bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous anaerobic treatment of autoxidized bark extracts in laboratory‐scale columns
Authors: Field J. , Leyendeckers M. J. H. , Sierra‐Alvarez R. , Lettinga G. , Habets L. H. A.
Published: December 2004

Debarking wastewaters of the forest industry contain high concentrations of tannins that are inhibitory to methane bacteria. The tannins can be polymerized to nontoxic colored compounds by the applications of an autoxidation pretreatment, enabling the anaerobic treatment of easily biodegradable components in the wastewater. The continuous anaerobic treatment of untreated and autoxidized pine bark extract was studied in laboratory‐scale columns packed with a granular sludge bed. The autoxidation doubled the conversion efficiency of bark extract COD to methane (from 19 to 40%). After 5 months of operation, anaerobic treatment of the autoxidized extracts was feasible at high influent concentrations (14 g COD/L) and loading rates (26 g biodegradable COD/L · d) with 98% elimination of the biodegradable fraction. The detoxification pretreatment polymerized the toxic tannins to poorly biodegradable high molecular weight tannins and humic compounds which were not eliminated during anaerobic treatment. Although the original tannins of the untreated extract were eliminated by 60%, they were not biodegraded to volatile fatty acids and methane but instead were transformed to phenolic degradation intermediates (phenol, p‐cresol, 3‐phenyl‐propionate, and carboxycyclohexane). Therefore, the autoxidation pretreatment did not decrease the content of readily biodegradable substrates which accounted for 53% of the extract COD. The recalcitrant COD expected in the effluents of reactors treating autoxidized debarking waste‐water can be effectively separated by calcium precipitation prior to anaerobic treatment.
The interaction of xylanases with commercial pulps
Authors: Senior D. J. , Mayers P. R. , Saddler J. N.
Published: December 2004

When purified xylanases from Trichoderma harzianum E58 or from a clone of Bacillus circulans were incubated with various low‐yield wood pulps, little of the original enzyme activity could be detected in the filtrate at the end of the reaction. Partial bleaching of the pulps prior to enzymatic treatment generally resulted in an increased recovery of the xylanase activity. It appears that both nonspecific adsorption and soluble inhibitors may be responsible for the loss of much of the xylanase activity. However, xylanases from Aureobasidium pullulans and Schizophyllum commune were not as inhibited by the pulps, and the activity of the latter enzyme actually increased after incubation with several high‐yield pulps. Although a lignin preparation from spent sulfite liquor at a concentration of 0.06 mg/mL could inhibit the xylanase activity of T. harzianum and B. circulans by 65% and 50%, respectively, xylanases from Thermoascus aurantiacus, S. commune, and A. pullulans were activated at similar lignin concentrations. At higher concentrations these latter xylanases were also inhibited. Water‐soluble lignins extracted from a variety of pulps and used at a lignin concentration of 2.5 μg/mL resulted in inhibition of more than 65% of the original activity of the xylanase from T. harzianum. Kinetic studies showed that lignin from spent sulfite liquor resulted in noncompetitive inhibition of this enzyme.
Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures
Authors: Facchini Peter J. , Dicosmo Frank
Published: December 2004

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.

Glass fiber mats were incorporated in to a 4.5‐L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (μ = 0.06 day−1) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor‐and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor‐and shake‐flask‐cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.
Morphology and clavulanic acid production of Streptomyces clavuligerus: Effect of stirrer speed in batch fermentations
Authors: Belmar‐Beiny M. T. , Thomas C. R.
Published: December 2004

Streptomyces clavuligerus ATCC 20764 was grown from spore‐inocuia on a glycerol, malt extract, bacteriological peptone medium in 5‐L batch fermentations at 490, 990, and 1300 rpm. Dry cell weights, clavulanic acid production, and the morphological parameters main hyphal length, total hyphal length, number of tips, and hyphal growth unit were measured. Growth and productivity were hardly dependent on stirrer speed. After early growth fragmentation of long, highly branched mycelia to shorter, less branched fragments occurred. This was followed by regrowth and, at 1300 rpm, a second fragmentation phase. The effect of increasing stirrer speed was to accelerate the initial fragmentation phase. It was clearly possible to obtain the same biomass concentration and clavulanic acid liter, with different morphologies depending on stirrer speed. This shows that for this fermentation at least there is no direct link between morphology and productivity and, hence, that it might be possible to manipulate them independently to improve fermentor performance.
Oxidation of hydrogen sulfide by flocculated Thiobaccillus denitrificans in a continuous culture
Authors: Ongcharit Chawan , Sublette Kerry L. , Shah Y. T.
Published: December 2004

In a continuous fermentation, significant advantages may be gained by immobilization of microbial cells. Immobilization allows cells to be retained in the fermenter or to be readily recovered and recycled. Therefore, the hydraulic retention time and the biomass retention time are decoupled. A novel cell immobilization has been developed for the immobilization of autotrophic bacteria by coculture with floc‐forming heterotrophic bacteria with growth of the latter limited by the availability of organic carbon. The result is an immobilization matrix which grows along with the immobilized autotroph. We have previously demonstrated the utility of this approach by immobilizing the chemoautotroph Thiobacillus denitrificans in macroscopic floc by coculture with floc‐forming heterotrophs from an activated sludge treatment facility. Floc with excellent settling characteristics were produced. These floc have now been used to remove H2S from a gas stream bubbled through continuous cultures. The stoichiometry and kinetics of H2S oxidation by immobilized T. denitrificans were comparable to that reported previously for free‐cell cultures. Oxygen uptake measurements indicated the growth of both T. denitrificans and the heterotrophs although the medium contained no added organic carbon. Continuous cultures with total biomass recycle were maintained for up to four months indicating the long‐term stability of the commensal relationship between the immobilized autotroph and the heterotrophs which composed the immobilization matrix. It was observed that at any given H2S loading the biomass concentration reached a maximum and leveled out. The ultimate biomass concentration was dependent upon the H2S feed rate.
Adsorption of γ‐globulin, a model protein for antibody, on colloidal particles
Authors: Kondo Akihiko , Oku Shinya , Higashitani Ko
Published: December 2004

The effect of surface properties on the adsorption of bovine γ‐globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2‐hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2‐hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of γ‐globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for γ‐globulin at alkaline pH, while PS showed high affinities for γ‐globulin over the whole range of pH and ionic strength. The γ‐globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of γ‐globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.
Effect of operating variables on biofilm formation and performance of an anaerobic fluidized‐bed bioreactor
Authors: Sreekrishnan T. R. , Ramachandran K. B. , Ghosh P.
Published: December 2004

The effect of various operating variables such as initial inoculum circulation, dilution rate, chemical oxygen demand (COD) loading rate, and quantity and quality of inoculum on the process of film formation on sand surface and reactor performance were studied using synthetic glucose based wastewater. It was found that the film formation process is favored by a high dilution rate, a large quantity of inoculum, and an inoculum having high methane producing capacity. Experimental observations indicate that the biofilm formation process is initiated by methanogenic bacteria.
The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae
Authors: Chen H. C. , Wilde F.
Published: December 2004

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic‐producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6–8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one‐third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).
Flotation of Streptomyces pilosus after lead accumulation
Authors: Sadowski Z. , Golab Z. , Smith R. W.
Published: December 2004

The flotation of Streptomyces pilosus was studied in the absence and presence of adsorbed Pb(II) ions on the cell walls and with and without using sodium dodecylsulfate (SDS) as collector. Adsorption of SDS onto the organism in the absence and presence of Pb(II) ions was also investigated. It was found that in the absence of adsorbed Pb(II), very little flotation took place with or without the presence of SDS although SDS adsorbed to some extent on the organism. The presence of Pb(II) on the bacterium's cell walls significantly increased flotation even in the absence of the collector. Addition of SDS, however, further increased both flotation rate and recovery.
A semimechanistic mathematical model for growth of Rhizopus oligosporus in a model solid‐state fermentation system
Authors: Mitchell David A. , Do Duong D. , Greenfield Paul F. , Doelle Horst W.
Published: December 2004

A Semimechanistic mathematical model is developed which describes the growth of Rhizopus oligosporus in a model solid‐state fermentation system. Equations are presented for the release of glucoamylase, the diffusion of glucoamylase, the hydrolysis of starch, the generation and diffusion of glucose, and the uptake of glucose and conversion into new biomass. Good agreement of the model with the experimental data was obtained only after the glucoamylase diffusivity and the maximum specific glucose uptake rate were altered from their originally determined values. The model recognizes the distributed nature of the solid‐state fermentation and therefore is able to predict the concentration profiles of the system components within the substrate. The model provides an insight into the possible rate‐limiting steps in solid‐state fermentation—the generation of glucose within the substrate and the resulting availability of glucose at the surface.
1‐Monoglyceride production from lipase‐catalyzed esterification of glycerol and fatty acid in reverse micelles
Authors: Hayes Douglas G. , Gulari Erdogan
Published: December 2004

Glycerol‐fatty acid esterification has been conducted with lipase from R. delemar in water/AOT/isooctane reverse micellar media, with the major product being 1‐monoglyceride, a useful food‐emulsifier. 1,3‐diglyceride was also synthesized, but to a much lesser extent. For a given set of initial conditions, the reaction productivity, measured in terms of the initial product formation rate, V0, and the final or equilibrium concentration of product, is optimal for a particular concentration of each surfactant, fatty acid, glycerol, and water. Many of these optimal values correlate well with a “critical” region on the phase diagram. Also, results indicate lipase‐catalyzed esterification stops due to the achievement of kinetic equilibrium expect for a few cases where enzyme deactivation is severe. Dynamic light scattering was employed to examine the influence of water, glycerol, and fatty acid on micellar and interfacial structure. Results from this technique indicate enzyme kinetic are linked to interfacial phenomena and the presence of substrates at the interfacial region.
Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism
Authors: Nollert M. U. , Diamond S. L. , McIntire L. V.
Published: December 2004

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel‐plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.
Properties of microfiltration membranes: Flux loss during constant pressure permeation of bovine serum albumin
Authors: Bowen W. Richard , Gan Quan
Published: December 2004

An experimental study of permeation of dilute BSA solutions (filtration) at microfiltration membranes has been carried out. Most measurements were made with capillary pore aluminum oxide membranes, with some comparative measurements with tortuous and capillary pore polymeric membranes. In all cases, a continuous and substantial decrease in the rate of permeation with time was observed. This decrease in permeation with time was observed. This decrease in permeation rate was due neither to concentration polarization nor to protein adsorption alone. However, it could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of protein on the walls of the pore. The maximum calculated thickness of the deposited layers was 55nm on the walls of 200‐nm diameter pores. This phenomenon is quite different to adsorption of protein at such surfaces, this latter giving only sub‐monolayer or monolayer protein coverage under the conditions studied.
Lipase kinetics: Hydrolysis of triacetin by lipase from Candida cylindracea in a hollow‐fiber membrane reactor
Authors: Guit R. P. M. , Kloosterman M. , Meindersma G. W. , Mayer M. , Meijer E. M.
Published: December 2004

The aptitude of a hollow‐fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis‐Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow‐fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.
Dynamics of induced CAT expression in E. coli
Authors: Bentley William E. , Davis Robert H. , Kompala Dhinakar S.
Published: December 2004

The dynamics of chemically induced chloramphenicolaceyl‐transferase (CAT) expression are determined in batch cultures of Escherichia coli DH5αF'IQ [pKK262‐1]. This article is directed towards understanding the coupling of induced cloned‐protein synthesis and reduced cell growth which are balanced in the optimal system. Experimental results indicate that the best inducer (IPTG) concentration is near 1.0 mM when added during midexponential growth. Lower concentrations cause only weak induction, whereas higher concentrations cause sufficiently strong induction that cell growth is suppressed. Induction at the onset of the stationary phase results in high expression but is accompanied by stimulated protease activity. Also, cell mass yield is adversely affected by enhanced protein synthesis. A structured metabolic model is shown to predict the responses of instantaneous growth rate and productivity which result from protein overexpression. This model can be employed to predict alternative reactor strategies and operating conditions necessary for the design of efficient bioprocess.
Long‐term perfusion culture of hybridoma: A “grow or die” cell cycle system
Authors: de la Broise Denis , Noiseux Manon , Lemieux Réal , Massie Bernard
Published: December 2004

In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1–2.2 days), antibody, productivity per cell produced (30–38 mg/109 cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar “grow or die” life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.
Differential product release (DPR) of proteins from yeast: A new technique for selective product recovery from microbial cells
Authors: Huang R.‐B. , Andrews B. A. , Asenjo J. A.
Published: December 2004

A novel method has been developed for the separation of bioproducts from yeast cells. The method uses a combination of physical, chemical, and biological agents such as lytic enzymes, osmotic supports, and spheroplast stabilizers. Using this technique, products (proteins and enzymes) can be released from specific cell locations at different process states; it has thus been celled differential product release (DPR). The wall‐associated proteins are released first and the lytic enzyme is removed together with the wall proteins at this stage. Secondly, the cytosol products are released by a mild procedure during which the organelles remained intact. Finally, the organelle proteins are solubilized. In each stage, specific proteins are released while others are kept inside the different cell compartments. This method can be used with relatively high yeast concentrations (up to 145 g dry wt/L) and gives higher product recoveries and much higher selectivity than mechanical disruption.
Protein partitioning at the isoelectric point: Influence of polymer molecular weight and concentration and protein size
Authors: Forciniti D. , Hall C. K. , Kula M. R.
Published: December 2004

We report the partition coefficient, Kp′ at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which Kp is less than 1, but increases for chymotrysinogen for which Kp is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 104 to Dx 1.1 × 105 and then slightly decrease from Dx 1.1 × 105 to Dx 5 × 105. The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two‐phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.
Inactivation and stabilization of stabilisins in neat organic solvents
Authors: Schulze Birgit , Klibanov Alexander M.
Published: December 2004

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45°C. The half‐life of subtilisin BPN' in dimethyl‐formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert‐amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time‐dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert‐amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl‐enzymes.
On the enthalpy of formation of Escherichia coli K‐12 cells
Authors: Battley Edwin H.
Published: December 2004

An examination is made of five methods for obtaining values of the enthalpy of formation of a unit mass of living Escherichia coli K‐12 cells. The values obtained by these methods ranged from −88.95 kJ to −99.55 kJ, the gross average being 96.01 kJ, per unit carbon formula weight equivalent of living, hydrated cells. Although theoretically the growth of this organism in a microcalorimeter should provide the best value, the value obtained by this method (−88.95 kJ per UCFW equivalent) is not in close agreement with those of the other four methods, the values from which form a cluster averaging −97.8 ± 1.0 kJ (−23.4 ± 0.2 kcal)/UCFW equivalent. Calculations using this value indicate that the enthalpy change accompanying anabolism (as this is represented) is zero, or very nearly so, and that the heat of growth is that from catabolism alone.
Solubilizing water involved in protein extraction using reversed micelles
Authors: Ichikawa Sosaku , Imai Masanao , Shimizu Masaru
Published: December 2004

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di‐2‐ethylhexyl sodium sulfosuccinate (C20H37O7Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under CKCI = 1.0 × 102 mol · m−3 in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein‐containing system was larger than in the protein‐free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under CKCI = 1.0 × 102 mol · m−3, i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.
In vitro culture of hybridoma cells in agarose beads producing antibody secretion for two weeks
Authors: Cadic Ch. , Dupuy B. , Pianet I. , Merle M. , Margerin Ch. , Bezian J. H.
Published: December 2004

A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose‐embedded C6 glioma cells with 31P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m2/ mL of gel). It was possible to seed 20‐fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.
Continuous butanol/isopropanol fermentation in down‐flow column reactor coupled with pervaporation using supported liquid membrane
Authors: Matsumura Masatoshi , Takehara Shigemasa , Kataoka Hiroshi
Published: December 2004

Continuous butanol/isopropanol fermentation with immobilized Clostridium isopropylicum was performed in a downflow column reactor using molasses as the substrate. In order to prevent product inhibition and at the same time obtain high concentration of the products, the column reactor was coupled with a pervaporation module using a supported liquid membrane. The liquid membrane was prepared with oleyl alcohol nontoxic to the microorganism. In comparison with the continuous fermentation without product removal, the specific butanol production rate was 2 times higher. The butanol concentration in the permeate was 230 kg/m3, which was about 50 times higher than that in the culture broth. A numerical investigation suggested a further increase in the productivity by improving the module construction.
Biocatalytic synthesis of disaccharide high‐intensity sweeterner sucralose via a tetrachlororaffinose intermediate
Authors: Bennett Christopher , Dordick Jonathan S. , Hacking Andrew J. , Cheetham Peter S. J.
Published: December 2004

A second high‐yielding bioorganic synthesis of the highintensity sweetener sucralose (4,1′,6′‐trichloro‐4,1′,6′‐trideoxygalactosucrose) is described. This procedure involves the chemical chlorination of raffinose to form a novel tetrachloroaffinose intermediate (6,4′,1″,6″‐tetrachloro‐6,4′,1″,6″‐tetradeoxygalactoraffinose; TCR) followed by the enzymic hydrolysis of the α‐1‐6 glycosidic bond of TCR to give sucralose and 6‐chlorogalactose. Commercial enzyme preparations and microorganisms were screened to select α‐galactosidases which have high catalytic activity on this compound. The most active enzyme was produced by a strain of Mortierella vinacea and had a maximum rate of 118 μmol sucralose/g dry weight cells/h, which was approximately 5% of the activity toward raffinose, and a Km of 5.8 mM toward TCR. The enzyme could be used in the form of mycelial pellets in a continuous packed bed column reactor. The reaction was also studied in a water‐immiscible hydrophilic organic solvent, such as methyl isobutyl ketone, to overcome the poor aqueous solubility of TCR and to increase volumetric productivity. Synthesis of raffinose was achieved from saturated aqueous solutions of galactose and sucrose using a selected α‐galactosidase from Aspergillus niger. When raffinose is used as a starting material for sucralose synthesis, this route has fewer steps than either the preceeding method using glucose‐6‐acetate as an intermediate or the complete chemical synthesis from sucrose. The relative merits of the two bioorganic routes and the utility of such methods to synthesize new sugars are discussed.
Simulation of L/A control in fed‐batch culture of baker's yeast
Authors: Dantigny Philippe , Lakrori Maqo
Published: December 2004

L/A controllers have extended their use from continuous to fed‐batch fermentation where the control is applied from the start of an initial batch phase. As opposed to proportional integral derivative (PID) controllers where even a startup procedure is recommended prior to fed‐batch, the L/A controller is not upset by an early connection. It is easily retuned continuously by means of ethanol measurements and can cope with a large range of output conditions. The performance of an L/A algorithm, which uses biomass concentration as the controlled variable, is assessed through simulation. The self‐contained algorithm is relatively simple with no greater intrinsic complexity than modern PID stand alone controllers.
Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: I. Steady‐state analysis
Authors: Bibila Theodora A. , Flickinger Michael C.
Published: December 2004

Steady‐state simulations using our previously developed structured kinetic model of antibody synthesis and secretion by hybridoma cells are used here in conjunction with factorial design analysis to identify intracellular parameters important in determining the specific antibody secretion rate and predict the dependence of this rate on cell specific growth rate. Simulation results suggest that the specific growth rate, the assembly rate of the heavy and light chains and the heavy‐ and ‐chain gene dosage can significantly affect the rate of antibody secretion. Based on these results, environmental and/or genetic manipulation approaches are proposed for maximizing the specific antibody secretion rate and the antibody volumetric productivity in large‐scale antibody production systems.
Modeling high‐biomass‐density cell recycle fermentors
Authors: Monbouquette Harold G.
Published: December 2004

Since intrinsic models, which take into account cell volume fraction, follow from proper application of the law of conservation of mass to a multiphase system, the intrinsic modeling approach should be used whenever biomass occupies a significant volume fraction of the culture. A recent report11 offers the first comparison of intrinsic and nonintrinsic model predictions to actual experimental data gathered from a high‐density yeast recycle fermentor. Here, the analysis of Jarzebski et al.11 has been carried further to show that the improper nonintrinsic model predicts a steady‐state culture glucose concentration that differs from that given by the fundamentally correct intrinsic model by over 60% at the optimal, bleed stream flow rate. In addition, a revised formulation for an intrinsic ethanol mass balance is presented.
Effect of adsorbents on degradation of toxic organic compounds by coimmobilized systems
Authors: Siahpush Ali R. , Lin Jian‐Er , Wang Henry Y.
Published: December 2004

The use of coimmobilized systems for treatment of toxic organic compounds has been proposed. The proposed approach combines the use of adsorbents and laboratory identified microorganisms immobilized in a protective permeable barrier to achieve a greater degree of control over the remediation process. This study was launched to understand the effect of adsorbents and changes in adsorption on the degradation of toxic compounds by coimmobilized systems. The specific case studied involved the degradation of pentachlorophenol (PCP) by Arthrobacter (ATCC 33790) coimmobilized with powdered activated carbon within calcium alginate capsules.

The design parameters studied included adsorbent content and type as well as the effect of solution pH and surfactant concentration on adsorption and biodegradation. It was found that the equilibrium adsorption behavior of PCP was strongly influenced by solution pH and surfactant concentration. A mathematical model was developed that combined the physical processes of mass transfer and adsorption with biological degradation of PCP. The model was used to predict the effect of various parameters on the degradation of PCP. Based on model predictions, the degradation of PCP. Based on model predictions, the degradation of PCP was strongly dependent on variations in adsorbent capacity and affinity for this contaminant.
Accurate pseudoanalytical solution for steady‐state biofilms
Authors: Sáez Pablo B. , Rittmann Bruce E.
Published: December 2004

An extremely accurate pseudoanalytical solution for the flux of substrate into a steady‐state biofilm is developed. The standard deviations between the substrate fluxes computed from the pseudoanalytical solution and the numerical solution were less than 2.6%. Additional advantages of the pseudoanalytical solution are that it has no inaccuracies around S = 1 and it is composed of single continuous functions applicable to the whole S region.
Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells
Authors: Porro Danilo , Martegani Enzo , Ranzi Bianca Maria , Alberghina Lilia
Published: December 2004

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli β‐galactosidase gene under the control of the upstream activating sequence of the GAL1–10 genes and the GAL4 activator gene release part of β‐galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of β‐galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey‐based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.
Hydrolysis of liquefied corn starch in a membrane reactor
Authors: Sims Kevin A. , Cheryan Munir
Published: December 2004

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first‐order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (Vm) was 3.86 g glucose/L min and the apparent Michaelis constant (K) was 562 g/L. The K value for the continuous reactor was 2–7 times greater than that obtained in a batch reactor.

Kinetic data were fit to a model based on the Michaelis‐Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.
Removal of dissolved metals by plant tissue
Authors: Scott Charles D.
Published: December 2004

Various types of microbial biomass have been shown to adsorb metals dissolved in aqueous media. It has now been demonstrated that certain plant tissues are also effective for this type of adsorption process. In particular, tomato and tobacco roots harvested from field‐grown plants were shown to adsorb Sr from an aqueous solution of SrCl2. Distribution coefficients in excess of 550 were measured and the adsorption isotherms at 25°C could be fitted to Langmuir‐type expressions. The bioadsorbent could be regenerated and metals recovered by either a reduction in the pH to less than 2.0 or by use of a concentrated chloride salt solution.
Effect of matrices on affinity purification of protein C
Authors: Kang Kyung , Ryu Dewey , Drohan William N. , Orthner Carolyn L.
Published: December 2004

One of the critical problems in scale‐up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.

We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross‐linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)‐Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.

A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm × 10 cm column.
Culture instability of auxotrophic amino acid producers
Authors: Kiss Robert D. , Stephanopoulos Gregory
Published: December 2004

The long‐term dynamic characteristics of the L‐lysine producer Corynebcterium glutamicum in continuous culture were studied over a range of specific growth rates. The double‐auxotroph parent strain was found to be susceptible to a back mutation, or reversion, which negated the regulatory bypass that allows this strain to accumulate L‐lysine in culture but also gives rise to a L‐threonine auxotrophic requirement. Consequently, the revertant cells no longer over‐produced L‐lysine, nor were they limited in their growth by the low levels of L‐threonine in the medium. All continuous culture experiments were enventually taken over by these revertants. The instability of the culture was found to be primarily due to the growth rate differential between the two competing populations, the (productive) parent auxotrophs and the (nonproductive) revertants. A deterministic mathematical model of the culture dynamics, incoroporating two limiting‐substrate balances, satisfactorily described the takeover profiles. A linear stability analysis of the model equations identified that although long‐term culture demise is inevitable, the dimensionless ratio of the two limiting substrates controls the rate of takeover by nonproductive cells. The anslysis further demonstrated the importance of proper medium design in delaying the onset of takeover in cultures of this double‐auxotroph strain. The theoretical medium design criterion was then confirmed experimentally by the stabilization of a fed‐batch culture against revertant takeover for an extended fermentation time.
Fermentation kinetics of recombinant yeast in batch and fed‐batch cultures
Authors: Patkar Anant , Seo Jin‐Ho
Published: December 2004

Fed‐batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned‐gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed‐batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy‐efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.
Solubilization and growth of Candida pseudotropicalis in water‐in‐oil microemulsions
Authors: Pfammatter N. , Hochköppler A. , Luisi P. L.
Published: December 2004

Some new aspects of microbiology in water‐in‐oil microemulsions are investigated using Candida pseudotropicalis in a hexadecane solution containing Tween85/Span80 (each 5% wt:wt) as surfactant, and limited amount of water (up to 3%, vol:vol), Microemulsion solutions containing cells up to 10 mg fresh weight per milliliter can be prepared, which display a greater time stability and a much smaller light scattering than aqueous suspensions having the same cell concentration. This is ascribed to a lower aggregation tendency of the cells in the microemulsion environment. It is also shown that C. pseudotropicalis cells are able to grow (up to a factor of approximately 6–7 within a few days) in the microemulsion system containing nutrient medium in the aqueous microphase; but they are also able to grow at the expense of the hexadecane. This is proved with radioactive‐labeled hexadecane by measuring the increase of radioactivity in the cells as well as the emission of 14CO2. The growth rate of the cells is then compared with the growth rate of cellular proteins during cell reproduction in the microemulsion system. Two regimes are observed: a first one, in which cells growth rate and protein growth rate proceed parallel to each other; and a second one (established after 0.5–1 day) characterized by depletion of proteins in the microemulsion system. The implications of these findings for cell metabolism in microemulsion and for possible biotechnological applications are discussed.
Effect of low serum concentrations (0%–2.5%) on growth, production, and shear sensitivity of hybridoma cells
Authors: van der Pol L. , Bakker W. A. M. , Tramper J.
Published: December 2004

In a stirred culture of hybridoma cells, the effects of serum reduction from 2.5% to 0% on growth and monoclonal antibody porduction have been investigated. The shear sensitivity of cells from the same culture has been tested in a bubble column. Serum reduction does not greatly affect viable‐cell concentrations, but cell specific monoclonal‐antibody production rate shows a decreasing trend. A gradual increase in sensitivity for sparging, which is nor the result of a long‐term biological effect, has beeen measured in a bubble column at decreasing fetal calf serum concentrations. Finally, the hypothetical killing‐volume model describing the death rate of insect cells in bubble columns has now been completely validated for the pertinent hybridoma‐cell line.