bluuebunny/crossref_metadata_2025 huggingface dataset:
A new biosensor for specific determination of glucose or fructose using an oxidoreductase of Zymomonas mobilis
Authors: Park Je‐Kyun , Kim Hak‐Sung
Published: December 2004

Cells of Zymomonas mobilis were permeabilized with toluene in order to utilize the enzymes, glucose–fructose oxidoreductase and gluconolactonase, inside the intact cells. Permeabilized cells were immobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on pH electrode. The biosensor developed was used for specific determination of glucose or fructose by detecting the production rate of hydrogen ion. Optimum conditions for biosensor response were pH 6.2 and temperature of 39 °C. The biosensor was highly specific and reproducible, and calibration curves for glucose and fructose were excellent, being linear up to 5 and 50 g/L, respectively.
A balanced DO‐stat and its application to the control of acetic acid excretion by recombinant Escherichia coli
Authors: Konstantinov Konstantin , Kishimoto Michimasa , Seki Tatsuji , Yoshida Toshiomi
Published: December 2004

During fed‐batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130–14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30–35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production — 8 g/L phenylalanine. A novel control strategy, called a balanced DO‐stat. was developed to prevent acetic acid excretion. It represents a model‐independent two‐loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO‐stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by‐products.
Foam fractionation of globular proteins
Authors: Brown Lynn , Narsimhan Ganesan , Wankat P. C.
Published: December 2004

Foam fractionation of bovine serum albumin (BSA) was studied as a model system for potato wastewater. The effects of feed concentration, superficial gas velocity, feed flow rate, bubble size, pH, and ionic strength on the enrichment and recovery of BSA were investigated in a single‐stage continuous foam fractionation column. Enrichments ranged from 1.5 to 6.0 and recoveries from 5 to 85%. The feed concentrations were varied from 0.01 to 0.2 wt %, and enrichments were found to increase with lower feed concentrations. Enrichments also increased with lower superficial gas velocities and larger bubble sizes. At sufficiently low feed flow rates, enrichment was found to increase with an increase in the flow rate, eventually becoming insensitive to the feed flow rate at higher values. The pH was varied from 3.5 to 7.0 and ionic strength from 0.001M to 0.2M. The effects of pH and ionic strength were found to be coupled with bubble size. A minimum bubble size was found at pH 4.8, the isoelectric point of BSA, resulting in a minimum in the enrichment. Bubble size, and thus enrichment, was found to increase as the ionic strength decreased from 0.2M to 0.01M. Previous models1,2 for the hydrodynamics of foam column were extended for a singlestage continuous foam fractionation column for the prediction of enrichment and recovery. The model assumed adsorption equilibrium, infinite surface viscosity, and bubbles of the same size. Though coalescence was formally accounted for in the model by considering bubble size as a function of foam height, calculations for the experimental runs were performed only for the case of no coalescence. Quantitative predictions of enrichment and recovery could not be made with a single representative bubble size because of the broad inlet bubble size distribution as well as broadening of the distribution as a result of coalescence. The experimental enrichments were higher and recoveries were lower than the model predictions, the discrepancy being more pronounced at lower feed concentrations because of increased coalescence. The higher enrichments are due to the predominant effect of internal reflux as a result of coalescence whereas the lower recoveries are a result of detrimental effects of broadening bubble size distributions.
Intramolecular esterification by lipase powder in microaqueous benzene: Factors affecting activity of pure enzyme
Authors: Yamane Tsuneo , Ichiryu Takayuki , Nagata Masahiro , Ueno Asayo , Shimizu Shoichi
Published: December 2004

Various factors affecting the catalytic activity of pure lipase of Pseudomonas fluorescens in microaqueous benzene were investigated with respect to lactonization of 15‐hydroxypentadecanoic acid. Without deposition of the enzyme or of the enzyme plus activity enhancer (additive) on celite powder, the pure enzyme was very poorly dispersed in the microaqueous benzene, resulting in very low activity. The enzyme immobilized on celite powder exhibited the highest activity at a free water content of ca. 0.083%. When a sugar alcohol such as erythritol, arabitol, or sorbitol was added before lyophilization with approximate proportion of 3 g/g enzyme, marked increases in the enzyme activity were observed at a shifted optimal free water content, i.e., 0.04%. Inclusion of phosphotidylcholine resulted in a somewhat higher activity than in the system of enzyme plus celite only. Addition of lactose, bovine serum albumin, casein, dextran, polyvinyl alcohol, phosphate, or NaCl all caused a decrease in the enzyme activity. From the effects of the additives examined, it is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water‐immiscible organic solvent: (1) optimum moisture content, (2) disperser (support particles having enough surface area on which the enzyme is thinly deposited), and (3) activity enhancer (additive) at optimum concentration The importance of noting the purity of the enzyme preparation is emphasized when its catalysis in an organic solvent is investigated.
Computer‐aided synthesis of biochemical pathways
Authors: Mavrovouniotis Michael L. , Stephanopoulos George , Stephanopoulos Gregory
Published: December 2004

The synthesis of biochemical pathways satisfying stoichiometric constraints is discussed. Stoichiometric constraints arise primarily from designating compounds as required or allowed reactants, and required or allowed products of the pathways; they also arise from similar restrictions on intermediate metabolites and bioreactions participating in the pathways. An algorithm for the complete and correct solution of the problem is presented; the algorithm satisfies each constraint by recursively transforming a base‐set of pathways. The algorithm is applied to the problem of lysine synthesis from glucose and ammonia. In addition to the established synthesis routes, the algorithm constructs several alternative pathways that bypass key enzymes, such as malate dehydrogenase and pyruvate dehydrogenase. Apart from the construction of pathways with desired characteristics, the systematic synthesis of pathways can also uncover fundamental constraints in a particular problem, by demonstrating that no pathways exist to meet certain sets of specifications. In the case of lysine, the algorithm shows that oxaloacetate is a necessary intermediate in all pathways leading to lysine from glucose, and that the yield of lysine over glucose cannot exceed 67% in the absence of enzymatic recovery of carbon dioxide.
Crossflow microfiltration of animal cells
Authors: Maiorella Brian , Dorin Glenn , Carion Antonio , Harano David
Published: December 2004

Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s−1 (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m2 h (depending on cell size) can be sustained during 20‐fold concentration from 2.5 × 106 cells/ml, while maintaining high cell viability.
Uptake of cadmium and zinc by the algaChlorella vulgaris: II. Multi‐ion situation
Authors: Ting Y. P. , Prince I.G. , Lawson F.
Published: December 2004

Many microorganisms are capable of sequestering and concentrating heavy metals from their aqueous environment. While much research has beep carried out on the uptake of single species of metal ions, little attention seems to have been given to the study of multimetal ion systems. A mathematical model has previously been developed to describe the uptake of individual metal species by a microorganism. The model proposes two sequential processes: an initial rapid uptake due to cellular surface adsorption and a subsequent slow uptake due to membrane transport of the metal into the cells. This article extends the treatment by considering the uptake of two metal species together, cadmium and zinc, under different experimental conditions. The results are discussed in terms of possible mechanistic interactions.
In situ recalibration of probes of dissolved gas concentrations in bioreactors without effect on metabolism and growth
Authors: Iversen J. J. Lønsmann
Published: December 2004

This article describes a general method for the in situ recalibration of a probe for dissolved gas concentrations without changing any parameter but the gas composition. Recalibration of an oxygen probe is used as an example.

The recalibration procedure can be used for any probe, provided the culture's rate of metabolism and growth is independent of the substrate concentration in the range being measured by the probe during recalibration. The method was tested using a galvanic oxygen electrode in an oxygen‐consuming culture of Klebsiella oxytoca and an oxygen‐evolving culture of Anabaena variabilis. The recalibration caused no detectable effect on the metabolism and growth of the cultures, provided the dissolved oxygen tension was above 3μM.
Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities
Authors: Laluce Cecilia , Palmieri Mauricio Cesar , Lopes da Cruz Rose Cristina
Published: December 2004

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38–40°C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35°C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35–38°C.
Fermentation of lactose to ethanol with recombinant yeast in an immobilized yeast membrane bioreactor
Authors: Jeong Yong S. , Vieth W. R. , Matsuura T.
Published: December 2004

A novel concept of membrane bioreactor in which living cells are sandwiched between ultrafiltration (UF) and reverse osmosis (RO) membranes was applied for lactose fermentation to ethanol by genetically engineered yeast cells. The productivity of the Lactophile 13B strains was higher than that of the Lactophile 13D strains. In both cases performance data similar to those for glucose fermentation to ethanol by Saccharomyces cerevisiae were obtained. However, the operational stability of recombinant yeast cells was improved in the new bioreactor in comparison to the stability of these cells in a shake flask.
A study of the energetics and economics of microalgal mass culture with the marine chlorophyte Tetraselmis suecica: Implications for use of power plant stack gases
Authors: Laws E.A. , Berning J.L.
Published: December 2004

The marine phytoplankter Tetraselmis suecica was grown in shallow outdoor flumes for a period of approximately 6 months at the Natural Energy Laboratory of Hawaii. In full sunlight, gross production rates were 15–20 g C m−2 d−1. The corresponding photosynthetic efficiencies (PE's) were 9–10%. Respiration losses removed about half the gross production. The CO2 utilization efficiencies of 96 ± 11% were achieved by bubbling CO2 into the culture with the use of a counterflow sump system. Adding the CO2 in the form of carbonated water resulted in utilization efficiencies of 81 ± 11%. Archimedes screws proved superior to both paddle wheels and propellers as a means of circulating the water in the flumes. Insertion of foil arrays into the flumes to effect systematic mixing of the culture significantly enhanced production. The enhancement was greater when the foils were oriented at a small angle relative to the horizontal than when they were oriented at the same angle relative to the vertical. Light modulation effects are implicated as the probable cause of most of the enhancement. Substitution of electric power plant stack gases for pure CO2 resulted in no significant change in the production of T. suecica grown in chemostat culture.
Microfluorimetric analysis of spatial and temporal patterns of immobilized cell growth
Authors: Kuhn Robert H. , Peretti Steven W. , Ollis David F.
Published: December 2004

Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high‐resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse‐labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass‐transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr‐alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.
Industrial scale harvest of proteins from mammalian cell culture by tangential flow filtration
Authors: van Reis Robert , Leonard Lee C. , Hsu Chung C. , Builder Stuart E.
Published: December 2004

An industrial‐scale methods for harvest of biologically active proteins form mammalian cell culture has been developed using tangential flow filtration. A robust and economical process capable of processing approximately 5000 L conditioned media/h with protein yields in excess of 99% has been achieved. A completely contained system has been designed in which total cell number and viability are maintained throughout the process. The process has successfully been implemented at 1.25 × 104 L scale for the recovery of kilogram quantities of pharmaceutical proteins such as recombinant tissue type plasminogen activator (rt‐PA).
Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase
Authors: Park Sunghoon , Hanna Leslie , Taylor Robert T. , Droege Michael W.
Published: December 2004

Methanotrophs have promising applications in bioremediation and in the production of fuel‐related chemicals due to their nonspecific enzyme, methane monooxygenase (MMO). The optimal conditions for cell growth and production of the soluble from of MMO (sMMO) were determined from batch cultivations of an obligatory methanotrophs, Methylosinus trichosporium OB3b, in shake flasks and a 5‐L bioreactor. It was confirmed that a copper deficiency is essential for the formation of the cytoplasmic sMNO. Optimum cell growth without added copper was observed at pH 6.0–7.0, temperature of 30–34°C, and phosphate concentration of 10–40 mM. In the bioreactor experiments, external CO2 addition eliminated the long lag period observed in the absence of added CuSO4, i.e., prior to the exponential cell growth phase. When methane was continuously supplied, the profile of the cell growth showed two different phases depending on the availability of nitrate, an initial fast exponential growth phase (specific growth rate, μ = 0.08 h−1) and a later slow growth phase (μ = 0.008 h−1). The cell density at the transition from a fast to a slow growth rate was proportional to the initial medium nitrate concentration in the range 5–20 mM and cell yield was estimated to be 7.14 g dry cell wt/g N. Whole‐cell sMNO activity remained essentially constant regardless of the growth rate unit cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMNO activity was observed. The lower sMNO activity could be restored by supplying additional iron to the bioreactor culture. Cell yield on iron was estimated to be 1.3 × 103 g dry cell wt/g Fe.
Proliferation of anchorage‐dependent contact‐inhibited cells: I. Development of theoretical models based on cellular automata
Authors: Zygourakis K. , Bizios R. , Markenscoff P.
Published: December 2004

We report the development of new class of discrete models that can accurately describe the contact‐inhibited proliferation of anchorage‐dependent cells. The models are based on cellular automata, and they quantitatively account for contact inhibition phenomena occurring during all stages of the proliferation process: (a) the initial stage of “exponential” growth of cells without contact inhibition; (b) the second stage where cell colonies form and grow with few colony mergings; and (c) the final stage where proliferation rates are dominated by colony merging events. Model prediction are presented and analyzed to study the complicated dynamics of large cell populations and determine how the initial spatial cell distribution, the seeding density, and the geometry of the growth surface affect the observed proliferation rates. Finally, we present a model variant that can simulate contact‐inhibited proliferation of asynchronous cell populations with arbitrary cell cycle–time distribution. The latter model can also compute the percentage of cells that are in a specific phase of their division cycle at a given time.
Proliferation of anchorage‐dependent contact‐inhibited cells. II: Experimental results and validation of the theoretical models
Authors: Zygourakis K. , Markenscoff P. , Bizios R.
Published: December 2004

This study establishes that the cellular automata models developed in an earlier article capture the essential features of the proliferation process for anchorage‐dependent contact‐inhibited cells. Model predictions are in excellent agreement with experimental data obtained with bovine pulmonary artery endothelial cells. The models are particularly suitable for predictive purposes since they have no adjustable parameters. All model parameters can be easily obtained from a priori measurements. Our studies also show that proliferation rates are very sensitive to the spatial distributions of seed cells. The adverse effects of seeding heterogeneities become more pronounced as a cell population approaches confluency and they should be accounted for in experimental studies attempting to assess the response of cells to external stimuli.
Production of L‐phenylacetyl carbinol by biotransformation: Product and by‐product formation and activities of the key enzymes in wild‐type and ADH isoenzyme mutants of Saccharomyces cerevisiae
Authors: Nikolova P. , Ward O. P.
Published: December 2004

The capacities of yeast wild‐type and mutants strains known to lack specific ADH isoenzymes to produce L‐phenylacetyl carbinol (PAC) and benzyl alcohol in biotransformation trials were also investigated. Pyruvate decarboxylase activity, responsible for PAC formation and ADH activity, which can participate in reduction of benzaldehyde to benzyl alcohol, was also determined in each strain. In addition, the capacity of each strain to produce ethanol was investigated. Mutant strains lacking all of the isoenzymes, ADH‐I, ADH‐II, and ADH‐III, still exhibited some ADH activity and were capable of production of benzyl alcohol and ethanol.
Growth and stoichiometry of a Catharanthus roseus cell suspension culture grown under nitrogen‐limiting conditions
Authors: Rho Denis , André Gérald
Published: December 2004

The uptake of carbohydrate and nitrate by Catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. Biomass production was similar when using either 6, 12, 18, or 24 mM nitrate as the nitrogen source and 20 g L−1 sucrose as the carbon source. In all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. Apparent biomass yields, YX/S and YX/N were 0.49 g biomass g−1 glucose equivalent and 0.23 g biomass mmol−1 nitrate, respectively. The determination of the cellular carbon‐to‐nitrogen ration (C/N ration) resulted in the identification of three district growth phases: an active growth phase, and accumulation phase, and a biomass decline phase (endogenous metabolism). The onset of the last two phases was correlated with nitrate and sugar of the last two phases was correlated with nitrate and sugar exhaustion, respectively. Balanced stoichiometric equations describing the active growth and accumulation phases were proposed based on elemental composition and ash content of the biomass. The stoichiometric equation related to the accumulation phase predicts that the available sugars are stored as starch‐ and lipid‐like materials.
On the thermodynamics of microbial growth processes
Authors: Sandler Stanley I. , Orbey Hassan
Published: December 2004

In this article, we provide a rigorous thermodynamic analysis of microbial growth process, clarify the role of the generalized degree of reduction concept as it is used in both stoichiometric equations and as a characterizing factor for thermophysical properties, and introduce a classification method to account for errors when using the generalized degree of reduction to estimate the energy and free energy contents of molecules. We maintain the advantages of using the generalized degree of reduction while correcting for the large errors in the principle of energy regularity, especially for small molecules and for nitrogen‐source compounds. As a result, we obtain more accurate energy balances (heat loads) and second law constraints, and are able to clarify contradictory statements in the literature as to whether nonphotosynthesis fermentation process can produce oxygen or absorb rather than produce heat. Indeed, the answers to such questions become evident using the classification system introduced here.
Kinetics of enzymatic hydrolysis of olive oil in biphasic organic‐aqueous systems
Authors: Tsai Shau‐Wei , Wu Guang‐Huei , Chiang Chen‐Li
Published: December 2004

A theoretical model for the lipase‐catalyzed hydrolysis of high‐concentration olive in biphasic isooctane‐aqueous systems has been proposed and confirmed by experiments. The enzymatic reaction of the Michaelis‐Menten type that occurred at the interface between organic and aqueous phases was assumed in deriving the rate equations, from which the maximum reaction rate could be obtained by carefully adjusting the volume ratio between the two phases to the optimal value. Equilibrium conversions higher than 98% for 0.1 g/L olive oil in isooctane were attained for systems with volume ratios up to one. Fractions of lipase and lipase‐substrate complex adsorbed at the interface increased and seemed to approach to asymptotic values asthe rotation speed of impeller increased. Activity of the lipase showed no apparent change between 26 and 37°C, but decrease rapidly with temperature above 43°C. The methodology presented in this work might be used to find kinetic parameters for reactor design and scaleup.
Optimization of a two‐stage recombinant fermentation process: The dilution rate effect
Authors: Hortacsu Amable , Ryu Dewey D. Y.
Published: December 2004

The effects of dilution rates on the performance of a two‐stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growht rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time‐dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over‐all productivity is 0.40 h−1. This coincides with the optimal specific growht rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, μ2(t) = 0.0007t, in the course of the fermentation time.
Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and β‐xylosidase while maintaining low protease production
Authors: Smith David C. , Wood Thomas M.
Published: December 2004

A growth medium was developed for maximal production in batch culture of extracellular xylanase and β‐xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild‐type strain. The optimum pH for the production of xylanase and β‐xylosidase was 4.0. The best temperature of xylanase production was 30°C; 35°C was optimal for β‐xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5‐fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL−1) but β‐xylosidase titre was increased 4.7‐fold to 1.5 U mL−1. When corn steep liquor was used as the sole nitrogen source, xylanse and β‐xylosidase titres were further increased by 1.5‐ and 1.9‐fold, respectively. Of the carbon sources investigated, ball‐milled oat straw or oat spelt xylan produced the highest titres of xylanse and β‐xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or β‐xylosidase induced by either oat straw for xylanse and β‐xylosidase was 2% and the optimum spore inoculum was between 106 and 107 spores/mL−1 final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL−1 when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.
Application of photon correlation spectroscopy as a technique for detecting culture contamination
Authors: Jepras R. I. , Perkins E. A. , Rarity J. , Carr R. J. G. , Clarke D. J. , Atkinson T.
Published: December 2004

The application of photon correlation spectroscopy (PCS) to detect culture contamination in chemostats was studied. It was found that the presence of a given particle size in a population of particles of a different size could be detected, but this ability was strongly dependent on particles of a different size could be detected, but this ability was strongly dependent on particle size difference and was most sensitive when contaminants are larger than the host. The inherent polydisparity of actively growing and dividing microbial cells negates any advantage in the use of multi‐angle PCS to detect contaminants.
Utility of culture redox potential for identifying metabolic state changes in Amino acid fermentation
Authors: Kwong Simon C. W. , Rao Govind
Published: December 2004

We investigated the relationship of dissolved oxygen and culture redox potential (CRP) on amino acid production. Corynebacterium glutamicum ATCC 14296 was used for all experiments. The fermentation can be divided into a growth phase and a production phase. Our results indicate that in order to get higher amino acid production, a lower oxygen supply during the exponential phase is favored. A higher oxygen supply rate appears to be necessary during the production phase. Culture redox potential (CRP) was used to monitor the fermentation. CRP readings were observed to drop to a characteristic minimum value as the metabolic state changed from a growth to production phase. This was evidenced by the commencement of amino acid production and a simultaneous uptake of lactate. Upon lactate exhaustion, the CRP increased abruptly. At the same time, maximal amino acid yields were observed. By the use of minimum CRP as an indication of metabolic phase changes, the agitation rate was changed to increase oxygen supply during the production phase. This significantly increased amino acid production. These results show that culture redox potential measurements can be used to monitor and optimize amino acid production by process manipulation.
Recovery of L‐lysine from dilute water solutions by liquid pertraction
Authors: Boyadzhiev L. , Atanassova I.
Published: December 2004

Kinetics of liquid membrane (Pertraction) recovery of L‐lysine from dilute aqueous solutions is studied in a tow‐compartment glass cell. A 5% (vol) solution of the cation exchange carrier di(2‐ethylhexyl)phosphoric acid in n‐decane was used as intermediate, membrane liquid. The third stripping phase was 1/v hydrochloric acid. The reaction mechanism and stoichiometry were defined, and on the basis of the proposed mathematical model of the process and the experimental data obtained, the mass transfer coefficients were evaluated. It was found that overall transfer rate is controlled by the eddy diffusion of transported species in the donor and membrane liquids. The results proved the feasibility of the pertraction process for recovery and concentration of L‐lysine from its dilute aqueous solutions.
Large‐Scale, High‐density freezing of hybridomas and its application to high‐density culture
Authors: Ninomiya Nobutaka , Shirahata Sanetaka , Murakami Hiroki , Sugahara Takuya
Published: December 2004

Large‐scale, high‐density freezing of hybridomas was studied to apply frozen cells to start high‐density culture. We showed here that hybridomas can be frozen at 1.5 × 108 cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large‐scale freezing to store 25 mL of cell suspension with a cell density, 1.5 × 108/mL. The number of cells stored in a bag (3.0 × 109 cells) was enough to start a high‐density culture at a 10 times higher cell density (6.0 × 106 cells/mL) than normal inoculation, and the cells proliferated to 107 cells/mL within 2 days. These results indicate that the large‐scale freezing method is useful for large‐scale culture of mammalian cells.
Effect of immiscible organic solvents on activity/stability of native chymotrypsin and immobilized‐stabilized derivatives
Authors: Blanco Rosa M. , Halling Peter J. , Bastida Agatha , Cuesta Carmen , Guisáun Jose M.
Published: December 2004

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one‐point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the “exact composition of the aqueous phase.”

High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin‐agarose derivatives are much more stable against these water‐immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized‐stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.
Enzyme‐Catalyzed oxidation of cholesterol in physically characterized water‐in‐oil microemulsions
Authors: Hedström Gun , Slotte J. Peter , Molander Ove , Rosenholm Jarl B.
Published: December 2004

The enzymatic conversion of cholesterol to cholestenone by cholesterol oxidase (Brevibacterium sp.)in reversed micelles in a system composed of AOT/isooctane/water/cholesterol has been examined. The catalytic activity of the enzyme was correlated with the physicochemical properties of water in water‐in‐oil (w/o) microemulsion systems. In a system consisting of 3 wt % AOT in isooctane, reversed micelles started to form as the [H2O]/[AOT] (e.g., the w0) ratio increased above 4–5. The formation of reversed micelles with a core of neat (bulk) water was verified from determinations of both the partial molar volume of water and the scissors vibration of water [with Fourier transform infrared (FTIR) spectroscopy] in the w/o microemulsion systems. A plot of enzyme activity vs. w0 indicated that the hydration of enzyme molecules per se was not sufficient to give rise to catalytic activity. Instead, it appeared that the formation of an aqueous micellar core was necessary for full activation of the enzyme. Based on micelle size distribution analysis, it was estimated that about one micelle per one thousand contained an enzyme molecule. Since the apparent reaction rate could be markedly enhanced by increasing the enzyme/water ratio, we conclude that the number of enzyme‐containing micelles was an important rate‐limiting factor in the system.
Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions
Authors: Arcidiacono S. , Kaplan D. L.
Published: December 2004

The isolation of chitosan from a fungal source offers the potential of a product with controlled physicochemical properties not obtainable by the commercial chemical conversion of crustacean chitin. A variety of culture and processing protocols using Mucor rouxii were studied for their effects on biomass yield and chitosan molecular weight. Weight‐averaged molecular weight determined by gel permeation chromotography ranged from 2.0 × 105 to approximately 1.4 × 106 daltons. The chitosan yield ranged from 5% to 10% of total biomass dry weight and from 30% to 40% of the cell wall. Of the culture parameters studied, length of incubation and medium composition effected biomass production and molecular weight. Modification of the processing protocol, including the type and strength of acid, and cell wall disruption in acid prior to refluxing were used to optimize the efficiency of chitosan extraction.

The degree of deacetylation of fungal and commercial chitosans was compared using infrared spectrometry, titration, and first derivative of UV absorbance spectrometry. The chitosan obtained directly from the fungal cell wall had a higher degree of deacetylation than commercial chitosan from the chemical conversion process.
Development of the optimal inoculation conditions for microcarrier cultures
Authors: Forestell Sean P. , Kalogerakis Nicolas , Behie L. A. , Gerson Donald F.
Published: December 2004

The environmental conditions under which anchorage‐dependent mammalian cells are grown are not necessarily those under which a culture should be initiated. Cell attachment is a physical process, and those factors which affect forces involved in cell attachment differ from the biological factors which affect cell growth. We have conducted an extensive experimental study to define clearly the optimal environmental conditions for MRC‐5 cell attachment onto microcarriers. These inoculation conditions are particularly important when the serial propagation of mammalian cells on microcarriers is considered as in a human vaccine production process. The conditions which were investigated are: initial serum content (% v/v), initial pH, inoculation level (cells/bead), agitation rate (rpm), and the concentration of microcarriers (g/L). The initial distribution of attached cells was found to have a significant affect on the overall efficiency of anchorage‐dependent cell cultures, and was used to evaluate attachment efficiency. Based on the experimental results, we propose an optimized protocol for the inoculation of microcarrier cultures.
Population balance between producing and nonproducing hybridoma clones is very sensitive to serum level, state of inoculum, and medium composition
Authors: Chuck Alice S. , Palsson Bernhard O.
Published: December 2004

Secreting and nonsecreting hybridoma populations derived from the murine hybridoma cell line 167.4G5.3 were each grown in batch culture in low serum and serum‐free media. Under serum‐free conditions, a secreting population gained on a predominantly nonsecreting population and competed with the existing antibody‐deficient cells effectively. It was found that this competition was sensitive to state of inoculum and medium composition. We conclude that the competition between a secreting and nonsecreting, or more generally, a producing and nonproducing, population is important; the appearance of the latter may not be a significant setback in terms of expected product titer.
Monoclonal antibody production in dialyzed continuous suspension culture
Authors: Linardos Theodor I. , Kalogerakis Nicolas , Behie Leo A. , Lamontagne Louis R.
Published: December 2004

Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5‐L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d−1. Dailysis tubing with a molecular weight cut‐off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start‐up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.
Development of a helical‐ribbon impeller bioreactor for high‐density plant cell suspension culture
Authors: Jolicoeur M. , Chavarie C. , Carreau P. J. , Archambault J.
Published: December 2004

A double helical‐ribbon impeller (HRI) bioreactor with a 11‐L working volume was developed to grow high‐density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear‐thinning for concentrations higher than 12 to 15 g DW · L−1. A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW · L−1, in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (kLa ∼4 to 45 h−1) from agar suspensions of 30 g DW · L−1 to water and for mixing speeds ranging from 120 to 150 rpm. These high surface kIa values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High‐density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (≥10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate (∼0.4 d−1) and attained the same high biomass concentrations (∼25 to 27 g DW · L−1, 450 to 500 g filtered wet biomass · L−1, and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale‐up potential of this bioreactor configuration is discussed.
Metabolic characterization of a L‐lysine‐producing strain by continuous culture
Authors: Kiss Robert D. , Stephanopoulos Gregory
Published: December 2004

Continuous culture experiments with the L‐producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L‐lysine exhibited a maximum with respect to specific growth rate, with an initial growth‐associated behavior up to specific growth rates of about 0.1 h−1, and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h−1. The productivity dropped at specific growth rates larger than about 0.2 h−1. The yield of L−lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L‐lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L‐lysine with an intrinsic yield, or efficiency, of about 0.41 mol L‐lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L‐lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L‐lysine fermentations.
Acetic acid formation in escherichia coli fermentation
Authors: Han Keehyun , Lim Henry C. , Hong Juan
Published: December 2004

Acetic acid formation in Escherichia coli fermentation has been studied in continuous cultures. Experimental results suggest that the limited capacity of the oxidative metabolism (perhaps the limited capacity of TCA cycle) may be responsible for acetic acid formation. At low growth rates, both anabolic and catabolic requirements may be satisfied by the oxidative metabolism. However, at high growth rates these two demands may exceed the capacity of the oxidative metabolism alone. It is proposed that under these circumstances, E. coli reorganizes the oxidative metabolism to first meet the anabolic requisition and then supply the necessary amount of energy using both the remaining capacity of the oxidative metabolism and acetic acid formation metabolism. Escherichia coli selects acetic acid synthesis as the aerobic energy source because it generates the second largest amount of ATP and NADH2. According to our proposition, acetic acid formation could be reduced by decreasing the anabolic requirement, i.e., reducing glucose uptake, or by increasing the capacity of the oxidative metabolism. These two approaches were experimentally confirmed by observing reduced acetic acid formation by reducing the glucose uptake with a yeast extract addition and enhancing the capacity of oxidative metabolism with a methionine addition.
Growth of the obligate methylotroph Methylobacillus flagellatum under stationary and nonstationary conditions during continuous cultivation
Authors: Baev M. V. , Schklyar N. L. , Chistoserdova L. V. , Chistoserdov A. Y. , Polanuer B. M. , Tsygankov Y. D. , Sterkin V. E.
Published: December 2004

The growth characteristics of a chemostat culture of the obligate methylotrophic bacterium Methylobacillus flagellatum have been determined. Steady‐state cultures growing at a rate of 0.73–0.74 h−1, equal to the maximal growth rate, were obtained under oxyturbidostat cultivation conditions. The response of a chemostat culture to a pulse increase of methanol concentration was studied. It was shown that slow and rapidly growing cultures of M. flagellatum responded differently to pulse methanol addition. The growth characteristics of slow‐growing cultures decreased after methanol addition compared to those of stationary chemostat cultures. The growth characteristics of rapidly growing cultures were practically unchanged with and without pulse methanol addition.