bluuebunny/crossref_metadata_2025 huggingface dataset:
A single‐cell assay of β‐galactosidase in recombinant Escherichia coli using flow cytometry
Authors: Miao Fudu , Todd Paul , Kompala Dhinakar S.
Published: December 2004

A flow cytometric method was developed for the assay of β‐galactosidase in single Escherichia coli cells. A new fluorogenic substrate for β‐galactosidase, C12FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular β‐galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained β‐galactosidase–positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid‐bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular β‐galactosidase along with a rapid increase in the fraction of plasmid‐free cells. Once the cells lose the plasmid, they no longer produce β‐galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid‐bearing cells can be distinguished from the nonfluorescent, plasmid‐free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between β‐galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction. © 1993 John Wiley & Sons, Inc.
Pressure affects enzyme function in organic media
Authors: Kim Jungbae , Dordick Jonathan S.
Published: December 2004

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme‐bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. © 1993 John Wiley & Sons, Inc.
Lipase‐Catalyzed acyiation of sugars solubilized in hydrophobic solvents by complexation
Authors: Ikeda Isao , Klibanov Alexander M.
Published: December 2004

By complexing glucose and other mono‐ and disaccharides with phenylboronic acid, they were solubilized in many organic solvents. Pseudomonas sp. lipoprotein lipase readily acylated such solubilized sugars in these solvents (no reaction was observed without phenylboronic acid, i.e., when sugars are insoluble in the reaction medium). Solubilized D‐glucose was enzymatically acylated with vinyl acrylate on a preparative scale, and the resultant 6‐O‐acryloylglucose was chemically polymerized to yield a sugar‐based polyacrylate with a molecular weight of 14,000 Da. © 1993 John Wiley & Sons, Inc.
Factors affecting bio‐oxidation of sulfide minerals at high concentrations of solids: A review
Authors: Bailey A. D. , Hansford G. S.
Published: December 2004

Bio‐oxidation has proved to be a viable process for the oxidative pretreatment of refractory gold‐bearing sulfides. Generally, the oxidation rate is maximal at 20% solids for high sulfide content materials [ca. 30% sulfur]. Low grade ores [1% sulfur] have been successfully oxidized at 55% solids, indicating a link between the sulfide grade of the material and the optimal solids concentration for operation. Concentrations of high solids have been reported to lower oxidation rates, increase lag times, and decrease the ultimate extent of oxidation. This review discusses the various factors that have been proposed as causes of these phenomena. The factors include oxygen and carbon dioxide availability, low bacteria–solids ratio; mechanical damage or inhibition of the bacteria, inhibition of bacterial attachment, and the buildup of toxic leach products or other detrimental substances such as some flotation reagents. © 1993 John Wiley & Sons, Inc.
Step‐fortifications of nutrients in mammalian cell culture
Authors: Jo Eui‐Cheol , Kim Dong‐II , Moon Hong Mo
Published: December 2004

A series of high‐density media for mammalian cell culture were developed by step‐fortifications of most nutrient components in RPMI‐1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 × 107 cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step‐fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step‐fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell‐line–specific responses to the step‐fortifications. The significant influence of nutritional fortifications on high‐density culture of mammalian cells was evaluated. © 1993 John Wiley & Sons, Inc.
Design of a biomedical reactor for plasma low‐density lipoprotein removal
Authors: Shefer Samuel D. , Payne Richard G. , Langer Robert
Published: December 2004

The purpose of this study was to design a biomedical reactor that reduces plasma cholesterol when incorporated in an in vivo extracorporeal system. Phospholipase A2, immobilized onto Agarose beads and housed inside the bioreactor, modifies plasma low density lipoprotein (LDL) into a form that is rapidly removed from circulation. In a packed bed reactor, the enzymatic conversion of LDL to the modified form (with plasma taken from hypercholesterolemic New Zealand white rabbits) was relatively low, 25% ± 6 for a single pass of plasma through the reactor. An extended bed reactor, a hybrid of fluidized and packed bed reactors, was then developed to increase the conversion. This reactor displays a single pass conversion of 60% ± 5 under optimal flow conditions. An evaluation of the flow rate through the reactor indicates that the system is limited by external mass transfer when employed under in vivo conditions. In addition, this system requires blood separation before the enzyme modification, which complicates the circuit control. Therefore, a new system was designed for in vivo use with rabbits. The resulting design, called the plasma separator reactor (PSR), combines plasma separation and enzymatic conversion in a single chamber. The PSR has three advantages over other studied systems: improved external mass transfer conditions, easy controlability, and simple set‐up procedures. Single pass conversion reached 52% ± 12 in suboptimal flow under simulated in vivo conditions. This reactor was also tested in vivo with hypercholesterolemic New Zealand white rabbits. A continuous conversion of up to 80% ± 6 of rabbit plasma phospholipids was observed during 90 min of blood circulation (5 mL/min). The decrease in total plasma cholesterol reached a level of 60% of the initial value and was observed to be a function of the bioreactor enzyme activity. © 1993 John Wiley & Sons, Inc.
Optimization of 2,3‐butanediol production by Klebsiella oxytoca through oxygen transfer rate control
Authors: Beronio Peter B. , Tsao George T.
Published: December 2004

Production of 2,3‐butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy‐coupled growth, during which cell growth and oxygen supply are coupled; and, energy‐uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3‐butanediol productivity occurs during the energy‐coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy‐coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasing the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3‐butanediol productivity of 18%. © 1993 John Wiley & Sons, Inc.
An energetic model for oxygen‐limited metabolism
Authors: Beronio Peter B. , Tsao George T.
Published: December 2004

Microbial production of 2,3‐butanediol by Klebsiella oxytoca occurs under conditions of an oxygen limitation. The extent to which substrate is oxidized to 2,3‐butanediol and its coproducts, (acetic acid, acetoin, and ethanol) and the relative flow rates of substrate to energetic and biosynthetic pathways are controlled by the degree of oxygen limitation. Two energetic relationships which describe the response to an oxygen limitation have been derived. The first relationship describes the coupling between growth and energy production observed under oxygen‐limited conditions. This allows calculation of energetic parameters and modeling of the cell mass and substrate profiles in terms of the degree of oxygen limitation only. The second relationship describes the average degree of oxidation and the rate of the end‐product flow. The model has been tested with both batch and continuous culture. During these kinetic studies, two phases of growth have been observed: energy‐coupled growth, which was described above; and, energy‐uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal culture performance with respect to 2,3‐butanediol productivity occurs during energy‐coupled growth. © 1993 John Wiley & Sons, Inc.
Diffusivity of Cu2+ in calcium alginate gel beads: Recalculation
Authors: Lewandowski Z. , Roe F.
Published: December 2004

Calculations of the diffusivity of Cu2+ in calcium alginate gel beads using the shrinking core model were checked by us. Corrected results are reported here. Diffusivity was still found to increase with increasing alginate concentration, but at a lower rate than reported in the cited paper. The diffusivity increased by a factor of 2 over the range of alginate concentrations studied rather than 10. The original data is included with sample calculations. © 1994 John Wiley & Sons, Inc.
Ammonia inhibition of hybridomas propagated in batch, fed‐batch, and continuous culture
Authors: Newland Mark , Kamal Mohammed Nazlee , Greenfield Paul F. , Nielsen Lars K.
Published: December 2004

The nature and temporal development of ammonia inhbition were investigated in batch, fed‐batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum‐containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 → 3.7 → 4.0 → 4.9 → 7.7 → 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady‐state specific growth kinetics or specific antibody productivity. © 1994 John Wiley & Sons, Inc.
Measurement of kLa by dynamic pressure method in pilot‐plant fermentor
Authors: Linek V. , Moucha T. , Doušová M. , Sinkule J.
Published: December 2004

The dynamic pressure method (DPM) is used for measurement of kLa in a 1‐m3 pilot scale fermentor in coalescing (distilled water) and noncoalescing (0.3 M Na2SO4 aqueous solution) batches. The method consists in recording oxygen concentration in a batch after a small pressure change (20 kPa) in the fermentor. The upward pressure change is brought about by temporary closing and subsequent throttling of outlet gas stream and the downward change by full reopening of the gas outlet. Absorption of pure oxygen yields the same kLa values as absorption of air. In noncoalescing batch, the downward kLa values are always higher than the upward values owing to spontaneous nucleation of bubbles. The experiments performed in a stirred cell confirm this behavior. Thus, only upward pressure change should be used for measurement. The correlation of kLa data measured in small (18‐L) and large (1000‐L) vessels based on power dissipated and superficial gas velocity are in a good agreement. Unlike the DPM, the classical dynamic methods yield, under the same conditions, excessively low values of kLa (the dynamic startup method) or fail to produce data at all (the dynamic method with interchange of air for N2). © 1994 John Wiley & Sons, Inc.
Transition state stabilization of subtilisins in organic media
Authors: Xu Zu‐Feng , Affleck Rhett , Wangikar Pramod , Suzawa Valerie , Dordick Jonathan S. , Clark Douglas S.
Published: December 2004

Electrostatic forces are among the stabilizing interactions that contribute to the high degree of enzyme‐transition state complementarity. The active‐site polarity, which can differ substaintially from that of water, is thus an important determinant of transition state stabilization. Here we pose the question of whether the rate of an enzymatic reaction proceeding through a charged transition state can be increased by increasing the active‐site polarity in an organic solvent. The active‐site polarity of subtilisin has been reduced by dehydration and suspension in a nonpolar solvent (tetrahydrofuran), and then increased by adding water to the solvent. Enhancing the local polarity substantially increasing the rate of catalysis, implicating polarity as an important factor in stabilizing the charged tetrahedral transition state. Studies with subtilisins whose active sites have been modified by site‐directed mutagenesis support the role of polarity in transition state stabilization. © 1994 John Wiley & Sons, Inc.
Population dynamics of human activated natural killer cells in culture
Authors: Pierson Bryce A. , Miller Jeffrey S. , Verfaillie Catherine , McGlave Philip B. , Hu Wei‐Shou
Published: December 2004

The growth kinetics and population dynamics of recombinent interleukin‐2 (rlL‐2) stimulated human natural killer (NK) cell–enriched populations were studied in vitro. The NK‐enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3+ and CD5+ T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK‐enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK‐enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK‐enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte‐enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK‐enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56+/CD3− NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte‐enriched population were predominantly CD56+/CD2−. Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.
The in vitro growth of a three‐dimensional human dermal replacement using a single‐pass perfusion system
Authors: Halberstadt Craig R. , Hardin Robert , Bezverkov Kelly , Snyder Dale , Allen Lynn , Landeen Lee
Published: December 2004

A human dermal replacement has been developed by seeding human neonatal dermal fibroblasts onto a biosorbable polyglactin (polyglycolide/polylactide) mesh and culturing in a bioreactor. The mesh provides the proper environment for the cells to attach, grow in a three‐dimensional array, and establish a tissue matrix over a 2‐ to 3‐week culture period. The dermal replacement has been characterized and found to contain a variety of naturally occurring dermal matrix proteins, including fibronectin, glycosaminoglycans, and collagen types I and III. To efficiently and reproducibly produce this dermal tissue equivalent, a closed, single‐pass perfusion system was developed and compared with a static process. In the single‐pas perfusion system, growth medium (containing ascorbic acid) was perfused around the 4 × 6 in. pieces of mesh at specific flow rates determined by nutrient consumption and waste production rates. The flow rates used for this system indicate that a diffusion‐limited regime exists with a mean residence time greater than 1 h for essential nutrients and factors. By controlling glucose concentrations in the system to a delta of 0.70 g/L from the inlet to the outlet of the bioreactor, it took 6 fewer days to grow a tissue similar to that produced by the static system.
Stereotypic culture systems for liver and bone marrow: Evidence for the development of functional tissue in vitro and following implantation in vivo
Authors: Naughton Brian A. , Román Julia San , Sibanda Benson , Weintraub Jory P. , Kamali Vafa
Published: December 2004

Stromal cell‐associated liver cell and bone marrow (BM) culture on three‐dimensiional nylon screen or polyglycolic acid (PGA) felt templates conveys certain functional advantages to the parenchyma of these tissues. Hepatic parenchymal cells (PC) manifest long‐term (∼2 month) expression of liver‐specific activities including cytochrome P450 enzyme activity and the synthesis of albumin, fibrinogen, transferrin, and other proteins. PC also undergo proliferation in association with stromal cells that were pre‐established on these templates. PC mitoses are directly proportional to available space within the template for their expansion indication that geometric or sterotypic parameters influence the growth of these cells in vitro. BM cultured on a similar template exhibits long‐term multilineage hematopoietic expression and limited expansion of progenitor cell numbers. Progenitor cell concentration within the cultures can be substantially enhanced if these cells are liberated from co‐culture and reseeded onto a template containing fresh stromal cells. BM and liver cel cultures established on felt composed of bioresorbable PGA filaments was grafted into various sites in rats. Liver co‐cultures generated sinusoids and other liver‐like structures in situ; active hematopoietic blasts were observed at sites of BM co‐culture grafts. Biodegradable polymer constructs may prove useful for certain clinical applications as vehicles for the delivery of tissues that were engineered in culture.
Effects of biofilm structures on oxygen distribution and mass transport
Authors: de Beer Dirk , Stoodley Paul , Roe Frank , Lewandowski Zbigniew
Published: December 2004

Aerobic biofilms were found to have a complex structure consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. The oxygen distribution was strongly correlated with these strutures. The voids facilitated oxygen transport from the bulk liquid through the biofilm, supplying approximately 50% of the total oxygen consumed by the cells. The mass transport rate from the bulk liquid is influenced by the biofilm structure; the observed exchange surface of the biofilm is twice that calculated for a simple planar geometry. The oxygen diffusion occurred in the direction normal to the cluster surfaces, the horizontal and vertical components of the oxygen gradients were of equal importance. Consequently, for calculations of mass transfer rates a three‐dimensional model is necessary. These findings imply that to accurately describe biofilm activity, the relation between the arrangement of structural components and mass transfer must be undrstood. © 1994 John Wiley & Sons, Inc.
Phase fluorometric sterilizable optical oxygen sensor
Authors: Bambot Shabbir B. , Holavanahali Raja , Lakowicz Joseph R. , Carter Gary M. , Rao Govind
Published: December 2004

We report here on a low‐cost, optical oxygen sensor as an attractive alternative to the widely used amperometric Clark‐type oxygen electrode for measuring dissolved oxygen tensions in cell cultures and bioreactor. Our sensor is based on the defferential quenching of the fluorescence lifetime of chromophore in response to the partial pressure of oxygen. This is measured as a phase shift in fluorescence emission from the chromophore due to oxygen quenching when excited by an intensity modulated beam of light. In this article we demonstrate the advantages of lifetime‐based optical methods over both intensity based optical methods and amperometric electrodes. Our sensor is particularly suitable for measuring dissolved oxygen in bioreactors. It is autoclavable, is free of maintenance requirements, and solvents the problems of long‐term stability, calibration drifts, and reliable measurement of low oxygen tensions in dense microbial cultures that limit the utility of Clark‐type elcectordes. © 1994 John Wiley & Sons, Inc.
Co‐immobilized Nitrosomonas europaea and Nitrobacter agilis cells: validation of a dynamic model for simultaneous substrate conversion and growth in κ‐carrageenan gel beads
Authors: Hunik Jan H. , Bos Cees G. , van den Hoogen Marijke P. , De Gooijer Cornelius D. , Tramper Johannes
Published: December 2004

A dynamic model for two microbial species immobilized in a gel matrix is presented and validated with experiments. The model characterizes the nitrification of ammonia with Nitrosomonas europaea and Nitrobacter agilis co‐immobilized in K‐carrageenan gel beads. The model consists of kinetic equations for the microorganisms and mass transfer equations for the substrates and products inside and outside the gel beads. The model predicts reactor bulk concentrations together with the substrate consumption rate, product formation, and biomass growth inside the gel beads as a function of time. A 50–day experiment with immobilized cells in a 3.3‐dm3 air‐lift loop reactor was carried out to validate the model. The parameter values for the model were obtained from literature and separate experiments. The experimentally determined reactor bulk concentrations and the biomass distribution of the two microorganisms in the gel beads were well predicted by the model. A sensitivity analysis of the model for the given initial values indicated the most relevant parameters to be the maximum specific growth rate of the microorganisms, the diffusion coefficient of oxygen, and the radius of the beads. The dynamic model provides a useful tool for further study and possible control of the nitrification process. © 1994 John Wiley & Sons, Inc.
Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene‐divinylbenzene) carrier and its application to the degrading of 2‐chlorophenol
Authors: Ruckenstein Eli , Wang Xiao‐Bai
Published: December 2004

Porous poly(styrene‐divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene–divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon‐limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1‐day‐old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene–divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2‐chilorophenol in a 2‐L bioreactor. The porous poly(styrene‐divinylbenzene) particle (diameter ≅ 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2‐chlorophenol than the freely suspended mycelial pellets. © 1994 John Wiley & Sons, Inc.
Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: II. Sensitivity analysis
Authors: Torres Néstor V.
Published: December 2004

Steady state sensitivity analysis of a model of carbohydrate metabolism and anaplerotic synthesis of oxalacetate were, in Aspergillus niger under conditions of citric acid accumulation, carried out. The flux and metabolite concentration control structure of the system obtained shows that the hexokinase/substrate transport step is the main controlling step of the pathway. The quantitative contribution of the other enzyme catalyzed or transport steps are also discussed. These results allow the design of a proper strategy of biotechnological manipulation aimed at improvement of the process. © 1994 John Wiley & Sons, Inc.
Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth
Authors: Dedhia Neiley N. , Hottiger Thomas , Bailey James E.
Published: December 2004

Excessive production of acetate is a problem frequently encountered in aerobic high‐cell‐density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five‐fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl‐‐‐D‐thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high‐cell‐density fermentations. © 1994 John Wiley & Sons, Inc.
Interactions of microbial biofilms with toxic trace metals: 1. Observation and modeling of cell growth, attachment, and production of extracellular polymer
Authors: Hsieh Ke Ming , Murgel George A. , Lion Leonard W. , Shuler Michael L.
Published: December 2004

Adsorbent surfaces in natural and engineered systems are frequently modifies by bacterial attachment, growth of a biofilm, and bacterial production of extracellular polymer. Attached cells or sorbed polymers may alter the metal‐binding characteristics of the supporting substratum and influence metal partitioning. The interdependent behavior of toxic trace metal partitioning and biofilm development requires description of the interaction between cell growth with its accompanying polymer production and metal speciation. In this article, the first of a two part series, a mechanistic model is developed to describe the growth of a film‐forming bacterium which adheres to a substratum through the production of extracellular biopolymers. Each bacterial cell was modeled as a two‐component structure consisting of active cell mass and biopolymer. The biopolymer component was further divided into cell‐associated and dissolved categories to distinguish which remained naturally bound to cell surfaces from that which did not. Use of this structured model permitted independent description of the dynamics of cell growth, and polymer production, both of which may influence trace metal behavior. Employing parameters obtained from independent experiments as well as published values, the model satisfactorily predicts experimental observations of bacterial growth, attachment and detachment, biopolymer production, and adsorption of polymer onto solid (glass) surfaces. The model stimulated transient and steady‐state biofilm systems equally well. In the second article in this series, we describe how this model may be extended and utilized to make predictions of the behavior of transient and steady‐state biofilm systems in the presence of a toxic transition metal(Pb). © 1994 John Wiley & Sons, Inc.
Saccharification and fermentation of Sugar Cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway
Authors: Doran Joy B. , Aldrich H. C. , Ingram L. O.
Published: December 2004

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g−1 acid‐treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid‐treated bagasse L−1), a combination of 20 FPU cellulase g−1 bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L−1. Alternatively, almost 40 g ethanol L−1 was produced with 10 FPU cellulase g−1 bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU−1 of commercial cellulase. © 1994 John Wiley & Sons, Inc.
Biodegradation process development using a bacterial cytochrome in vivo
Authors: Horowitz Jeffrey B. , Vilker Vincent L.
Published: December 2004

Pseudomonas putida PpG786 that contains the inducible enzyme system cytochrome P‐450cam is considered for use as specialized biomass fore detoxification of hazardous hydrocarbons. The test substrate 1,2‐dibromochloropropane (DBCP) is used to assess the organohalide degradation activity of P. Putida PpG786. Activity was found to be a strong function of intracellular heme content, variables which affect the culturing and processing of the cells, and oxygen tension in the degradation incubation medium. The lifetime for maintaing active biomass in chemostat washout operation, after including substrate was removed and then restarted, was also studied. These results indicate that initial activity of the P. Putida biomass is high enough, and decays slowly enough, so that industrial wastewater treatment at the operating conditions of a sequencing batch reactor (SBR) could remove hazardous compounds. © 1994 John Wiley & Sons, Inc.
Cyclic AMP regulation of tylosin biosynthesis and secondary metabolism in Streptomyces fradiae
Authors: Tata Murthy , Menawat Anil S.
Published: December 2004

Adenosine 3′:5′ cyclic monophosphate seems to regulate antibiotic biosynthesis and secondary metabolism in tylosin‐producing cultures of Streptomyces fradiae C373.1. A dose‐dependent response is observed by exogenous additions of dibutyryl cyclic AMP (cAMP), and is related to the nutritional status of the culture. Addition of cAMP to cultures growing in nutritionally lean media caused higher cumulative antibiotic tigers and some cellular differentiation compared with the control. In nutritionally rich media, a qualitatively different behavior resulted: an almost instantaneous shift toward secondary metabolism occurred. The response is characterized by extensive cellular differentiation with little growth and only a trace of antibiotic production. The possible role of cyclic AMP n the regulation of tylosin biosynthesis and secondary metabolism and its relation to specific nutrient limitations in synthetic, defined media in Streptomyces fradiae is discussed. © 1994 John Wiley & Sons, Inc.
A new method for studying microaerobic fermentations. I. A theoretical analysis of oxygen programmed fermentation
Authors: Lidén Gunnar , Frazén Carl Johan , Niklasson Claes
Published: December 2004

An experimental method for studying microaerobic fermentation, called oxygen programmed fermentation, is introduced. The method if based on a chemostat. The mathematical equations governing the dynamics of the system are derived and simulations are made for two principally different cases: a purely respirative organism, and an organism capable of fermentation during oxygen limitation. It is shown that at a suitably chosen ramp rate, the dissolved oxygen concentration in the broth can be made to decrease almost linearly. It is suggested that the greatest use of oxygen programmed fermentation will be in initial experiments. Compared with chemostat studies, a scan of different oxygenation rates will provide a time‐saving method of finding the interacting regions for metabolic transitions. Furthermore it is shown that the methods makes it possible to study cell physiology at condition which would normally lead to washout. © 1994 John Wiley & Sons, Inc.
Affinity precipitation of an antibody by ligand‐modified phospholipids
Authors: Powers Daniel D. , Carbonell Ruben G. , Kilpatrick Peter K.
Published: December 2004

A new method for the selective precipitation of proteins is applied to the isolation and purification of an antibody. Ligand‐modified phospholipids (LMPs) are solubilized by the nonionic ethoxylated alcohol detergent, resulting in small (50 to 100 Å) micellar aggregates of LMPs and surfactant. When introduced into protein solutions containing an antibody for which the LMP has specific affinity, the ligand binds to the protein. Hydrophobic interactions between phospholipid tail groups bound to the protein molecules result in an insoluble precipitate. Polyclonal and monoclonal antibiotin antibody (pABA and mABA) are shown to be selectively precipitated using ratios of dimyristoylphosphatidylethanolamidobiotin (DMPE‐B) to ABA ranging from 1:1 to 19:1. The kinetics and yield of the precipitation achieve a maximum at a ratio of DMPE‐B to ABA of approximately 7:1. The kinetics and magnitude of the turbidity change are modeled using the Mie theory of light scattering coupled with the smoluchowski theory of aggregation. The kinetics are shown to be enhanced significantly by the addition of salt. In particular, the addition of 0.5 M ammonium sulfate salt increases the rate of precipitation by more than an order of magnitude. It is demonstrated that pABA can be recovered with total activity yields of 60% to 70% from mixtures containing nonspecific lgG antibodies in very high purity (>99%). © 1994 John Wiley & Sons, Inc.
Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion
Authors: Oh Duk Jae , Koller Manfred R. , Palsson Bernhard O.
Published: December 2004

The establishment of prolific long‐term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10‐ to 30‐fold increase in total cell number and in the number of colony forming units‐granulocyte/macrophage (CFU‐GMs), and a four‐ to tenfold increase in the number of long‐term culture initiating cells (LTC‐ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion‐based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100‐fold and the CFU‐GM expansion exceeded 30‐fold over a 27‐day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long‐term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC‐IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long‐term maintenance and replication of human stem cells ex vivo. © 1994 John Wiley & Sons, Inc.
Linear constrain relations in biochemical reaction systems III. Sequential application of data reconciliation for sensitive detection of systematic errors
Authors: van der Heijden R. T. J. M. , Romein B. , Heijnen J. J. , Hellinga C. , Luyben K. C. A. M.
Published: December 2004

This article presents a method to test the presence of relatively small systematic measurement errors; e.g., those caused by inaccurate calibration or sensor drift. To do this, primary measurements—flow rates and concentrations—are first translated into observed conversions, which should satisfy several constraints, like the laws of conservation of chemical elements. This study considers three objectives:

Modification of the commonly used balancing technique to improve error sensitivity to be able to detect small systematic errors. To this end, the balancing technique is applied sequentially in time.

Extension of the method to enable direct diagnosis of errors in the primary measurements instead of diagnosing errors in the observed conversions. This was achieved by analyzing how individual errors in the primary measurements are expressed in the residual vector.

Derivation of a new systematic method to quantitatively determine the sensitivity of the error, is that error size at which the expected value of the chisquare test function equals its critical value.

The method is applied to industrial data demonstrating the effectiveness of the approach. It was shown that, for most possible error sources, a systematic errors of 2% to 5% could be detected. In given application, the variation of the N‐content of biomass was appointed to be the cause of errors. © 1994 John Wiley & Sons, Inc.
Multiple product inhibition and growth modeling of clostridium butyricum and klebsiella pneumoniae in glycerol fermentation
Authors: Zeng A.‐P. , Ross A. , Biebl H. , Tag C. , Günzel B. , Deckwer W.‐D.
Published: December 2004

The inhibition potentials of products and substrate on the growth ofClostridium butyricum and Klebsiella pneumoniae in the glycerol fermentation are examined from experimental data and with a mathematicalmodel. Whereas the inhibition potential of externally added and self‐produced 1,3‐propanediol is essentially the same, butyric acid produced by the culture is more toxic than that externally added. The same seems to apply for acetic acid. The inhibitory effect of butyric acid is due tothe total concentration instead of its undissociated form. For acetic acid, it cannot be distinguished between the total concentration and the undissociated form

The inhibition effects of products and substrate in the glycerol fermentation are irrespective of the strains, and, therefore, the same growth model can be used. The maximum product concentrations tolerated (critical concentrations C*pi) are 0.35 g/Lfor undissociated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol, 71.4 g/L for 1,3‐propanediol, and 187.6 g/L for glycerol, which are applicable to C. butyricum and K. pneumoniae grown under a variety of conditions. For 55 steady‐states, which were obtained from different types of continuous cultures over a pHrange of 5.3–8.5 and under both substrate limitation and substrate excess, the proposed growth model fits the experimental data with an average deviation of 17.0%. The deviation of model description from experimental values reduces of 11.4% if only the steady‐states with excessive substrate are considered. © 1994 John Wiley & Sons, Inc.
Production of somatic embryos in a helical ribbon impeller bioreactor
Authors: Archambault Jean , Williams Robert D. , Lavoie Luc , Pépin Marie‐France , Chavarie Claude
Published: December 2004

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11‐L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures (∼8–13 SE · mL−;1). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate (∼0.05 VVM, kL a ∼ 6.1 h−;1) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO (∼60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (−5–10%) inhibited biomass and SE production. The best production of SE (∼44 SE · mL−1 or ∼757 SE · g dw−1 · d−1) was achieved by operating the bioreactor at 60 rpm while controlling DO at ∼20%by surface oxygenation only (0.05 VVM, kL a ∼ 1.4 h−;1). This production was found to be a biomass production/growth‐associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. © 1994 John Wiley & Sons, Inc.
Effect of modified glucose uptake using genetic engineering techniques on high‐level recombinant protein production in escherichia coli dense cultures
Authors: Chou Chih‐Hsiung , Bennett George N. , San Ka‐Yiu
Published: December 2004

Reduction of acetate excretion using a modified cellular glucose uptake rate was examined. An Escherichia coli strain bearing a mutationin ptsG, a gene encoding enzyme II in glucose phosphotransferase system (PTS), was constructed and characterized. The growth rate of the mutant strain was slower than its parent in glucose defined medium, butwas not affected in complex medium. Experimental results using this mutant strain showed a significant improvement in culture performance in simple batch cultivations due to reduced acetate excretion through the modified glucose uptake. Both biomass and recombinant protein productivity were increased by more than 50% with the ptsG mutant when compared to the parent strain. Recombinant protein productivity by the newly constructed strain at a level of more than 1.6 g/L was attained consistently in a simple batch bioreactor. © 1994 John Wiley & Sons, Inc.
Biosorption of mercury by the inactivated cells of pseudomonas aeruginosa PU21 (Rip64)
Authors: Chang Jo‐Shu , Hong Juan
Published: December 2004

Biomass of a mercury‐resistant strain Pseudomonas aeruginosa PU21 (Rip64) and hydrogen‐form cation exchange resin (AG 50W‐X8) were investigated for their ability to adsorb mercury. The maximum adsorption capacity was approximately 180 mg Hg/g dry cell in deionized water and 400 mg Hg/g dry cell in sodium phosphate solution at pH 7.4, higher than the maximum mercury uptake capacity in the cation exchange resin (100 mg Hg/g dry resin in deionized water). The mercury selectivity of the biomass over sodium ions was evaluated when 50 mM and 150 mM of Na+ were present. Biosorption of mercury was also examined in sodium phosphate solution andphosphate‐buffered saline solution (pH 7.0), containing 50mM and 150 mM of Na+, respectively. It was found that the presence of Na+ did not severely affect the biosorption of Hg2+, indicating a high mercury selectivity ofthe biomass over sodium ions. In contrast, the mercury uptake by the ion exchange resin was strongly inhibited by high sodium concentrations. The mercury biosorption was most favorable in sodium phosphate solution (pH 7.4), with a more than twofold increase in the maximum mercury uptake capacity. The pH was found to affect the adsorption of Hg2+bythe biomass and the optimal pH value was approximately 7.4. The adsorption of mercury on the biomass and the ion exchange resin appeared to follow theLangmuir or Freundlich adsorption isotherms. © 1994 John Wiley & Sons, Inc.
The influence of dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis
Authors: Suphantharika M. , Ison A. P. , Lilly M. D. , Buckland B. C.
Published: December 2004

The antibiotic, difficidin, and its hydroxylated derivative oxydifficidin, were synthesized by cultures of Bacillus subtilis grown on a complex medium. Maximum titers of about 200 and 130 mg/L, respectively, were obtained. In fermentations where the dissolved oxygen tension (DOT) was controlled, the maximum specific growth rate was only reduced below 5% air saturation. DOT had little effect on the volumetric rateof synthesis of oxydifficidin but greatly influenced the rate for difficidin, which was reduced at DOT values below 40% air saturation. © 1994 John Wiley & Sons, Inc.