherichia coli
Authors: Rodin J. B. , Lyberatos G. K. , Svoronos S. A.
Published: December 2004

Information on the response of a microbial culture to dynamic environmental conditions is necessary for the design of transient operation processes. However, most attempts at modelling culture response have been directed at describing the steady‐state behavior. Thus, there is a need for adequate dynamic models for process design. Simulations of nutrient shifts were completed using a “single‐cell” model for Escherichia coli. It was discovered that the specific mass growth rate and the specific number of cells growth rate were different under transient conditions, whereas at steady state (balanced growth) these rates are equivalent. Using these observations, a simple delay model to describe the transient behavior of the two growth rates is formulated and tested. The model contains as state variables only the readily measurable macroscopic quantities (biomass, cell number, and limiting nutrient). This model agreed well with the predictions of the single‐cell model.
Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: Batch and continuous studies
Authors: Mehta Perdeep K. , Ghose Tarun K. , Mishra Saroj
Published: December 2004

The DEAE‐cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono‐oxygenase activity (66 μmol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell‐to‐carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH2 required for the sustained activity of methane mono‐oxygenase. Maximum methanol level of 50 μmol/mg cells was obtained in the batch reactor. In a standard 50‐mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 μmol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH2. The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 μmol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half‐life t1/2 and thermal denaturation rate constant Kd were computed to be 108 h and 6.42 × 10−3 h−1, respectively.
Complex growth dynamics in batch cultures: Experiments and cybernetic models
Authors: Straight Jeffrey V. , Ramkrishna D.
Published: December 2004

The cybernetic framework developed by Ramkrishna and co‐workers is shown to encompass the regulation of nutrient transport processes as well as the effect of nutrient transport on the biotic phase. A structured model which accounts for both an abiotic or environmental phase and a biotic or cellular phase is proposed to describe bacterial growth on lactose as the limiting carbon and energy source. In the presence of lactose, competing uptake mechanisms are proposed. At low lactose concentrations, an energy‐requiring transport process is the preferred uptake mechanism. The coupling between cellular energetics and nutrient uptake results in an interesting intermittent growth phenomenon. As the concentration of lactose increases, a nonenergetic transport process is preferred and cellular growth ceases to be intermittent. Model simulations are compared with previously reported experimental results and exhibit good agreement over the entire range of initial lactose concentrations.
Papain kinetics in the presence of a water‐miscible organic solvent
Authors: Fernandez Mirtha M. , Clark Douglas S. , Blanch Harvey W.
Published: December 2004

The effects of various concentrations of a water‐miscible organic solvent [a 7:3 (v/v) mixture of N, N dimethylformamide and dimethylsulfoxide] on the kinetics of papain have been investigated. The parameters kcat and Km for the amidase and esterase activity of papain using N‐α‐benzoyl‐DL‐arginine‐p‐nitroanilide (BAPNA) and N‐α‐benzoyl‐L‐arginine ethyl ester (BAEE) as substrates were determined. For both types of activity, kcat initially increased (up to about 15% solvent), and then decreased with increasing concentrations of organic solvent. In contrast, Km increased sharply with the organic solvent concentration. Active site titration at 0 and 50% solvent indicated no change in the amount of active enzyme. Fluorometric measurements of the emission spectrum of papain did not indicate any major conformational changes with increasing concentrations of organic solvent.
Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose
Authors: Slininger P. J. , Branstrator L. E. , Bothast R. J. , Okos M. R. , Ladisch M. R.
Published: December 2004

Pichia stipitis NRRL Y‐7124 has potential application in the fermentation of xylose‐rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen‐limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K1a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (Cox). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (μ) was a Monod function of Cox. Specific oxygen uptake rate was proportional to μ by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady‐state COX depended only on D, while steady‐state biomass concentration was controlled by both D and K1a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of μ. The dying portion increased with E and X.
Critical assessment of gassing‐in methods for measuring kla in fermentors
Authors: Linek V. , Sinkule J. , Beneš P.
Published: December 2004

The reliability of dynamic measurement methods of kla in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen ⇌ air exchange and the newly presented variant using the oxygen‐enriched air (27 vol % O2) → air exchange. The testing consisted in comparing kla values determined from these methods with values determined from the steady‐state Na2SO3 feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na2SO4 solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low kla values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.
Biofilm detachment mechanisms in a liquid‐fluidized bed
Authors: Chang H. Ted , Rittmann Bruce E. , Amar D. , Heim R. , Ehlinger O. , Lesty Y.
Published: December 2004

Bed fluidization offers the possibility of gaining the advantages of fixed‐film biological processes without the disadvantage of pore clogging. However, the biofilm detachment rate, due to hydrodynamics and particle‐to‐particle attrition, is very poorly understood for fluidized‐bed biofilm processes. In this work, a two‐phase fluidized‐bed biofilm was operated under a constant surface loading (0.09 mg total organic carbon/cm2 day) and with a range of bed height (H), fluid velocities (U), and support‐particle concentrations (Cp). Direct measurements were made for the specific biofilm loss rate coefficient (bs)and the total biofilm accumulation (XfLf). A hydrodynamic model allowed independent determination of the biofilm density (Xf), biofilm thickness (Lf), liquid shear stress (τ), and Reynolds number (Re). Multiple regression analysis of the results showed that increased particle‐to‐particle attrition, proportional to Cp and increased turbulence, described by Re, caused the biofilms to be denser and thinner. The specific detachment rate coefficient (bs) increased as Cp and Re increased. Almost all of the 6, values were larger than predicted by a previous model derived for smooth biofilms on a nonfluidized surface. Therefore, the turbulence and attrition of bed fluidization appear to be dominant detachment mechanisms.
Inhibition of microbial growth and metabolism by excess turbulence
Authors: Toma M. K. , Ruklisha M. P. , Vanags J. J. , Zeltina M. O. , Lelte M. P. , Galinine N. I. , Viesturs U. E. , Tengerdy R. P.
Published: December 2004

Excess turbulence caused by high‐intensity stirring inhibited microbial growth and metabolism. In stirred tank bioreactors, the growth rate and lysine biosynthesis decreased in Brevibacterium flavum beyond 900 rpm, the growth rate of Trichoderma reesei on wheat straw beyond 150 rpm, and the growth rate of Saccharomyces cerevisae beyond 800 rpm. The term turbohypobiosis was introduced to describe this inhibition. Turbohypobiosis was characterized by a stress factor Fstr expressing the interaction of medium flow with microbial cells in local turbulent zones, dependent on the energy distribution of the stirring regime. Lysine synthesis was inhibited at significantly lower Fstr values than the growth of B. flavum. The main reason for the inhibition was shear effects causing decreased adenosine triphosphate (ATP) generation, lower O2 uptake, and lower specific growth rate of bacteria.
Culture of insect cells in helical ribbon impeller bioreactor
Authors: Kamen A. A. , Tom R. L. , Caron A. W. , Chavarie C. , Massie B. , Archambault J.
Published: December 2004

An 11‐L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf−9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface‐induced babble generation. Serum‐supplemented and serum‐free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h−1) to maximum cell densities of 5.5 × 106−6.0 × 106 cells. mL−1 with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum‐free medium culture with a recombinant baculovirus. Heterologous protein production of ∼35 μg per 106 cells was comparable to yields obtained in serum‐free cultures grown in spinner flasks and petri dishes. Average specific oxygen up‐take and carbon dioxide production rates of the serum‐free culture prior to infection as measured by on‐line mass spectroscopy were 0.20 μmol O2μ·(106 cells)−1 h−1 and 0.22 μmol CO2 · (106 cells)−1h−1 and increased by 30–40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by‐product generation.
Enzymatic resolution of (S)‐(+)‐naproxen in a continuous reactor
Authors: Battistel Ezio , Bianchi Daniele , Cesti Pietro , Pina Carlo
Published: December 2004

An enzymatic method for the continuous production of (S)−(+)−2−(6‐methoxy‐2‐naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen ethoxyethyl ester catalyzed by Candida cylindracea lipase. The reaction has been carried out in a continuous‐flow closed‐loop column bioreactor packed with Amberlite XAD−7, a slightly polor resin on which the lipase has been immobilized by adsorption. Various immobilization conditions as well as the properties of the immobilized lipase have been studied. The performance and the productivity of the bioreactor were evaluated as a function of the critical reaction parameters such as temperature, substrate concentration, and product inhibition. By using a 500‐mL column bioreactor, 1.8 kg of optically pure (S)‐(+)‐Naproxen were produced after 1200 h of continuous operation with a slight loss of the enzymatic activity.
Kinetic parameters of a mixed culture oxidizing sulfide and sulfur with oxygen
Authors: Buisman Cees J. N. , Jspeert Peter I , Hof Anne , Janssen Albert J. H. , Hagen Robert Ten , Lettinga Gatze
Published: December 2004

Kinetic parameters of biological sulfide oxidation are described. The influence of the sulfide loading rate on growth yield and specific oxidation rate were investigated with free‐cell suspensions. It is concluded that at least two types of bacteria were present, namely, sulfate producers (type A) that grow at higher loading rates. Type A bacteria have a growth yield of 04 g dry S/mol S, while type B bacteria have a growth yield of 04 g dry S/mol S. Type A has a high affinity for sulfide and is inhibited by sulfide at sulfide concentrations exceeding 10 mg/L. Type B has a low affinity for sulfide and is not inhibited by sulfide, but by oxygen.
Size changes associated with metal adsorption onto modified bone gelatin beads
Authors: Petersen James N. , Davison Brian H. , Scott Charles D. , Blankship Stephen L.
Published: December 2004

Significant quantities of heavy metals will adsorb onto modified bone gelatin beads. As this adsorption occurs, the bead can undergo a substantial volume change. Research has shown that the equilibrium bead diameter was a function of the solution pH and the ion concentration in the solution. Here, we demonstrate that under certain conditions, the volume of the beads that absorbed the metal was only 35% of the bead volume when no metal was adsorbed. By taking advantages of these size changes, a fluidized‐bed separator can be operated such that natural segregation of loaded beads occurs. This phenomenon may facilitate the design of continous separators for the recovery and concentration of heavy‐metal‐contaminated waters. These concepts are demonstrated using Cu2+ adsorption onto such beads.
Amperometric determination of amino group on a solid support using glutaraldehyde
Authors: Ukeda Hiroyuki , Nakzono Yukika , Matumoto Kiyoshi , Osajima Yutaka
Published: December 2004

A simple amperometric method has been developed for the determination of amino groups on a solid support. The method is based on oxygen consumption during the reaction between the bifunctional reagent gluaraldehyde and the amino groups on the solid support. Oxygen consumption was monitored with a Clark oxygen electrode. AHSepharose 4B, Affi‐Gel 102, and Amino‐Celluofine were used as amino‐terminal solid supports. The concentration of amino groups on each support was calculated from a calibration curve of the standard compound. When hexamethylenediamine was used as the standard, the concentration of amino groups on AH‐Sepharose 4B and Affi‐Gel 102 by the present method showed good agreement with that by the conventional method. In the case of Amino‐Cellulofine, ethanolamine had to be selected as the standard. The relative standard deviation for six successive determinations of AH‐Sepharose 4B was 3.2%; the polymerization of glutaraldehyde showed no effect on the result. The spectral property of AH‐Sepharose 4B treated with glutataraldehyde showed no effect on the result. The spectral property of AH‐Sepharose 4B treated with lutaraldehyde suggested that the reaction mechanism of the present method was based on the oxygen consumption during the formation of the pyridinium salt on the support. A comparative study of the present method with a colorimetric method with Traut's regent is also included.
Protein immobilization to polystyrene via long poly(ethylene lycol) chains
Authors: Bergström Karin , Holmberg Krister
Published: December 2004

Human albumin has been attached to 24‐hole polystyrene plates via branched poly(ethylene lycol) (PEG) spacer arms. A tetraepoxude of PEG of molecular weight (1.4–1.5) × 104 g/mol was reacted with the protein in solution allowing approximately one‐third of the oxirane rings to react. The protein conjugate was then coupled to the long, cationic polymer poly(ethylene imine) (PEI), and the protein‐PEG‐PEI adduct was subsequently adsorption to unmodified polystyrene. Since the protein is linked to the surface via long, hydrophilic and nonchargedchains, interactions between the biomolecule and the surface is minimized.
Application of lipase to concentrate the docosahexaenoic acid (DHA) fraction of fish oil
Authors: Yadwad V. B. , Ward O. P. , Noronha L. C.
Published: December 2004

A commercial lipase preparation from Rhizopus niveus was used to concentrate the omega‐3 fatty acid, docosahexaenoic acid (DHA) component in fish oil. The DHA content of cod‐liver oil was 9.64% (w/w) of total fatty acids. Enzymatic digestion conditions were established which produced a DHA content in the monoglycerides fraction of 29.17% (w/w) of total fatty acid, triglyceride, and diglyceride components were 5.72, 9.95, and 15316%, respectively.
Spectrophotometric method for high biomass concentration measurements
Authors: Thatipamala R. , Hill G. A.
Published: December 2004

Continuous monitoring of concentration is important in the optimal control of bioreactors. Spectrophotometry provides a simple, accurate, and rapid way for measuring cell concentration of unicellular microorganisms, based on either Beer's law or calibration curves prepared using standard solutions. However, this method is limited to a low range of microbial concentrations, because Beer's law deviates significantly at high concentrations. In the present investigation, based on experimental work, a new technique is posed to monitor the concentration of microbial cells as high as 100 g DW/L. this is achieved by using a mixture of known concentration as reference, rather than the “ideal blank” with zero concentration of analyte. As a result, a new equation is developed that, although applied here only to microbial concentration, in principle can be used for monitoring the concentration of any optically sensitive material.
Biotransformation of dopamine to norlaudanosoline by Aspergillus niger
Authors: Hoover Larry K. , Moo‐Young Murray , Legge Raymond L.
Published: December 2004

Norlaundanosoline is a key intermediate in the synthesis of the benzylisoquinoline alkaloids providing the upper isoquinoline portion of the morphinan skeleton. This study evaluates the feasibility of using Aspergillus niger as an in situ biotransformation system to produce norlaudanosoline from dopamine. A. niger was chosen because monoamine oxidase can be readily induced in this organism. Monoamine oxidase catalyzes the conversion of dopamine to 3,4‐dihydroxyphenylacetaldehyde. In the presence of dopamine, this aldehyde will then undergo a spontaneous Picket‐Spengler condensation to form norlaudanosoline. Fermentation condition to form norlaudanosoline. Fermentation conditions were optimized for the production monoamine oxidase by using a two‐stage process consisting of a growth stage and an induction stage. pH control was found to be important, and at pH 4.5 dopamine accumulation in the cells was high as was the level of monoamine oxidase. With pH control at 4.5, up to 21% of the cellular dopamine was converted to norlaudanosoline. It is proposed that with further protein engineering improvements, this system may prove suitable for the in situ bio‐transformation of dopamine to norlaudanosoline.
Mathematical model for apical growth, septation, and branching of mycelial microorganisms
Authors: Yang H. , King R. , Reichl U. , Gilles E. D.
Published: December 2004

A mathematical model for apical growth, septation, and branching of mycelial microorganisms is presented. The model consists of two parts: the determinstic part of the model is based on fundamental cellular and physical mechanisms; it represents the kinetics for growth of hyphal tips and septation of apical as well as intercalary compartments. In regard to random occurrences of hyphal growth and branching, the stochastic part deals with branching processes, tip growth directions, and outgrowth orientations of branches. The model can explain the morphological development of mycelia up to the formation of pellets. The results, as predicted by the model, correspond very closely to those observed in experiments. In addition, some unmeasured states can be ascertained, such as the distribution functions of hyphal length (biomass) and tips along pellet radii.
Correlating the effect of pretreatment on the enzymatic hydrolysis of straw
Authors: Koullas D. P. , Christakopoulos P. , Kekos D. , Macris B. J. , Koukios E. G.
Published: December 2004

Avicell, Alkali‐treated straw cellulose (ATSC), and wheat straw were ball‐milled to reduce crystallinity; wheat straw was delignified by hot (120°C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (Xmax), and the enzymic hydrolysis time corresponding to 50% of Xmax (t1/2). An empirical correlation between Xmax and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.
Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper
Authors: Park Jin Won , Takahata Yasuyuki , Kajiuchi Toshio , Akehata Takashi
Published: December 2004

Effects of five types of nonionic surfactant having a polyoxyethylene glycol (POG) group on enzymatic hydrolysis of used newspaper were studied. The surfactants examined in this work always enhanced the saccharification. The optimum surfactant concentration was 0.05% (wt/substrate wt) in the case of POG(21) sorbitane oleic ester. Among the surfactants, POG phenyl ether types showed the highest enhancement effect, for example, with two times higher conversion at 80 h than that without surfactant. Using POG nonylphenyl ether series, the effects of surfactant were considered from the point of the HLB (hydrophile–lypophile balance) value. The GFC (gel filtration chromatography) analysis of free enzyme quantity were also done to study the effect of surfactant on enzyme adsorption onto substrate. As the HLB value increased, the free enzyme quantity and the conversion both increased. It appears that surfactants help the enzyme to desorb from the binding site on the substrate surface after the completion of saccharification at that site.
Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis
Authors: Reichl U. , King R. , Gilles E. D.
Published: December 2004

Many filamentous bacteria and fungi tend to form pellets, or mixtures of dispersed mycelium and pellets in liquid fermentation broths. In some cases, a specific kind of morphology is required for optimum product yield. When quantitative analysis and characterization of the pellet morphology are needed, an image processing system can be used. It allows a fast and reproducible analysis of the frequency distribution of pellet size, mean pellet size, contents of pellets, or their shape. The use of such a system allows for an on‐line analysis. For a demonstration of the method, results of two fermentations of Streptomyces tendae are shown.
Alginate as immobilization material: III. Diffusional properties
Authors: Martinsen Anita , Storrø Ivar , Skjårk‐Bræk Gudmund
Published: December 2004

The rate of diffusion of serum albumin (MW 6.9 · 104 D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of ∼23 kJ · mol−1.
Evaluation of production of recombinant human interleukin‐2 in fluidized bed bioreactor
Authors: Kratje Ricardo B. , Wagner Roland
Published: December 2004

The production of recombinant human interleukin‐2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 × 108 mL−1 intrasphere volume were reached while the interleukin‐2 production rate was 0.75 mg L−1 d−1. The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble‐free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow‐fiber bioreactor with the same cell line.
Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme
Authors: Miyawaki Osato , Yano Toshimasa
Published: December 2004

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half‐life period of the reactor activity after one‐shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes–which is of more general use.
An extension of the pressure‐test technique for immobilized biocatalyst preparations
Authors: Vlaev S. D. , Valeva M.
Published: December 2004

Based on an analysis of variables in the Ergun equation for pressure drop in granular beds, the technique of pressuretesting of immobilized biocatalyst preparations is reexamined from the point of view of evaluation of biocatalyst mechanical properties in packed bed reactors. The variation of biocatalyst particle size in the course of bioreactor performance was found to be a convenient estimate of mechanical strength for the biocatalyst particles. Based on this finding, an extension of the pressure‐test technique with particle size analysis was proposed as an opportunity to carry out differential analysis of biocatalyst structure, regardless of time and position. An example of bed destruction is given to support the suggested characterization analysis.
Characterization of the mutant lytic state in lambda expression systems
Authors: Padukone N. , Peretti S. W. , Ollis D. F.
Published: December 2004

The two propagative phases of bacteriophage lambda, lysogeny and lysis, can be used in concert to enhance productivity of recombinant expression systems. Lambda vectors carrying mutations to prevent both cell lysis and λ DNA packaging in the lytic state have been shown to yield 100% stability of the product gene in lysogeny and to produce up to 15% of total cell protein as product β‐galactosidase in a mutant lytic state.14 Despite these mutations, partial lysis of the culture was observed following induction of the cells from a lysogenic phase into the lytic state. To understand better the phage‐host cell interactions and to investigate the possible cause(s) of lysis in these highly productive expression systems, we have made a detailed study of the suppressor‐free system JM105(NM1070). We have found high levels of product (15% of total cell protein as β‐glactosidase) to be due chiefly to a high‐copy number of λ DNA in the mutant lytic state. There is partial lysis of the culture even in this suppressor‐free system caused by a low‐level natural suppression of the amber mutation in gene S of NM1070, resulting in accumulation of λ endolysin. We have also monitored changes in cell growth and morphology upon induction of the lysogen. There is a slight increase in cell number that follows a linear relationship with time and a 25‐fold increase in cell volume during recombinat protein production in the mutant lytic state.
Noninvasive evaluation of somatic embryogenesis
Authors: Harrell R. C. , Bieniek M. , Cantliffe D. J.
Published: December 2004

Callus Suspension Cultures of Ipomea batates Poir. cv. White Star were grown in an airlift bioreactor. A machine vision system was used to monitor nondestructively callus growth during a 10 day culture period. Growth data obtained with this system included the overall reactor population and population estimates for the 200–1200‐μm fractions at 200‐μm intervals. A model of callus growth was developed to explain the mechanics of callus enlargement. The model was based on the assumptions that (1) the calli could not subdivide or shrink, (2) there was a fixed percentage of the initial population for each fraction that was nonviable, and (3) growth rates did not vary with time during the culture period. It was determined that the growth rates and nonviable ratios decreased as fraction size increased.
Reductive biotransformations of organic compounds by saccharomyces cerevisiae: Study of oxidoreductases involved using electrophoretic zymograms
Authors: Young C. S. , Ward O. P.
Published: December 2004

The role of oxidoreductases in reduction of carbonyl compounds was investigated by application of zymogram techniques. Eight bands were observed using ethanol with nicotinamide adenine dinucleotide (NAD) as coenzyme. Bands observed with lactic acid and (R)‐(‐)‐phenyl‐1,2‐ethanediol with nicotinamide adenine dinucleotide phosphate (NADP) had similar Rm values. 2‐Hydroxyvalerate and malate manifested bands having similar Rm values and were active with both NAD and NADP. Based on their structural similarity and identical Rm values, oxidation of 1,4‐cyclooctanediol (band #2) and cis‐1,5‐cyclooctanediol may be due to a common enzyme. The PAGE‐zymogram technique may be used on a preparative scale to facilitate purification and full characterization on the observed stained bands.
Continuous photoautotrophic cultures of the eukaryotic alga chlorella vulgaris can exhibit stable oscillatory dynamics
Authors: Javanmardian Minoo , Palsson Bernhard O.
Published: December 2004

Sustained oscillations in cell concentration, average per cell DNA content, and average cell size were found in continuous photoautotrophic cultures of Chlorella vulgaris at low dilution rates (0.1/day). The period of oscillation was approximately 10 days. DNA histograms determined by flow cytometry exhibited reproducible pattern through consecutive oscillations. At the maximum cell concentration during an oscillation, the DNA histograms showed that the majority of the cells were not replicating their chromosomes, and most of the culture was comprised of single cells in G0/G1 phase. The cells then initiated DNA replication; however, because of the long generation time, the cell concentration decreased to a minimum, and at the same time the average per cell DNA content reached its maximum value. At this point the cells began to divide, and the cell concentration increased until it reached its maximum value at the beginning of the next oscillation. Calculations based on the supplied nutrients and comparison to biomass generation showed that the oscillatory behavior in continuous photoautotrophic cultures of C. vulgaris was not due to nutrient limitation, but most likely was due to the secretion of compounds that alter cell cycle kinetics. The oscillatory behavior disappeared when the dilution rate was increased to 0.3/day and the culture reached a stable steady state.
Application of empirical design methodologies to the study of the influence of reaction conditions and N‐α‐protecting group structure on the enzymatic X‐Phe‐Leu‐NH2 dipeptide synthesis in buffer/dimethylformamide solvents systems
Authors: Calvet S. , Clapés P. , Vigo J. P. , Xaus N. , Jorba X. , Mas R. M. , Torres J. L. , Valencia G. , Serralheiro M. L. , Cabral J. M. S. , Empis J. M. A.
Published: December 2004

The influence of five different N‐terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the α‐chymotrypsin‐catalyzed synthesis of the dipeptide derivative X‐Phe‐Leu‐NH2 was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N‐α protection choice. Boc and Z were the most reactive N‐α groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.
Quantification of cell culture factors affecting recombinant protein yields in baculovirus‐infected insect cells
Authors: Lindsay David A. , Betenbaugh Michael J.
Published: December 2004

An experimental study was undertaken to quantify the effects of infection cell density, medium condition, and surface aeration on recombinant protein yields in insect cells. In the absence of surface aeration and fresh medium, insect cells generated higher product yields (on a per cell basis) when infected with recombinant baculovirus at low cell densities, LCD (3 × 105−4 × 105 cells/mL), than at high cell densities, HCD (>0.9 × 106 cells/mL), for two distinct baculovirus types. Surface aeration of a HCD culture infected in spent medium improved β‐glactosidase yields 5‐fold over the nonaerated case. Surface aeration and medium replenishment improved β‐galactosidase yields of a HCD culture by 20‐fold (compared to a 1.6‐fold improvement for a LCD culture), resulting in cultures with productivties that were independent of the cell density at infection.