bluuebunny/crossref_metadata_2025 huggingface dataset:
Solventogenesis in Clostridium acetobutylicum fermentations related to carboxylic acid and proton concentrations
Authors: Hüsemann Michael H. W. , Papoutsakis Eleftherios T.
Published: December 2004

The mechanism primarily implicated in the solventogenesis process in batch fermentations of Clostridium acetobutylicum is examined in considerable detail. A variety of fermentations with or without pH control in the pH range of 3.7–6 have been carried out in order to examine which of a host of suspect parameters correlate with the initiation of solventogenesis. The parameters that did not correlate are the external (pH0) and intracellular (pHi) pH, and ΔpH, and the external or intracellular butyrate and acetate concentrations. Undissociated butyric acid (UBA) correlated well with the initiation of solventogenesis. A linear relationship between UBA and butanol concentrations was found at the onset of solventogenesis in all fermentations examined. The intercept of this linear relationship was 6–13mM UBA for the pH0 range of 3.7–5 and approximately zero for pH0 at or above 6. The required minimal UBA was interpreted as a dependency of the solventogenesis process on both H+ and butyrate concentrations. Undissociated acetic acid was found not to correlate with the initiation of solventogenesis. Addition of acetoacetate (AA) and propionate enhanced the effect of UBA on the solventogenesis process. The action of a nonmetabolizable (FCCP) and a metabolizable (AA) uncoupler on the ΔpH, pH0, pHi, and solventogenesis were also studied in order to gain further understanding of the solventogenesis mechanism.
Removal of lignin and reuse of cellulases for continuous saccharification of lignocelluloses
Authors: Tanaka Mitsuo , Fukui Masahide , Matsuno Ryuichi
Published: December 2004

Method of the removal of lignin and reuse of cellulases for a continuous saccharification of lignocelluloses were investigated. Only lignin could be separated from hydrolysates by differences in the settling velocity; it was removed from the saccharification process by flocculation with chitosan without loss of cellulases. The ultra‐filtration membrane PM10 (Amicon) could be used for recovery of cellulases, but the membrane UH‐1 (Toyo Roshi) was better for this purpose, because no cellulases leaked from the membrane, and the amount of cellulase adsorbed to the membrane was less. The cellulases were inactivated by vigorous agitation of the solution in an ultra‐filtration device. The loss of cellulase activity by such agitation increased with agitation time, but could be controlled by recovery at a low speed of agitation, so the cellulases could be reused.
Continuous processing of toxic organics in a fluidized‐bed GAC reactor employing carbon replacement
Authors: Pfeffer John T. , Suidan Makram T.
Published: December 2004

The fluidized‐bed, granular activated carbon (GAC) anaerobic reactor has been shown to be an effective process for the continuous long‐term treatment of wastewaters that contain biodegradable or nonbiodegradable toxic organic compounds. With loadings of 10 g COD/kg GAC day, COD removal of 94% was achieved. The anaerobic biofilm that develops on the GAC reduces the load on the carbon by converting the biodegradable organics to methane and carbon dioxide. Approximately 50% of the COD applied to the reactor was converted to methane, thereby reducing carbon requirements. Successful operation of the system requires that a carbon replacement schedule be maintained that will keep the bulk concentrations of toxic adsorbable compounds below their toxic threshold. As long as toxic substances can be adsorbed by the carbon, they will not inhibit the anaerobic biofilm. If nonadsorbable toxic compounds are present, processing must be included to reduce these materials to concentrations below their threshold level.
Mixed substrate‐metabolism in an obligate methanol utilizer: Chemostat studies
Authors: Samuelov Nissim S.
Published: December 2004

In Pseudomonas C, an obligate RMP‐methylotroph, externally added formic acid and multi‐carbon intermediates are co‐metabolized in the obligate presence of methanol. Cell yield and methanol‐carbon conversion to cell mass are optimal in a chemostat culture growing on a mixture of energy sources and methanol as the sole limiting carbon source. Growth on methanol as the sole, obligate energy source and an additional carbon source (e.g. pyruvate) is less efficient. Under these energy limited conditions, the cyclic oxidation pathway is predominantly operated.
A model for continuous fermentations with amylolytic yeasts
Authors: Pasari Ajay B. , Korus Roger A. , Heimsch Richard C.
Published: December 2004

A two‐stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single‐stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two‐stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single‐stage, pure cultures. In the two‐stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.
An algorithmic approach to constructing the on‐line estimation system for the specific growth rate
Authors: Shimizu Hiroshi , Takamatsu Takeichiro , Shioya Suteaki , Suga Ken‐Ichi
Published: December 2004

The objective of this article is to propose an algorithm for the on‐line estimation of the specific growth rate in a batch or a fed‐batch fermentation process. The algorithm shows the practical procedure for the estimation method utilizing the macroscopic balance and the extended Kalman filter. A number of studies of the on line estimation have been presented. However, there are few studies discussing about the selection of the observed variables and for the tuning of some parameters of the extended Kalman filter, such as covariance matrix and initial values of the state.

The beginning of this article is devoted to explain the selection of the observed variable. This information is very important in terms of the practical know‐how for using technique. It is discovered that the condition number is a practically useful and valid criterion for number is a practically useful and valid criterion for choosing the variable to be observed.

Next, when the extended Kalman filter in applied to the online estimation of the specific growth rate, which is directly unmeasurable, criteria for judging the validity of the estimated value from the observed data are proposed. Based on the proposed criterial, the system equation of the specific growth rate is selected and initial value of the state variable and covariance matrix of the system noises are adjusted. From many experiments, it is certified that the specific growth rate in the batch or fed ‐batch fermentation can be estimated accurately by means of the algorithm proposed here. In these experiments, that is, when the cell concentration is measured directly, the extended Kalman filter using the convariance matrix with a constant element can estimate more accurately values of the specific growth rate than the adaptive extended Kalman filter does.
Multiple forms of endo‐1,4‐β‐D‐glucanase in the extracellular cellulase of Penicillium pinophilum
Authors: Bhat K. Mahalingeshwara , Wood Thomas M.
Published: December 2004

The influence of the composition of the growth medium on the production of endo‐1,4‐β‐D‐glucanase (CM‐cellulase) activity by P. pinophilum was studied in shake flask cultures using Avicel PH101 as the carbon source. It was observed that the culture conditions had a profound effect on the level of endoglucanase (CM‐cellulase) produced by P. pinophilum. However, isoelectric focusing of the endoglucanase activity obtained from shake flask and fermenter cultures using the same growth medium revealed that the enzyme system found in both cultures was identical qualitatively, and contained seven or eight different endoglucanase components. All the endoglucanase components appeared simultaneously in the early stages of culture and prolonged incubation resulted only in an increase in the concentration of these enzymes. Protease levels were found to be low in both types of culture but were particularly so in the growth medium which contained corn steep liquor. The proteases were unable to release low molecular weight peptides when P. pinophilum cellulase protein was used as a substrate. The results were interpreted to indicate that the multiplicity of endoglucanase components found in cultures of P. pinophilum is most likely the result of expression of a number of specific genes rather than by post‐secretional modification of one or more endoglucanase(s) synthesized by the fungus.
Water‐soluble nonionic surfactants for affinity bioseparations
Authors: Guzman R. , Torres J. L. , Carbonell R. G. , Kilpatrick P. K.
Published: December 2004

Reversible competitive inhibitors of the three enzymes β‐galactosidase, trypsin, and serum cholinesterase have been covalently attached to nonionic ethoxylated surfactants. The binding of the resulting affinity‐derivatized surfactants to the respective enzymes has been quantified by measuring Michaelis‐Menten inhibition constants with kinetic assays. The surfactant‐inhibitor of serum cholinesterase, octaethylene glycol monohexadecy ether pyridinium (C16E8‐PYR), was adsorbed in aqueous solution to an octadecyl‐bonded reverse‐phase silica packing in a 2 × 0.2 cm stainless steel test column. The ability of the test column to function as a high‐performance affinity chromatography (HPAC) column was determined by applying a mixture of bovine serum albumin and cholinesterase (4:1 w/w). Virtually all of the cholinesterase bound and was eluted by applying a gradient in ionic strength. The applied cholinfesterase was recovered with a yield of over 90% and an 11‐fold purification. An aliquot of raw horse serum was then purified in the same fashion with a yield of 84% and a 280‐fold purification. The surfactant‐inhibitor was easily removed from the column with an alcohol wash for sterilization, cleaning, or application of a different affinity ligand. Moreover, the ligand density on the column can be easily manipulated by adsorbing mixtures of derivatized and underivatized surfactants. Leakage of ligands from the support seems to be minimal since the cholinesterase affinity column was operated efficiently after being exposed to 24,000 column volumes of buffer. The application of this technique to high‐capacity, high‐throughput reversible affinity purifications is limited only by the ability to identify suitable ligands.
Degradation of fluorene in soil by fungus Phanerochaete chrysosporium
Authors: George Elias J. , Neufeld Ronald D.
Published: December 2004

During investigation of biodegradation in soil, we have found that classical or standard techniques for introduction of compounds and the growth of fungus into soil are ill‐defined and inadequate. In response to this deficiency, a method for controlled introduction of extractable compounds and for the growth of fungus in soils has been developed. This method was successfully used to study the degradation of fluorene in soil by the fungus Phanerochaete chrysosporium.
Stabilization of restriction endonuclease Bam HI by cross‐linking reagents
Authors: Dubey A. K. , Bisaria V. S. , Mukhopadhyay S. N. , Ghose T. K.
Published: December 2004

Bacillus amyloliquefaciens H produces a restriction endonuclease enzyme BamHl which is heat labile even at low temperatures. Studies were conducted to enhance thermal stability of BamHl using cross‐linking reagents, namely, glutaraldehyde, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3′‐dithiobispropionimidate (DTBP). Reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. However, the DMA‐, DMS‐, and DTBP‐cross‐linked preparations of BamHI exhibited significant improvement in thermal stability. Studies on thermal denaturation of the cross‐linked enzyme preparations revealed that these do not follow a true first‐order kinetics A possible deactivation scheme has been proposed in which the enzyme has been envisaged to go through a fully active but more susceptible transient state which, on prolonged heat exposure, exhibits a first‐order decay kinetics. At 35°C, which is close to the optimum reaction temperature of 37°C for BamHl activity, the half‐line of DMA‐, DMS‐, and DTBP‐cross‐linked preparations were 4.0, 5.25, and 5.5 h, respectively, whereas the native enzyme exhibited a half‐line of 1.2 h only. The apparent values of deactivation rate constants for native, DMA‐, DMS‐, and DTBP‐cross‐linked BamHl were 1.13, 0.39, 0.29, and 0.26 h−1, respectively, at the same temperature, and the apparent values of activation energies for denaturation of native, DMA‐, DMS‐, and DTBP‐cross‐linked BamHl were 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. The DTBP‐cross‐linked Bam HI was, therefore, the best heat‐stable preparation among those tested. The unusually low values of activation energies for denaturation of Bam Hl represent their highly thermolabile nature compared to other commonly encountered enzymes such as trypsin, having activation energies of more than 40 kcal/mol for their denaturation.
The effect of concentration of phenols on their batch methanogenesis
Authors: Wang Yi‐Tin , Suidan Makram T. , Pfeffer John T. , Najam Issam
Published: December 2004

The biodegradability of phenol and six other phenolic compounds (o‐, m‐, and p‐cresol, 2‐, 3‐, and 4‐ethylphenol) was examined in batch methanogenic cultures. The effect of concentration of these alkyl phenols on the anaerobic biodegradation of phenol was also evaluated. The inoculum used in this study was cultivated in a continuous flow laboratory fermenter with phenol as the primary substrate. Phenol, at initial concentrations as high to 1400 mg/L was completely degraded to methane and carbondioxide after 350 hours incubation. Complete degradation of m‐ and p‐cresol was also observed while the ethylphenols and o‐cresol were not significantly degraded.

At initial concentrations exceeding 600 mg/L, phenol inhibited the phenol‐degrading microorganisms but not the methanogens. At about 600 mg/L, cresols reduced the rate of phenol degradation to 50% of that observed in a control culture containing only 200 mg/L phenol. Ethylphenols were more inhibitory than cresols. Phenol degrading microorganisms were more susceptible to inhibition by cresols and ethylphenols than were the methanogens. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.
On‐line monitoring of cell mass in mammalian cell cultures by acoustic densitometry
Authors: Kilburn D. G. , Fitzpatrick P. , Blake‐Coleman B. C. , Clarke D. J. , Griffiths J. B.
Published: December 2004

Acoustic resonance densitometry (ARD) provides a highly reproducible and stable method for on‐line measurement of culture biomass density. The technique provides a direct determination of changes in relative density of culture medium and cell mass. At cell concentrations higher than 106 cells mL−1this method can replace cell counts and provide a continuous measure of total cell mass. In cultures of hybridomas or U937 human lymphoma cells, the ARD value correlates well with cell number except when the average cell size changes during culture. It is argued that cell mass determined by ARD rather than cell number should be used as the basis for measurements of specific biological activity.
Two‐Level factorial screening of new plasmid/strain combinations for prodution of recombinant‐DNA products
Authors: Emborg Claus , Jepsen Pia Knak , Biedermann Kirsten
Published: December 2004

This article treats the basic problem of selection of experimental conditions for microbiological experiments for evaluation of newly isolated bacterial strains, mutants, or plasmid/strain combinations. For this purpose shake flask experiments in a 210‐4confounded factorial design at resolution IV with four blocks of 16 flasks were used. The design was used for testing of two new strain/plasmid combinations (E. coli MT 102/403‐SD2 and W 3110/403‐SD2) i.e., both strains with the same plasmid 403‐SD2. Both strains were integrated in the design, so both strains were tested with nine factors (temperature, aeration, glucose, initial pH, pH regulation, reduced aeration, chloramphenicol, acetate, and glycerol). With both strains the interaction between initial pH and reduced aeration had a significant influence on the yield of the recombinant‐DNA product nuclease. There was more than a factor of 10 between lowest and highest yield of product. In this interactive system the strains reacted differently. MT 102/403‐SD2 had highest yields at high initial pH (8.4) and no reduction in aeration, whereas W 3110/403‐SD2 had highest yields of nuclease at low initial pH (7.4) and reduced aeration (rubber stopper inserted after cultivation for 12 h). These data (and previous work) clearly demonstrate that it is impossible to suggest a simple set of experimental conditions for testing of new plasmid/strain combinations. It is clear that the exclusive application of a standardized growth technique e.g., LB‐medium at 37°C at an unspecified and uncontrolled aeration level, may lead to wrong conclusions on properties and potentials of now plasmid/strain combinations and may lead to rejection of useful strains or plasmids.
The degradation of lignocellulosics by extremely thermophilic microorganisms
Authors: Donnison A. M. , Brockelsby C. M. , Morgan H. W. , Daniel R. M.
Published: December 2004

Five extremely thermophilic cellulose‐degrading isolates obtained from New Zealand thermal springs were tested for their ability to degrade a number of natural lignocellulosic substrates. Degradation by three of the isolates was generally similar to that by the moderate thermophile, Clostridium thermocellum but occurred at a higher temperature. The New Zealand isolates were also found to grow on xylan as sole carbohydrate source, which probably extends their attack to hemicellulose.
Role of some whey components on mass transfer in ultrafiltration
Authors: Taddei C. , Daufin G. , Aimar P. , Sanchez V.
Published: December 2004

Ultrafiltration through Carbosep M4 mineral membrane of protein solutions of decreasing complexity (whey before and after centrifugation or clarification, β‐lactoglobulin) was studied. Mathematical models were used to explain variations in flux with time. Taking into account variations in protein retention and hydraulic resistance of the membrane during ultrafiltration, proteins and lipoproteins were found to be involved not only in the polarization layer (reversible fouling leading to a difference in the osmotic pressure), but also in irreversible fouling by adsorption. Morever, the presence of particles (e.g., inorganic precipitates) in whey explains the build‐up of a deposit over and within the membrane which contributes to the decline in flux after 1 h ultrafiltration. The relative importance of these phenomena was quantified.
An approach in mathematical modeling of an upflow packed‐bed reactor for enzymatic hydrolysis of wheat straw
Authors: Gonzàlez G. , Caminal G. , de Mas C. , Lòpez‐Santín J.
Published: December 2004

The behavior of a packed‐bed reactor for enzymatic hydrolysis of pretreated wheat straw has been described by means of a mathematical model. The flow pattern has been evaluated by residence time distribution experiments. Small deviations from ideal plug flow behavior were found using the dispersion model. The kinetic model proposed for enzymatic hydrolysis of cellulosic fraction of pretreated wheat straw has been derived from batch experimental data. Variations of enzyme concentration throughout the straw bed have been approximately described using a ramp variation of adsorbed enzyme. The final explains qualitatively the experimental results.
Lactose hydrolysis by immobilized β‐galactosidase in capillary bed reactor
Authors: Peterson R. S. , Hill C. G. , Amundson C. H.
Published: December 2004

The hydrolysis of lactose by immobilized β‐galactosidase was studied in a continuous‐flow capillary bed reactor operating at 30°C. Solutions containing 50, 100, and 150 g lactose and 0.5 g sodium acetate/L were fed to the reactor. Lactose conversions ranging from 24% to greater than 99% were achieved at reactor space times ranging from 0.06 to 6.3 min. These conversion data were successfully modeled in terms of a plug flow reactor model and a form of Michaelis–Menten kinetics which included competitive inhibition by both the alpha and beta forms of galactose.
Biospecific adsorption of lysozyme onto monoclonal antibody ligand immobilized on nonporous silica particles
Authors: Liapis A. I. , Anspach B. , Findley M. E. , Davies J. , Hearn M. T. W. , Unger K. K.
Published: December 2004

It is very important to understand the equilibrium and dynamic characteristics of biospecific adsorption (affinity chromatography) for both scientific and application purposes. Experimental equilibrium and dynamic column data are presented on the adsorption of lysozyme onto antibody immobilized on nonporous silica particles. The Langmuir model is found to represent the equilibrium experimental data satisfactorily, and the equilibrium association constants and heats of adsorption have been estimated for two systems with different ligand densities. The effects of nonspecific interactions are more pronounced in the system with low‐density ligand. The dynamic interaction kinetic parameters are estimated by matching the predictions of a fixed‐bed model with the experimental breakthrough curves. The agreement between theory and experiment is good for the initial phases of breakthrough, where the mechanism of biospecific adsorption is dominant. In the later phase (saturation neighborhood) of breakthrough, the effects of nonspecific interactions appear to be greater in the low‐density ligand system. The kinetics of the nonspecific interactions were estimated from the data of the later phase of breakthrough and were found to be considerably slower than those attributed to biospecific adsorption.
A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension cultures
Authors: Batt Brian C. , Kompala Dhinakar S.
Published: December 2004

A structured kinetic model is developed to describe the dynamics of hybridoma growth and the production of monoclonal antibodies and metabolic waste products in suspension culture. The crucial details of known metabolic processes in hybridoma cells are incorporated by dividing the cell mass into four intracellular metabolic pools. The model framework and structure allow the dynamic calculation of the instantaneous specific growth rate of a hybridoma culture. The steady state and dynamic simulations of the model equations exhibit excellent agreement with experimentally observed trends in substrate utilization and product formation. The model represents the first to include any degree of metabolic detail and structure in describing a hybridoma culture. In so doing, it provides the basic modeling framework for incorporating further details of metabolism and can be a useful tool to study various strategies for enhancing hybridoma growth as well as viability and the production of monoclonal antibodies in suspension cultures.
Oxygen diffusivity in gel beads containing viable cells
Authors: Kurosawa Hiroshi , Matsumura Masatoshi , Tanaka Hideo
Published: December 2004

This article proposes a simple steady‐state method for measuring the effective diffusion coefficient of oxygen (De) in gel beads entrapping viable cells. We applied this method to the measurement of De in Ca‐ and Ba‐alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of ±7% and found not to be influenced by cell density (0–30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca‐alginate gel beads was superior to that of the Ba‐alginate gel beads, and the De in the Ca‐alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca‐alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.
Alginate concentration: A key factor in growth of temperature‐sensitive baculovirus‐infected insect cells in microcapsules
Authors: King G. A. , Daugulis A. J. , Goosen M. F. A. , Faulkner P. , Bayly D.
Published: December 2004

The desire to increase cell density and product concentration has been the primary driving force for the development of better animal cell culture processes. In the technique used in our laboratory—microencapsulation—insect cells (Spodoptera frugiperda), infected with a temperature‐sensitive mutant of the Autographa californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate–polylysine (PLL) microcapsules which had a controlled membrane molecular‐weight cutoff and an intracapsular alginate concentration which was ca. 16% lower than that obtained in the commercially available single‐membrane system. Cell culture experiments indicated that the intracapsular alginate concentration appears to be a key factor in achieving good cell growth. It was possible to obtain intracapsular cell densities of 8 × 107 cells/mL capsules and virus concentrations to 109 IFU/mL capsules. The virus litre in the supernatant was ca. 300 times lower, indicating that virtually all of the virus was retained within the capsules.
Effects of paddle impeller geometry on power input and mass transfer in small‐scale animal cell culture vessels
Authors: Aunins John G. , Woodson Bruce A. , Hale Timothy K. , Wang Daniel I. C.
Published: December 2004

Process scaleup for stirred‐tank animal cell cultures such as suspension and microcarrier cultures often begins at the bench scale in small spinner vessels. In order to initiate process development under the proper conditions, it is essential to know the physical conditions under which the cells are grown. In this article, power inputs and surface oxygen transfer rates to culture medium in 500‐mL Corning spinner vessels were determined as a function of the impeller geometry, impeller height, and agitation speed. The results obtained indicate that power dissipation dependency differs from literature correlations and may compromise scale up at constant power input from these vessels. These results are of general utility to researchers using small‐scale vessels.
Continuous microbial desulfurization of coal—application of a multistage slurry reactor and analysis of the interactions of microbial and chemical kinetics
Authors: Uhl W. , Höne H.‐J. , Beyer M. , Klein J.
Published: December 2004

Microbial desulfurization of coal by pyrite oxidizing bacterial enrichment cultures has been studied in air‐agitated slurry reactors of 4‐ and 20‐L volumes. Batch experiments showed that inoculation with an active bacterial culture is essential to minimize the lag phase, although a considerable number of pyrite oxidizing bacteria was found on the coal prior to desulfurization. For detailed investigations of kinetics, energy requirements, and technical applicability, a bioreactor equipment consisting of a cascade of eight stages was developed and operated continuously. Microbial desulfurization of coal—monitored by measuring the axial profile of dissolved iron concentration, real and maximum oxygen consumption rates, and cell concentration—at pulp densities to 30% was performed over a period of 200 days without any disturbances concerning the aeration system, fluidization, transport of solids and microbial growth. At a pulp density of 20%, a pyrite conversion of 68% was achieved after the third reactor stage at a total residence time of five days in the first three stages. The kinetics of pyrite degradation were found to be well described by a rate equation of first order in pyrite surface area concentration if the pyrite is directly accessible for microbial attack. Rate constants were determined to 0.48 mg pyrite/(cm2 day) in the first and to 0.24 mg pyrite/(cm2 day) in the following reactor stages. Kinetic models taking into account adsorption/desorption as well as growth kinetics failed to describe the observed reaction rates. However, a model treating pyrite degradation and microbial growth kinetics formalistically seems to be applicable when backmixing between the reactor stages can be avoided. The advantage of a multistage reactor in comparison to single‐stage equipment was shown by calculation. To obtain a pyrite conversion of 68%, a three‐stage reactor would require only 58% of the volume of single‐stage equipment.

Measurement of oxygen consumption rates proved to provide quickly and easily measurable parameters to observe microbial coal desulfurization in technical scale: the real oxygen consumption rate is correlated to the pyrite oxidation rate and the maximum oxygen consumption rate is correlated to the concentration of viable cells. The Yo/s coefficient for the amount of oxygen consumed per mass unit of pyrite oxygen was determined to approximately 0.33 in comparison to 1.0 which can be calculated from stoichiornetry. This could yet not be explained. Chemical leaching experiments as well as sulfur analyses of desulfurized coal samples showed that the microorganisms play the main role in degradation of pyrite from coal and that pyrite oxidation by ferric iron can be neglected.
Cellular plasmid content and cloned‐gene expression: Some useful equations
Authors: Satyagal Vasuki N. , Agrawal Pramod
Published: December 2004

Cellular plasmid content determines the yield of closed‐gene product, affects plasmid replication, and influences the behavior of the culture by dictating the extent of metabolic burden on the host cell and plasmid segregation at cell division. Hence, it is a variable of primary importance in the study of recombinant cell cultures. In this article, equations are developed to enable the conversion of experimental determinations of cellular plasmid content into theoretically important units. The importance of different units in characterizing plasmid effect on cell response is highlighted. Also, equations that relate cellular plasmid content to cloned‐gene expression are developed and successfully tested on several sets of data from literature.
Encapsulation of mammalian cell with chitosan‐CMC capsule
Authors: Yoshioka Toshimitsu , Hirano Ryogo , Shioya Toshiaki , Kako Masatoshi
Published: December 2004

Viable hybridoma cells were encapsulated. The capsules were formed in one step by placing a drop of cell suspension mixed with negatively charged carboxymethylcellulose (CMC) into a positively charged chitosan solution through the interpolymeric ionic interaction between two oppositely charged polymers. These capsules were found to have a mean diameter of about 1. 5 mm and wall thickness of 3 μm. The cells grew in the capsules using supplemented DMEM/F12 (four kinds of growth factor). The maximum cell density in encapsulating cell culture reached 1 × 107 cells/ml, 10 times higher than that obtained in the free cell culture. The maximum monoclonal antibody concentration in the free cell culture was 15μg/mL, but that in the capsule was 45μg/mL The antibody produced by the cell was concentrated about four times higher inside than outside of the capsules.
Immobilization of Arthrobacter simplex in a thermally reversible hydrogel: Effect of temperature cycling on steroid conversion
Authors: Park Tae Gwan , Hoffman Allan S.
Published: December 2004

Arthrobacter simplex cells, which convert the steroid hydrocortisone to prednisolone, have been entrapped in a thermally reversible hydrogel. Such hydrogels exhibit a lower critical solution temperature (LCST) wherein the gel shrinks and deswells when it is warmed through its LCST, and then reversibly expands and reswells when it is cooled below the LCST. The immobilized cell‐hydrogel system has been thermally cycled between two temperatures, each below the LCST. The upper temperature was selected to be just below the LCST, where the gel deswells but does not collapse, as it does at the LCST. The thermal cycling acts like a “hydraulic pump” which enhances mass transfer of the substrate (hydrocortisone) in and the product (prednisolone) out of the gel, thereby increasing steroid conversion dramatically relative to isothermal operation at either the upper or lower temperature. The increased conversion can also be due in part to reduced product inhibition. Mass transfer resistance and product inhibition are among the most serious problems in immobilized biocatalyst technology and thermal cycling of LCST hydrogels is both a novel and useful approach to minimizing these problems.
Dynamic analysis of genetic control and regulation of amino acid synthesis: The tryptophan operon in Escherichia coli
Authors: Sen Asok K. , Liu Wei‐Min
Published: December 2004

A mathematical model of the tryptophan operon is analyzed to investigate the regulatory effects of feedback repression and the demand for tryptophan in the cell. In this model, feedback repression is considered to be a two‐step process. First, the endproduct tryptophan combines with the inactive repressor produced by the regulatory genes to yield an active complex. This complex subsequently binds to the operator and prevents transcription of the structural genes into mRNA. The demand for tryptophan in the cell is modeled by a hyperbolic saturation function of the Michaelis‐Menten type. Results are obtained for the expression of the tryptophan operon in Escherichia coli and their applicability to tryptophan production by microbial fermentation is discussed. It is shown that, depending on the strain level of the operon and the rate of utilization of tryptophan in the cell, an overproduction of tryptophan can be achieved under stable operating conditions; in other circumstances, the operon may become stable or unstable, and may lead to a periodic synthesis.
A liquid emulsion membrane process for the separation of amino acids
Authors: Itoh H. , Thien M. P. , Hatton T. A. , Wang D. I. C.
Published: December 2004

The method of using liquid emulsion membranes featuring the cation carrier D2EHPA [di‐(2‐ethylhexyl) phosphoric acid] for the separation of L‐phenylalanine is examined. Results from experiments performed under various conditions are discussed and an optimal condition for separation is determined. The selectivity of the liquid emulsion membrane system is discussed. The effects of impurities such as sodium chloride, glucose, lactic acid, and L‐tryptophan on the transport of L‐phenylalanine are evaluated. It is shown that the liquid emulsion membrane system is a potential operation not only to separate L‐phenylalanine but also concentrate it with great efficiency.
Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma
Authors: Ramírez O. T. , Sureshkumar G. K. , Mutharasan R.
Published: December 2004

A mouse–mouse hybridoma was grown in serum‐free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 × 106 cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 × 106 cells/ml, whereas it was only 0.2 × 106 cells/ml and 1.3 × 106 cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.
Batch and continuous cultivation of Pseudomonas fluorescens at increased pressure
Authors: Onken Ulfert
Published: December 2004

The effect of increased total pressure and partial pressures of oxygen and carbon dioxide on the growth of Pseudomonas fluorescens was investigated in an airlift reactor. In batch cultivations bacterial growth was completely inhibited with air at 8 bars total pressure. The same effect was observed with aeration by pure oxygen at 1.15 bars. Carbon dioxide partial pressure did not show inhibitory effects. Continuous experiments confirm the assumption that growth inhibition at higher total pressure is caused by the increase in oxygen partial pressure. Incubation of P. fluorescens at higher oxygen partial pressure led to an increase of bacterial productivity during subsequent continuous cultivation at ambient pressure (1 bar) with air. Maximum productivity was increased by about 75% after aeration with pure oxygen. This effect is probably the result of metabolic adaption of the bacterial cells to high oxygen partial pressure.
Ecoengineering high rate anaerobic digestion systems: Analysis of improved syntrophic biomethanation catalysts
Authors: Thiele Jurgen H. , Wu Wei‐Min , Jain Mahendra K. , Zeikus J. Gregory
Published: December 2004

High performance biomethanation granules with operational specific COD removal rates of 7 kg COD removed/kg SS/d were obtained by ecoengineering conventional, granular, UASB digester sludge using a designed protocol of starvation and selection on a defined volatile fatty acid (VFA) based mineral medium. Addition of low (0.15 mM) sulfate levels to this VFA medium increased the maximum shock‐load COD removal rate of the ecoengineered biomethanation granules to 9 kg COD/kg SS/d with specific acetate, propionate, and butyrate removal rates of 111, 28, and 64 mol/g SS/d. Addition of moderate (26 mM) calcium levels inhibited growth and altered the structure of granules. The general cellular, growth, stability, and performance features of these ecoengineered granules are described and discussed in relation to their use as improved biomethanation starter cultures.
Hydrodynamics and mass transfer in two‐phase aqueous extraction using spray columns
Authors: Sawant S. B. , Sikdar S. K. , Joshi J. B.
Published: December 2004

Spray columns can be used to isolate and purify proteins using the two‐phase aqueous extraction technique based on polyethylene glycol (PEG) and dextran. The fractional dispersed phase (PEG) holdup and overall mass transfer coefficients were measured in a 9.7 mm i.d. spray column. We found that the dispersed phase holdup increased with increasing PEG phase velocity. The overall mass transfer coefficients for bovine serum albumin, normalized for the PEG holdup, were found to be independent of the PEG phase velocity. This result was expected, since true mass transfer coefficients do not vary with phase velocity.
A bioenergetic model of a mixed production fermentation
Authors: Haughney H. A. , Nauman E. B.
Published: December 2004

A bioenergetic model has been developed for the fermentation of glucose by Bacillus polymyxa. This model uses energy balances to determine which pathways are utilized by the substrate. The model can predict substrate consumption, biomass formation, and the product distribution for this fermentation. The products are carbon dioxide, water, 2,3‐butanediol, and ethanol, where ethanol represents lumped anaerobic products.
Cell volume measurement as an estimation of mammalian cell biomass
Authors: Frame Kelly K. , Hu Wei‐Shou
Published: December 2004

Measurements of volume distributions and dry weight are made on hybridoma cells in culture. The volume of viable hybridoma cells is significantly larger than that of nonviable cells. During exponential growth, the volume of the viable hybridoma cells is found to be significantly larger than that during other stages of batch culture. Proportionality is found between the volume of the cells and their dry weight, indicating that the volume data can be used in conjunction with cell concentration data as a practical technique for indirect measurement of the biomass concentration present in a culture. Comparison of dry weight concentrations in continuous culture to predictions from the volume data shows very good agreement.
Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation
Authors: Park Chang‐Ho , Okos Martin R. , Wankat Phillip C.
Published: December 2004

Acetone–butanol–ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12‐ and 20‐in.‐long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18–6.43 to 4.50–4.90 in the first inch of the reactor. The pH dropped further to 4.36–4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.
Indirect electrochemical reduction of methemoglobin: Design of the process
Authors: Labrune P. , Bergel A. , Comtat M.
Published: December 2004

Methemoglobin can be reduced on a platinum cathode using flavin mononucleotide as an oxido‐reduction mediator. The process requires the utilization of a filter‐press cell with compartments separated by a semi‐permeable membrane. Analysis of the various constraints imposed by the process itself and by the nature of the molecules involved shows that the electrolysis cell must operate at a low temperature, in strictly anaerobic conditions, in series with a storage tank, and with fluid circulation rates lower than approximately 0.8 m/s. A process has been designed that takes into account these imperatives and enables volumes of solution of the order of 200 cm3 to be processed. It enables optimization of the flow rates used as well as of the methemoglobin/flavin ratio and is the forerunner of an industrial reactor.
High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor
Authors: Lee Youl Lark , Chang Ho Nam
Published: December 2004

A recombinant Escherichia coli HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle fermentor. The strain was very stable throughout the whole experiment. The main inhibitory by‐product was acetic acid, and cell growth ceased when its concentration was above 14 g/L Cell density could be increased up to 145 g/L dry weight without experiencing by‐product inhibition by regulating glucose concentration in the fermentor and by using total membrane recycle. Acetic acid formation was negligible not only when cells were cultured in medium containing no glucose but also when glucose was limited. Dissolved oxygen control as well as glucose limitation was an indispensable condition for minimizing acetic acid formation when the medium contained glucose. Low concentrations of accumulated acetic acid were reused when glucose was limited. Use of highly concentrated medium reduced the membrane surface area required for cell recycle greatly. The recycle fermentor could be operated in various operational modes including partial bleed and repeated recycle culture to give high productivity. Productivity of a repeated recycle system was over 10 times higher than that of a simple batch system.
Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: III. Hydrodynamic aspects
Authors: Vos Henk J. , van Houwelingen Cees , Zomerdijk Max , Luyben Karel Ch. A. M.
Published: December 2004

In Parts I and II of this series we described the modelling, design, and operation of a multistage fluidized bed reactor (MFBR) for immobilized biocatalysts. This article deals with those aspects of the MFBR which are different from single‐stage fluidized beds which are operated in batch mode with respect to the solids. The semicontinuous transport of the particles requires perfect mixing of the particles in the reactor compartments, because particles are mainly transported from the bottom of these compartments. A large spread in the physical properties of the biocatalyst particles, especially of both size and density, may cause the particles to segregate into layers with different diameter and/or density. This affects the efficient use of the biocatalyst. The properties of the particles are dependent on the immobilization method. The suitability of different methods for possible future application in the MFBR is therefore compared. Because of segregation, successful use of a biofilm catalyst with a nonuniform thickness of the biofilm is doubtful. Experiments in a small scale reactor (± 0.1 m diameter) demonstrated that perfect particle mixing is possible using commercially available biocatalyst particles of uniform density. Co‐immobilization of the biocatalyst with glass powder in a gel is a simple and effective method of increasing gel density. High density particles allow high liquid flow rates, and thus an improved external mass transfer can be achieved.

The distributor plates, which separate the reactor compartments, must allow unhindered transport of particles. Therefore, the holes in these plates must have a diameter of at least 4.5 times that of the largest particles which are present in the particle mixture used. Furthermore, the plates must be designed such that, when scaling‐up the reactor, a uniform liquid distribution over the cross‐sectional area of the reactor occurs. Large‐scale experiments were not carried out, but published correlations, indicate that particle mixing and a uniform liquid distribution can be accomplished in a large‐scale reactor under similar flow conditions.