bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilization of the serine protease from Thermomonospora fusca YX on porous glass
Authors: Gusek Todd W. , Tyn Myo T. , Kinsella John E.
Published: December 2004

As much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86°C) and pH optimum (9.4) for activity compared to soluble YX‐protease (80°C and pH 9.0, respectively). Immobilization improved enzyme thermo‐stability above 90°C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12°C). A continuous‐flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.
Diffusional limitations of immobilized Escherichia coli in hollow‐fiber reactors: Influence on 31P NMR spectroscopy
Authors: Briasco Catherine A. , Karel Steven F. , Robertson Channing R.
Published: December 2004

Escherichia coli cells were immobilized and grown in hollow‐fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using 35S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 °C were shown to be severe, with regions of starved cells occupying up to 80% of the cell‐containing region. Phosphorus‐31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume‐averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur‐35 autoradiographs of a reactor operated at 16°C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near‐normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.
Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors
Authors: Piret James M. , Cooney Charles L.
Published: December 2004

The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high‐molecular‐weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell‐side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 × 104 D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell‐side convective flow and its influence on the inoculum and high‐molecular‐weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.
The long‐term effects of ethanol on immobilized cell reactor performance using K. fragilis
Authors: Chen C. , Dale M. C. , Okos M. R.
Published: December 2004

The effects of ethanol on reactor performance were studied in a small, 5‐cm packed height, “differential” type immobilized cell reactor. Lactose utilizing yeast cells, Kluyveromyces fragilis, were absorbed to a porous adsorbant sponge matrix in a gas continuous reactor. Step changes in the feed ethanol concentration to the column (10–130 g/L) were used to test the reactor response over extended periods of time (about 30–50 h per dosage level) followed by a return to basal zero inlet ethanol feed. Effluent cell density and effluent cell viability were measured at intervals. An inhibitory response in ethanol productivity to feed dosage ethanol levels above 20 g/L was detected almost immediately, with a near steady state response noted within 2.5 h of initiating the dosage. Feed ethanol levels above 50 g/L resulted in a subsequent gradual decrease in reactor productivity over time, which was associated with a decrease in the fraction of viable shed cells in the reactor effluent. The reactor response to a step removal of the ethanol inhibition was also monitored. Quick and complete rebounding of the fermentation rate to the original basal rate was noted following dosage concentrations of under 50 g/L ethanol. Recovery rates slowed following ethanol dosage levels above 50 g/L. Viable shed cell density improved overtime during the slow recovery periods. Growth rates (as determined by shed cell density) were more strongly inhibited than productivity. Growth responded more slowly to changes in ethanol environment as growth rates at 30 h fell to about 40% of the rates measured 7.5 h after initiation of a dosage level. It is concluded that ethanol contributions to cell injury and death (and consequent ICR performance degradation) may be more important than ethanol inhibition of productivity rates in the long‐term operation of immobilized cell reactors at ethanol concentrations over 50 g/L.
Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: Hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture
Authors: Grosz Ron , Stephanopoulos Gregory
Published: December 2004

The growth and metabolism of Saccharomyces cerevisiae was studied in steady‐state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady‐state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady‐state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro‐aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.
On‐line prediction of fermentation variables using neural networks
Authors: Thibault Jules , Van Breusegem Vincent , Chéruy Arlette
Published: December 2004

This article presents an introduction to the use of neural network computational algorithms for the dynamic modeling of bioprocesses. The dynamic neural model is used for the prediction of key fermentation variables. This relatively hew method is compared with a more traditional prediction technique to judge its performance for prediction. Illustrative simulation results of a continuous stirred tank fermentor are used for this comparison. It is shown that neural network models are accurate with a certain degree of noise immunity. They offer the distinctive ability over more traditional methods to learn very naturally complex relationships without requiring the knowledge of the model structure.
Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers
Authors: Agerkvist Iréne , Enfors Sven‐Olof
Published: December 2004

The protein releases, the particle size distribution and the viscosity of disrupted E. coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M‐8TA and Microfluidizer M‐110 were determined. The effects of these parameters on separation of the cell debris from the protein solution by centrifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably different physical properties of the cell disintegrates which influences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disruption in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, increasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high‐pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disintegrate both in filtration and in centrifugation.
Effects of solvent molecular toxicity and microenvironment composition on the Δ1 dehydrogenataon activity of Arthrobacter simplex cells
Authors: Pinheiro H. M. , Cabral J. M. S.
Published: December 2004

Solvent selection tests were carried out for the Δ1 dehydrogenation of 6‐α‐methylhydrocortisone‐21‐acetate by Arthrobacter simplex cells in the presence of organic solvents. Solubility limits were determined for substrate and product in dry and water‐saturated solvents and solvent mixtures. Molecular toxicity levels were estimated by measuring the dehydrogenation activity decay of freely suspended cells incubated in solvent‐saturated aqueous media. Chloroform and n‐decan‐1‐ol were the best choice of solvent, for both solubility and catalytic stability. High concentrations of water‐soluble additives, such as monosodium glutamate, were found to greatly improve the retention of activity in chloroform‐saturated media.
Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta
Authors: Caldwell Steven R. , Raushel Frank M.
Published: December 2004

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 μmol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10‐unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1‐unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent Km for the immobilized enzyme was 3–4 times greater than the Km (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH–rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.
Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: Modeling studies of stationary and oscillatory states
Authors: Aon Miguel A. , Cortassa Sonia
Published: December 2004

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S1), in the presence of a second substrate, formate (S2), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S2 allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S1 which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S1) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (η) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S2/S1 ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of η was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high η predicting that higher biomass yields may be obtained through a variable, high, η by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.
Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum
Authors: Chun G.‐T. , Agathos S. N.
Published: December 2004

Process improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L‐valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L‐valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L‐valine during or after exponential growth. In contrast to the free cell cultures, the addition of L‐valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.
Recovery of copper and cobalt by biopolymer gels
Authors: Jang L. K. , Lopez S. L. , Eastman S. L. , Pryfogle P.
Published: December 2004

The recovery of copper from synthetic aqueous media circulating in a loop fluidized bed reactor operated batchwise was investigated by using the following biopolymer systems: (1) a viscous solution of sodium alginate (from kelp) dispensed directly into the reactor fluid containing dissolved copper (sulfate salt) at initial concentrations of 60–200 ppm, (2) partially coagulated calcium alginate spheres for absorbing dissolved copper at initial concentrations of 10–40 ppm, and (3) a mixture of green algae Microcystis and sodium alginate dispensed directly into the reactor fluid. The recovery of copper and cobalt, a strategic metal, from cobalt ore leachate was achieved by a two‐step approach: direct dispensing of sodium alginate to absorb the bulk of metals followed by the addition of partially coagulated calcium alginate spheres to “polish” the leachate. Metal binding capacity and conditional stability constant of each biopolymer system as well as the effective diffusivity of cupric ion in the matrix of biopolymer gels are reported.
Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid
Authors: Ryan Wen , Parulekar Satish J.
Published: December 2004

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid‐free cells were conducted in a well‐controlled bio‐reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid‐bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid‐free and plasmid‐bearing cells resulting from the extra metabolic burden on the plasmid‐bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi‐steady state representative of plasmid‐bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid‐free cells near the end of the quasi‐steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid‐bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid‐free cells. In continuous cultures, plasmid copy number during the quasi‐steady states increased with decreasing dilution rate from 50 (at 0.409 h−1) to 941 (at 0.233 h−1). Production of the plasmid‐encoded protein (β‐lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of β‐lactamase was observed at both low and high dilution rates.
A parametric study ot protease production in batch and fed‐batch cultures of Bacillus firmus
Authors: Moon Seung‐Hyeon , Parulekar Satish J.
Published: December 2004

Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen‐free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed‐batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.
The effect of an applied potential on the activity of carbonic anhydrase immobilized on graphite rods
Authors: Mclachlan K. L. , Crumbliss A. L.
Published: December 2004

The effect of both a positive and a negative applied potential on the p‐NPA hydrolysis activity of bovine carbonic anhydrase (BCA) immobilized on graphite rods has been investigated. Background experiments show that the pH‐activity profile for BCA free in solution is not affected by either a negative or a positive potential applied to graphite rods placed in the same solution. However, the activity of BCA immobilized by covalent attachment to a graphite rod is influenced by a potential externally applied to the graphite rod. An overall increase in activity (as determined by the initial rate of the p‐NPA hydrolysis reaction) is observed in the presence of a ‐0.2 V (Ag/AgCl) applied potential, while decreased activity is evident at +0.6 V (Ag/AgCl). This is indicative of an electrolyte anion effect rather than a local pH effect. In the presence of the specific anion inhibitors Cl− and SCN−, the relative BCA activity increases at ‐0.2 V (Ag/AgCl) and decreases at +0.6 V (Ag/AgCl) are consistent with the different BCA inhibition constants for Cl− and SCN−. Accelerated loss of immobilized BCA activity also accompanies the application of the external potentials, particularly at +0.6 V (Ag/AgCl). Results described here represent an early example of potentiostatic control of nonredox enzyme activity. Several possible mechanisms are discussed including specific anion inhibition, enzyme surface charge/charged support material interactions, and charged product inhibition. It is likely that a combination of such mechanisms is operational in this system. The implications of external potentials affecting the activity of immobilized enzymes in the design of stable immobilized enzyme electrodes are also discussed.
Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions
Authors: Ju Lu‐Kwang , Lee Jaw F. , Armiger William B.
Published: December 2004

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO2TM emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO2 emulsions with very fine emulsion particles (< 0.2 μm) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration‐based oxygen diffusion coefficients has also been discussed.
Fractal dimensions and porosities of Zoogloea ramigera and Saccharomyces cerevisae aggregates
Authors: Logan Bruce E. , Wilkinson Daniel B.
Published: December 2004

The fractal nature microbial aggregates is a function of the type of microorganism and mixing conditions used to develop aggregates. We determined fractal dimensions from length‐projected area (D2) and length‐number scaling (D3) relationships. Aggregates of Zoogloea ramigera developed in rotating test tubes were both surface and mass fractals, with fractal dimensions of D2 = 1.69 ± 0.11 and D3= 1.79 ± 0.28 (±standard deviation), respectively. When we grew this bacteria in a bench‐top fermentor, aggregates maintained their surface fractal characteristics (D2 = 1.78 ± 0.11) but lost their mass fractal characteristics (D3 = 2.99 ± 0.36). Yeast aggregates (Saccharomyces cerevisae) grown in rotating tests tubes had higher average fractal dimensions than bacterial aggregates grown under physically identical conditions, and were also considered fractal (D2 = 1.92 ± 0.08 and D3 = 2.66 ± 0.34). Aggregates porosity can be expressed in term of a fractal dimensions, but average porosities are higher than expected. The porosities of yeast aggregates (0.9250–0.9966) were similar to porosities of bacterial aggregates (0.9250‐0.9966) cultured under the same physical conditions, although bacterial aggregates developed in the reactor had higher average porosities (0.9857‐0.9980). These results suggest that that scaling relationships based on fractal geometry may be more useful than equations derived from Euclidean geometry for quantifying the effects of different fluid mechanical environments on aggregates morphology and characteristics such as density, porosity, and projected surface area.
Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch‐grown Saccharomyces cerevisiae
Authors: Larsson Christer , Blomberg Anders , Gustafson Lena
Published: December 2004

Energy balance calculations were performed for different physiological states during batch growth of Saccharomyces cerevisiae with glucose as carbon and energy source. For the different physiological states, energy recoveries close to one were obtained, which permitted a continuous control that the constantly changing growth process was quantified accurately. During the respiro‐fermantative phase of growth, during which glucose served as the carbon and energy source, a low‐heat‐yield value (ΔQx) of −8.6 kJ/g dry biomass formed was obtained. This low‐heat‐yield value was due to the mainly fermentative metabolism during the middle of this phase of growth. After a transition phase, the ethanol produced during the respiro‐fermentative growth was respired. During this respiratory phase, the heat yield values increased markedly, resulting in a lowest value of −42.7 kJ/g. The low‐heat‐yield values of the respiro‐fermentative growth is not a reflection of the most efficient metabolism of S. cerevisiae. On the contrary, during the middle of this phase, 74% of the energy input was dissipated as ethanol, 6% was dissipated as heat, and the energy conserved as biomass was just 13%, while during the early respiratory phase, 69% of the energy input was dissipated as heat, and 22% of the energy input was conserved as biomass. By mathematical modeling and direct monitoring on‐line of the rate of heat production, continuous calculations of (1) glucose consumption, and (3) biomass production were performed, and were shown to correlate closely with measured values for the continuously changing growth process.
Cybernetic modeling of iron‐limited growth and siderophore production
Authors: Alexander Matthew L. , Ramkrishna D.
Published: December 2004

The cybernetic modeling framework developed by Ramkrishna and co‐workers has been applied to a case of bacterial metabolite production, namely the production of siderophores (iron‐chelating agents) associated with iron‐limiting fermentation conditions. Experimental growth data showed that, even though final biomass levels were controlled by exhaustion of the carbon source, iron‐limiting conditions also affected the biomass yield. A structured model which includes the process of an iron‐limiting energy resource production was able to quantitatively account for this apparent dual‐substrate limitation over a wide range of batch and continuous operating conditions. The experiments data also showed quite large difference in iron uptake over the wide range of operating condition and iron levels investigated. The inclusion in the model of the processes of low and high (siderophore‐mediated) affinity iron transport, and siderophore production led to simulation results that were in good quantitative agreement with the siderophore, medium and cell iron levels, in both batch and steady‐state continuous culture operating conditions.
Production of monoclonal antibody using free‐suspended and immobilized hybridoma cells: Effect of serum
Authors: Lee Gyun Min , Varma Amit , Palsson Bernhard O.
Published: December 2004

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free‐suspended and immobilized S3H5/γ2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 ± 0.12 day−1 (±standard deviation), while that in medium containing 1% serum was approximately 0.73 ± 0.12 day−1. The specific MAb productivity was almost constant at 3.69 ± 0.57 μg/106 cells/day irrespective of serum concentration reached a maximum at ca. 1.8 × 106 cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free‐suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.
Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil
Authors: Malcata F. Xavier , Hill Charles G. , Amundson Clyde H.
Published: December 2004

A lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat‐sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35°C. For the reaction conditions used in this research, a pseudo‐zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis–Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell‐shaped (or Γ‐type) distribution. Data taken at five different pH values were fit assuming a Dixon–Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first‐order kinetics with a half‐life of 19.9 days at 35°C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped‐substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.
Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture
Authors: Robinson David K. , Memmert Klaus W.
Published: December 2004

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady‐state, continuous culture at dilution rates ranging from 0.21 to 1.04 day−1. The steady‐state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day−1. Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.
Expression of recombinant protein A from the lac promoter in Escherichia coli JM83 is not subject to catabolite repression when grown under specific conditions of continuous culture
Authors: Warnes Alan , Stephenson John R. , Fooks Anthony R. , Melling Jack , Brown Michael R. W.
Published: December 2004

Although widely used in experimental and industrial situations, genetically engineered plasmids containing the lac promoter from Escherichia coli are subject to catabolite repression when grown in glucose‐containing media. Several methods of overcoming this problem have been investigated by studying the expression of the protein A gene from Staphylococcus aureus under the control of the Escherichia coli lac promoter. When glycerol is used as a sole carbon source, the plasmid is unstable and is rapidly lost from the culture. When the bacteria are grown in chemostats under glucose limitation, the plasmid is maintained, even at high dilution rates, and the expression of protein A is similar to that observed when glycerol was used. The balance between metabolic load and protein A expression seems to be maintained by reducing the gene dose to a tolerable level. Depending on the metabolic conditions prevailing in the culture, this is achieved, either by reducing the copy number of the plasmid or in extreme cases by removing the plasmid altogether.
Observing protein synthesis, export, and tryptophan incorporation by front‐surface fluorescence
Authors: Lipton A. J. , Domach M. M.
Published: December 2004

Front‐surface detection of emission from fluorophores in the presence and absence of light‐scattering particles was contrasted to right‐angle and wave‐guide detection. We found that front‐surface detection was the least prone to the reabsorption, inner‐filtering, and scattering effects that can plague fluorescent measurements. Front‐surface detection was thus used to assess the use of protein and ANS fluorescence as a means of monitoring events in bacterial fermentations. Protein fluorescence appeared to track well changes in optical density during balanced growth. However, during the lag associated with diauxic growth and after exposure to ampicillin, protein fluorescence became decoupled from cellular growth in a manner consistent with prior observations and the known effect of ampicillin on cells. ANS proved to be nontoxic and capable of reporting the occurrence of protein release from cells. The spectral shifts of tryptophan indicated that the incorporation of tryptophan into cellular protein can be monitored.
Bioleaching of zinc sulfide concentrate by Thiobacillus ferrooxidans
Authors: Konishi Yasuhiro , Kubo Hideaki , Asai Satoru
Published: December 2004

The kinetics of the bioleaching of ZnS concentrate by Thiobacillus ferrooxidans was studied in a well‐mixed batch reactor. Experimental studies were made at 30°C and pH 2.2 on adsorption of the bacteria to the mineral, ferric iron leaching, and bacterial leaching. The adsorption rate of the bacteria was fairly rapid in comparison with the bioleaching rate, indicating that the bacterial adsorption is at equilibrium during the leaching process. The adsorption equilibrium data were correlated by the Langmuir isotherm, which is a useful means for predicting the number of bacteria adsorbed on the mineral surface. The rate of chemical leaching varied with the concentration of ferric iron, and the first‐order reaction rate constant was determined. Bioleaching in an iron‐containing medium was found to take place by both direct bacterial attack on the sulfide mineral and indirect attack via ferric iron. In this case, the ferric iron was formed from the reaction product (ferrous iron) through the biological oxidation reaction. To develop rate expressions for the kinetics of bacterial growth and zinc leaching, the two bacterial actions were considered. The key parameters appearing in the rate equations, the growth yield and specific growth rate of adsorbed bacteria, were evaluated by curve fitting using the experimental data. This kinetic model allowed us to predict the liquid‐phase concentrations of the leached zinc and free cells during the batch bioleaching process.
Degradation of microcrystalline cellulose: Synergism between different endoglucanases of Cellulomonas sp. ATCC 21399
Authors: Poulsen O. M. , Petersen L. W.
Published: December 2004

Three immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in “short fiber formation” when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.
Hydrolysis of penicillin G by combination of immobilized penicillin acylase and electrodialysis
Authors: Ishimura Fumihiro , Suga Ken‐Ichi
Published: December 2004

Phenylacetic acid, as inhibitory product, was formed from a hydrolysis of penicillin G by immobilized penicillin acylase. In this article, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance an efficiency of the reaction. When 268 and 537 mM of penicillin G solution were used as the substrate, the concentration of phenylacetic acid in the reaction mixture could be maintained at less than 81 and 126 mM, respectively, and eventually, 86% and 88% of phenylacetic acid produced were removed from the reaction mixture at the end of the hydrolysis, respectively. Times required to reach 96% and 94.8% conversion from 268 and 537 mM of initial penicillin G could be reduced to 65% and 64% respectively, by means of electrodialysis; while 3.0% and 4.3% of initial penicillin G of 268 and 537 mM were permeated out of the reaction chamber during the hydrolysis, respectively. However, a loss of penicillin G by permeation could be reduced from 4.3% to 3.4% by a repeated addition of penicillin G.
Biological method for protection of 6‐position of sucrose and its use in synthesis of disaccharide high‐intensity sweetener
Authors: Jones Joan D. , Hacking Andrew J. , Cheetham Peter S. J.
Published: December 2004

A general method for protecting the 6 primary hydroxyl position of sucrose is described. It involves the production of glucose‐6‐acetate by fermentation of glucose using a strain of Bacillus megaterium followed by conversion to sucrose‐6‐acetate as a kinetic product using a specially selected fructosyl transferase producted by a newly isolated strain of Bacillus subtilis. The sucrose‐6‐acetate was found to be more lipophilic than expected, and this property aided its purification by chromatography. Pure sucrose‐6‐acetate may then be chlorinated and subsequently deacetylated to give the high‐intensity sweetener 4,1′,6′‐trichlo‐4,1′,6′‐trideoxygalactosucrose (sucralose) in high yields. This process involves fewer steps than are required for chemical synthesis using trityl chloride and acetic anhydride. Related intensely sweet molecules which were synthesized by similar methods included 4,1′,6′‐trichloro, 4,1′,6′‐trideoxy L‐arabinosucrose, and 4,1′,6′‐trichloro‐4,6,1′,6′‐tetradeoxy‐galactosucrose. They were obtained from xylose and 6‐deoxyglucose, respectively, via the intermediates xylsucrose and 6‐deoxysucrose, formed by the reaction of the fructosyl transferase on the monosaccharide acceptors.
The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors
Authors: Wagner A. , Marc A. , Engasser J. M. , Einsele A.
Published: December 2004

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.
Organic solvents strip water off enzymes
Authors: Gorman Lu Ann S. , Dordick Jonathan S.
Published: December 2004

Exchange of enzyme‐bound H2O with T2O in aqueous solution followed by freeze drying provided tritiated water bound to chymotrypsin, subtilisin Carlsberg, and horseradish peroxidase. The desorption of T2O from these enzymes suspended in various organic solvents showed that all three enzymes lost enzyme‐bound water with peroxidase losing the most T2O of the three in solvents of moderate to high polarity. Polar solvent resulted in the highest degree of T2O desorption (e.g., methanol desorbed from 56%‐62% of the bound T2O), while nonpolar solvents resulted in the lowest degree of desorption (e.g., hexane desorbed from 0.4%–2% of the bound T2O). Desorption is nearly immediate with most of the desorbable T2O being released from the enzymes within the first 5 min. Both solvent dielectric and a measure of the saturated molar solubility of water in a given solvent provide accurate correlations between the properties of the organic solvents and the extent of T2O desorption. This investigation shows that water stripping from an enzyme into a nonaqueous medium does occur and can be significant in polar solvents.
Hydrolysis of lactose in skim milk by immobilized β‐galactosidase (bacillus circulans)
Authors: Bakken Andrew P. , Hill Charles G. , Amundson Clyde H.
Published: December 2004

A novel chemical reactor, consisting of β‐galactosidase from Bacillus circulans immobilized onto a ribbed membrane made from polyvinylchloride and silica, was used to hydrolyze the lactose constituent of skim milk. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of rate expressions based on a proposed enzymatic mechanism that includes the formation of oligosaccharides. High‐performance liquid chromatography (HPLC) methods were employed to monitor the concentrations of all species present in the effluent stream. For the experimental conditions used in this research, rate expressions which include the formation of trisaccharides, the inhibition effects of both the α and β anomers of galactose, and the corresponding mutarotation reaction are sufficient to model the reaction network.
Cell immobilization using PVA crosslinked with boric acid
Authors: Wu Kuo‐Ying Amanda , Wisecarver Keith D.
Published: December 2004

A new cell immobilization technique is described in which polyvinyl alcohol is crosslinked with boric acid, with the addition of a small amount of calcium alginate. The presence of the calcium alginate improves the surface properties of the beads, preventing agglomeration. A pure culture of phenol‐degrading Pseudomonas was immobilized in the PVA‐alginate beads. Phenol was successfully degraded in a fluidized bed of the beads, indicating that cell viability was maintained following the immobilization procedure. The PVA‐alginate beads proved to be very strong and durable, with no noticeable degradation of the beads after 2 weeks of continuous operation of the fluidized bed.
Control of a thiobacillus denitrificans bioreactor using machine vision
Authors: Camp Carl E. , Sublette Kerry L.
Published: December 2004

A PC‐based machine vision system has been used to continuously monitor changes in biomass concentration and to control the undesirable production of colloidal elemental sulfer (a reactor upset condition due to an excessive concentration of inhibitory sulfide substrate) in a bioreactor containing Thiobacillus denitrificans. A field of view of a video camera was established which contained regions of different background lighting. Mean values of the distribution of red, green, and blue intensity components within corresponding regions of a digital image image captured from the camera were used to monitr color changes associated with changes in biomass concentration, and to determine if the reactor was in an upset condition. The ration of red to blue intensity components was an important parameter in detecting the formatin of an elemental sulfur precipitant. Using a stepper motor‐driven pressure regulator, intelligent process control was performed by altering the hydrogen sulfide feed flow rate setpoint on the vision system measurements.