bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioconversion of wheat straw to ethanol: Chemical modification, enzymatic hydrolysis, and fermentation
Authors: Detroy R. W. , Lindenfelser L. A. , Sommer S. , Orton W. L.
Published: December 2004

Native wheat straw (WS) was pretreated with various concentrations of H2SO4 and NaOH followed by secondary treatments with ethylene diamine (EDA) and NH4OH prior to enzymatic saccharification. Conversion of the cellulosic component to sugar varied with the chemical modification steps. Treatment solely with alkali yield 51–75% conversion, depending on temperature. Acid treatment at elevated tempeatures showed a substantial decrease in the hemicellulose component, whereas EDA‐treated WS (acid pretreated) showed a 69–75% decrease in the lignin component. Acid‐pretreated EDA‐treated straw yielded a 98% conversion rate, followed by 83% for alkali–NH4OH treated straws. In other experiments, WS was pretreated with varying concentration of H2SO4 or NaOh followed by NH4OH treatment prior to enzymatic hydrolysis. Pretreatment of straw with 2% NaOH for 4 h coupled to enzymatic hydrolysis yield a 76% conversion of the cellulosic component. Acid–base combination pretreatment yielded only 43% conversions. A reactor column was subsequently used to measure modification–saccharification–fermentation for wheat straw conversion on a larger scale. Thirty percent conversions of wheat straw cellulosics to sugar were observed with subsequent fermentation to alcohol. The crude cellulase preparation yielded considerable quantities of xylose in addition to the glucose. Saccharified materials were fermented directly with actively proliferating proliferating yeast cells without concentration of the sugars.
Instabilities of product formation in a fed‐batch culture of Penicillium chrysogenum
Authors: Hegewald E. , Wolleschensky B. , Guthke R. , Neubert M. , Knorre W. A.
Published: December 2004

By investigation of a model of benzylpenicillin fermentation it was demonstrated that high fermentation productivity is connected with low stability. The increasing instability is not the result of external disturbances but of peculiarities inherent to the system. The main process variables and the essential parameters were selected by analysis of data obtained from fermentation runs in a pilot plant. By simulation on an analog computer it was shown that the proposed model gives a sufficient description of the kinetic behavior of the fermentation runs. By varying the initial conditions it was possible to obtain two types of fermentation runs. One type is distinguished by a high productivity connected with low initial concentrations of sucrose. An increase of the initial sucrose concentration also causes an increase of productivity. When a certain value of this concentration is exceeded a changeover to a faster growth of biomass takes place combined with the breakdown of productivity. The same phenomenon occurs at a defined initial concentration of sucrose if a disturbance of oxygen transfer rate takes place. Whether the culture stays in the one or in the other state after the disturbance has passed depends on the duration, the strength, and the beginning of the disturbance. In the pilot plant similar behavior can be observed. The sensitivity of fermentation processes to disturbances increases with the increase of productivity.
Hydrolysis of triglyceride by solid phase lipolytic enzymes of Rhizopus arrhizus in continuous reactor systems
Authors: Bell George , Todd John R. , Blain John A. , Patterson John D. E. , Shaw Charles E. L.
Published: December 2004

Continuous hydrolysis of triglyceride in organic solvent systems using Rhizopus arrhizus mycelia as a source of insolubilized lipase has been studied in packed‐bed and stirred‐tank reactors. Typically a packed bed reactor containing 1 g of mycelia fed at 1 mL/min with a solution of 2.5% (w/v) olive oil in di‐isopropyl ether gave a fatty acid yield of 45% at 30°C. The optimum water concentration was found to be 0.17% (w/v) except under conditions of high oil feed concentration and high yield where no optimum was established. No temperature optimum was observed over the range 20–55°C. Calculated activation energies of 13–20 kJ/mol, depending on temperature, were lower, while Km(app) values of 0.1–0.3M were higher than those for hydrolysis in conventional aqueous emulsion systems. No evidence of any significant diffusional limitation, which could account for these values, was obtained. The mycelia showed a loss of activity of 0.6–1.0%h at 30°C. The packed bed proved markedly superior to the stirred tank for this system.
Fed‐batch culture: Modeling and application in the study of microbiol energetics
Authors: Esener A. A. , Roels J. A. , Kossen N. W. F.
Published: December 2004

Microbial growth in the fed‐batch mode is described by a simple unstructured model. The model is found to be in good agreement with agreement with the experimental observation, except under highly transient conditions. Extensive experimental data were collected and the energetics of the bacterium Klebsiella pneumoniae is evaluated. It is shown that the fed‐batch culti vation is a powerful experimental tool in the study of microbial kinetics and energetics simultaneously. Methods for determining the maintenance requirements are shown and evaluated. The maintenance coefficients determined from fed‐batch data are systematically smaller than those reported for continuous culture systems. Results suggest a decrease in maintenance demands at low specific growth rates.
Pulse response in an immobilized‐enzyme column: Elution profiles in reversible and consecutive reactions
Authors: Adachi Shuji , Hashimoto Kenji , Miyai Kiyosato , Kurome Hirokazu , Matsuno Ryuichi , Kamikubo Tadashi
Published: December 2004

A pulse of substrate solution was applied to an immobilized‐enzyme column, in which the substrate was then converted by reversible or consecutive reactions. Immobilized glucose isomerase was used for the reversible reaction, and immobilized invertase and glucose oxidase for the consecutive reactions. The elution profiles of substrate and product were determined experimentally. These profiles were in good agreement with the ones predicted theoretically. The effect of some parameters on the elution profiles for reversible and consecutive reactions is discussed.
Purification of municipal wastewater by subsequent reverse osmosis and anaerobic digestion
Authors: Van Den Heuvel J. C. , Zoetemeyer R. J. , Boelhouwer C.
Published: December 2004

Experiments are described in which municipal wastewater is submitted to reverse osmosis. During a test period of two months a tubular apparatus, operated batchwise, maintained its original flux and rejection, the permeate being of good quality as a raw material for drinking water. The wastewater was concentrated 7.7 times. Continuous anaerobic digestion of the brine removed over 90% of the BOD evolving the expected amount of methane. The overall process is considered in its context of environmental and energetic aspects and is compared with a conventional treatment. This reveals clear perspectives for practical application in urban areas.
Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized‐enzyme complex for solubilizing vegetable protein in a recycle reactor
Authors: Adu‐Amankwa B. , Constantinides A. , Vieth W. R.
Published: December 2004

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.
Production of anchorage‐dependent cells—problems and their possible solutions
Authors: Jensen Mona D.
Published: December 2004

In vitro cell culture has proven to be a valuable procedure for the study of cellular function as well as the production of veterinary and human vaccines. In recent years, interest in cell culture technology has grown due to the need for production of interferon, monoclonal antibodies, various hormones, and other cell‐secreted products with potential medical uses. Many of these products have been successfully produced only be cells which require attachment to a surface in order to grow and function. This requirement places severe limitations on the economics and feasibility of large‐scale culture. In response, a number of new technologies have been proposed and developed in attempts to increase the surface area available within a given culture volume, to increase the cell density per surface area, to decrease medium and serum requirements per unit of product, to increase product production by control of the cellular environment, and to automate the production process. Some of the techniques and described along with their ability to control the environment of cells in culture.
A mathematical model of growth and alkaloid production in the submerged culture of Claviceps purpurea
Authors: Pažoutová Sylvie , Votruba Jaroslav , Řeháček Zdeněk
Published: December 2004

A mathematical model of the batch cultivation of Claviceps purpurea was formulated. The main attention was devoted to the effect of exocellular and intracellular phosphate on the growth of the mycelium and production of clavine alkaloid under experimental conditions without limitation by carbon and nitrogen sources. The method of nonlinear regression was used ot predict the optional technological regime of the phosphate addition in the batch culture at different time intervals of additions.
Fluid flow pattern in upflow reactors for anaerobic treatment of beet sugar factory wastewater
Authors: Heertjes P. M. , Kujivenhoven L. I , van der Meer R. R.
Published: December 2004

Residence‐time‐distribution experiments for the fluid in a 30‐m3 pilot plant and a 200‐m3 prototype upflow reactor were performed by means of continuous injection of an LiCl solution as a tracer in the influent of the reactor and measurement of the response of this stimulus on several location in the reactor and in the effluent. In a similar way as described in an article published earlier, models have been developed by use of the measured data of the fluid flow pattern which consisted of region of ideal mixing, plug flow, dead space, and short circuiting. It appeared that the fluid flow patterns in the two reactors were to a large extent analogous. For the pilot plant, three‐mixer models appeared to be appropriate while for the prototype reactor two‐mixer models have been found. This differences was a result of the difference in the heights of the sludge beds in the reactors: 2–3 m in the pilot plant and only 0.4 m in the prototype reactor, a result of too small an amount of sludge. Another differences was that, due to large amount of mud in the prototype reactor, a region of dead space occurred in the models for the fluid flow pattern in this reactor. The dimension of the prototype reactor have been chosen according to several recommendations obtained from work with the pilot plant (e.g., scale‐up should be done by increasing the cross section of the reactor; one influent point should be applied per 5 m2 bottom surface). The results presented here clearly show the value of these recommendations.
Statistical mechanical estimation of the free energy of formation of E. coli biomass for use with macroscopic bioreactor balances
Authors: Grosz Ron , Stephanopoulos Gregory
Published: December 2004

The need for the determination of the free energy of formation of biomass in bioreactor second law balances is well established. A statistical mechanical method for the calculation of the free energy of formation of E. coli biomass is introduced. In this method, biomass is modelled to consist of a system of biopolymer networks. The partition function of this system is proposed to consist of acoustic and optical modes of vibration. Acoustic modes are described by Tarasov's model, the parameters of which are evaluated with the aid of low‐temperature calorimetric data for the crystalline protein bovine chymotrypsinogen A. The optical modes are described by considering the low‐temperature thermodynamic properties of biological monomer crystals such as amino acid crystals. Upper and lower bounds are placed on the entropy to establish the maximum error associated with the statistical method. The upper bound is determined by endowing the monomers in biomass with ideal gas properties. The lower bound is obtained by limiting the monomers to complete immobility. On this basis, the free energy of formation is fixed to within 10%. Proposals are made with regard to experimental verification of the calculated value and extension of the calculation to other types of biomass.
Kinetics of growth of the hydrogen‐oxidizing bacterium Alcaligenes eutrophus (ATCC 17707) in chemostat culture
Authors: Siegel Robert S. , Ollis David F.
Published: December 2004

The hydrogen‐oxidizing bacterium, Alcaligenes eutrophus (ATCC 17707), was grown in chemostat culture with gas‐phase (hydrogen, oxygen, and carbon dioxide) and liquid‐phase (mineral nutrients) feedstreams; data were used to generate an analytical form for the specific growth rate equation. Model parameters obtained include Monod rate parameters for dissolved hydrogen and oxygen gases, yield coefficients, and specific maintenance rates under conditions of hydrogen or oxygen limitations. These values are similar to some obtained previously by Ohi et al. for another hydrogen bacterium. The observed increase in specific maintenance rates under hydrogen‐versus‐oxygen‐limited culture may be associated with hydrogenase deactivation by oxygen.
Studies on on‐line bioreactor identification. II. Numerical and experimental results
Authors: San Ka‐Yiu , Stephanopoulos Gregory
Published: December 2004

The on‐line bioreactor identification methodology of the preceding article (Part I) was tested with a series of numerical simulations and laboratory experiments. Results of these studies presented herein confirm the superior characteristics of the proposed estimator and its applicability to modelling studies, or on‐line bioreactor control. The sensitivity of the estimation scheme with respect to the respiratory quotient measurement is discussed, and suggestions to bypass these problems are offered.
Fermentation equations for propionic‐acid bacteria and production of assorted oxychemicals from various sugars
Authors: Papoutsakis Eleftherios Terry , Meyer Charles L.
Published: December 2004

Fermentation (stoichiometric) equations are derived for anaerobic fermentations of propionic‐acid bacteria (of both the Propionibacterium and acrylate pathways) and for production of various oxychemicals (butanol, acetone, isopropanol, butanediol, butyrate, acetate, propionate, succinate, lactate, and acrylate) from pentoses, hexoses, and cellobiose. The derivations of the equations are based on the fermentation biochemistries of the various bacterial classes. The validity of the equations is tested using fermentation data from the literature. The equations are shown to be valuable, among other uses, for calculating maximal yields and selectivities of the various fermentation products, as “gateway sensors” for monitoring of the fermentations, and for calculating the extents of the various intracellular reactions of the fermentation biochemistry.
High algal production rates achieved in a shallow outdoor flume
Authors: Laws E. A. , Taguchi S. , Hirata J. , Pang L.
Published: December 2004

A mass culture of Tetraselmis suecica grown in seawater enriched with only inorganic nutrients and CO2 in a shallow outdoor flume containing foil arrays to effect systematic vertical mixing achieved average daily production rates of over 40 g ash‐free dry wt (AFDW)/m2 over periods as long as one month when grown on a three‐day dilution cycle. Photosynthetic efficiencies associated with these high production rates averaged 8–11% based on visible irradiance. Operation of the system in a one‐, two‐, or four‐day dilution cycle resulted in lower photosynthetic efficiencies of 6–7%. A remarkable feature of the three‐day dilution cycle results was the fact that production on the third day after dilution averaged 60–70 g AFDW/m2, and corresponding photosynthetic efficiencies averaged 13–19%. The high production rates and photosynthetic efficiencies achieved on the third day after dilution may have reflected the nonequilibrium nature of the production cycle and, in particular, the fact that the adaptation of the cells to changing light condition lagged behind light condition in the culture.
Continuous cultivation of recombinant Escherichia coli: Existence of an optimum dilution rate for maximum plasmid and gene product concentration
Authors: Seo Jin‐Ho , Bailey James E.
Published: December 2004

Growth yield factors, plasmid stability, cellular plasmid content, and cloned gene product activity for Escherichia coli HB101 containing plasmid pDM246 were measured at several dilution rates in continuous culture. Cell mass yield per mass of glucose consumed declined with increasing dilution rate. There was no evidence of plasmid segregational instability in any experiments, none of which employed selective medium. Plasmid content per cell varied with population‐specific growth rate as observed in earlier batch experiments with the same strain. Plasmid content declined with increasing specific growth rate following indication of a maximum number of plasmids per cell at specific growth rates of ca. 0.3 h−1. Cloned gene product (β‐lactamase) activity exhibited a sharp maximum with respect to dilution rate in continuous culture. Qualitatively different results were observed in previous experiments in batch cultivation in which specific growth rate changes were effected by altering medium composition.
Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model
Authors: Chu I‐Ming , Papoutsakis Eleftherios Terry
Published: December 2004

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase‐plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single‐substrate, high‐dilution‐rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed‐substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.
Computer control of carbon–nitrogen ratio in Spirulina platensis
Authors: Walach Marek R. , Bazin Michael J. , Pirt S. John , Balyuzi Hushang H. M.
Published: December 2004

An on‐line computer was used to control the ratio of carbon to nitrogen in algal biomass. An indirect method of growth and biomass estimation was utilized. This was based on balancing the amount of CO2 carbon in and out of the algal bioreactor. It was shown that growth conditions govern the morphology and composition of Spirulina platensis. Cells grown under light limitation were narrower, had high levels of phycocyanin pigments, and were packed full of small lipid granules. Whereas cells grown under nitrogen limitation lost their characteristic blue‐green color, had reduced levels of phycocyanin, were fatter, and were packed full of larger lipid granules.
Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae
Authors: Mandenius C. F. , Mattiasson B. , Axelsson J. P. , Hagander P.
Published: December 2004

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO2 semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto–Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.
Lipase reaction in AOT‐isooctane reversed micelles: Effect of water on equilibria
Authors: Han Daeseok , Rhee Joon Shick , Lee Sun Bok
Published: December 2004

The effect of water on equilibria for hydrolytic reaction in reversed micelles has been investigated using lipase as a model enzyme. The effect of water on equilibria has been ignored for hydrolase reactions in an aqueous phase. In a reversed micellar system, however, the equilibrium of the lipase reaction was changed when water was added during the hydrolytic reaction. Furthermore, equilibrium fractional conversion is affected by the initial water concentration, being shifted to higher values with higher water concentrations, with other reaction conditions being held constant, indicating that the reaction should be regarded as a two‐substrate process. Equations corresponding to a two‐substrate, second‐order reversible model are derived and used for further analysis. The progress curves predicted from the rate equations agree very well with the experimental results under various reaction conditions. The values of the molar ratio of water to surfactant (R) which maximize the initial reaction rate and maximum fractional conversion is predictable from the derived rate equations and the resulting relationship between R and the kinetic constants.
Novel cationic triazine dyes for protein purification
Authors: Clonis Y. D. , Stead C. V. , Lowe C. R.
Published: December 2004

The effectiveness of a new immobilized cationic triazine dye was investigated alongside two new amphoteric triazine dyes and two well known anionic triazine dyes, Procion Red H‐3B and Procion Blue H‐B, as chromatographic media for binding four familiar proteases–trypsin, chymotrypsin, thrombin and carboxypeptidase‐B–as well as a typical oxidoreductase, lactate dehydrogenase, and human serum albumin. The new affinity adsorbent, CL‐Sepharose‐immobilized Cationic Dye, specifically binds trypsin‐like proteases such as trypsin, thrombin, and carboxypeptidase‐B, but none of the other proteins tested. In contrast, the amphoteric and anionic immobilized dyes bind all the other proteins tested in a similar fashion. The specificity of the cationic dye was exploited in the resolution of trypsin and chymotrypsin from a crude activated bovine pancreatic extract. The procedure described here affords trypsin with specific activity of 7400 units/mg with a 79% overall yield in a single step. The immobilized cationic dye, unlike previously reported adsorbents for trypsin, is inexpensive, readily synthesized, and displays a workable capacity of 4000 trypsin units or 0.55 mg protein/g moist weight gel (1.2 μmol dye/g moist weight gel) from a crude bovine pancreatic extract and, thus, is potentially amenable to process‐scale operations.
Conversion of α‐ketoglutarate into L‐glutamic acid with urea as ammonium source using multienzyme systems and dextran‐NAD+ immobilized by microencapsulation within artificial cells in a bioreactor
Authors: Gu Kang Fu , Chang Thomas Ming Swi
Published: December 2004

Urea could be effectively converted into L‐glutamic acid with semipermeable nylon‐polyethylenimine artificial cells containing L‐glutamic dehydrogenase (EC 1.4.1. 3), yeast alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1. 5) and soluble dextran‐NAD+. For batch conversion, the artificial cell suspension to total reaction volume ratios ranged from 1 in 5 to 1 in 60. From 22.6 to 53.4 μmol of L‐glutamic acid could be produced by 0.4 mL artificial cell suspension within 2 h. The corresponding conversion ratios were 56.5‐11. 1%. The L‐glutamic dehydrogenase multienzyme system showed a good storage stability: 66.0% of the original activity was retained after 1 month of storage at 4°C. A small bioreactor was prepared to contain 4.0 mL artificial cells. At a flow rate of SV = 1.5 h−1, the maximum conversion rate was 49.6 μmol L‐glutamic acid/p h. Thirty‐eight percent of the maximum activity was retained when continuously used for four days at 22°C. A kinetic analysis for the L‐glutamic dehydrogenase multienzyme system was studied. The Michaelis constants are as follows: α‐ketoglutarate is 0.838 mM; urea is 1.90 mM; dextran‐ NAD+ is 0.345 mM; and ethanol is 5.31 mM.
Surface immobilization of plant cells
Authors: Archambault J. , Volesky B. , Kurz W. G. W.
Published: December 2004

A novel technique has been developed to immobilize plant cells. The cells are deposited on a surface of manmade fibrous material that provides for strong binding of the plant tissue biomass growing in the submerged culture. The immobilized plant cells remain fully viable. Relatively uniform biomass loadings of up to 20 mg d.w. plant cells/cm2 support material have been attained. All plant cells from the inoculum suspension became attached within the first 24–48 h depending on the support matrix configuration and hydraulic culture conditions. The advantages and scale‐up potential of this technique are discussed and compared to other culturing modes.
A simple, leaky cell growth model for plant cell aggregates
Authors: Frazier George C.
Published: December 2004

A simple growth model is proposed for plant cell aggregates which accounts for leakage of a single intermediate metabolite from the aggregates to the medium. This model predicts a lag phase in the growth curve whose extent is determined by the intermediate metabolite leakage coefficient and its equilibrium distribution coefficient between the medium and the cell aggregates, the size of the inoculum relative to the system total water content, and the initial intermediate metabolite content in the medium. The model thus provides for an interaction between growing plant cells and their environment in a way that has heretofore been unquantified. Preliminary validation of the model has been made against literature data of Dioscorea deltoidea grown in batch suspension cell culture on sucrose, yielding a correlation coefficient of 0.997. The predicted glucose + fructose concentration in the medium agrees reasonably well with experimental measurements after ca, 3.5 days of culture, although a discrepancy exists between model prediction and experiment immediately after startup. Further validation of the model is suggested on this and other plant species.
Dual substrate removal by an axenic bacterial culture
Authors: Machado Ranjit J. , Grady C. P. Leslie
Published: December 2004

A pure bacterial culture capable of utilizing either L‐lysine or 2‐chlorophenol (2‐CP) as sole carbon source was isolated and used in continuous culture experiments to determine its response to dual substrate limitation by those two compounds. Dilution rate and feed composition were each set at three levels in a two factorial experimental design. The total chemical oxygen demand (COD) of the feed was fixed at 225 mg/L and its composition was varied by changing the ratio of lysine to 2‐CP. The effects of the two independent variables (dilution rate and feed composition) on the concentrations of cells, lysine, COD, and dissolved organic carbon (DOC) in the reactors were systematic whereas the effects on the 2‐CP concentration were less predictable. The concentrations of the two substrates responded to the two independent variables in a complex interactive manner which is not explained by existing models for dual, substitutable substrates. Rather, the results suggested that the prediction of the fate of a single organic component in a reactor receiving a multicomponent feed is a very difficult task.
Experimental investigation of G6P production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow‐fiber reactor
Authors: Ishikawa Haruo , Takase Susumu , Tanaka Takenori , Hikita Haruo
Published: December 2004

Enzymatic synthesis of glucose 6‐phosphate from glucose and ATP catalyzed by glucokinase from B. stearothermophilus and enzymatic regeneration of ATP from ADP and acetyl phosphate catalyzed by acetatekinase from B. stearothermophilus were simultaneously performed in an Ultrafiltration hollow‐fiber reactor of the multitubular heat‐exchanger type. Experimental results of space–time yield, the conversion, and ATP recycle number were in good agreement with the theoretical predictions based on the simple analytical model developed in the preceding article. The best results for space–time yield and conversion were Ys = 1.97 mol/m3 h and X = 92.8%, respectively, under the same conditions, and the best result for the ATP recycle number was NR = 2130 under conditions different from those above. However, Ys = 1.72 mol/m3 h, X = 81.2%, and NR = 1620 were the results when the space–time yield, conversion, and recycle number were at the highest in combination under the same conditions. Results of long‐term operation showed that the apparent remaining activity of the enzyme system was ca. 55% after continuous operation for 16 days, the decrease in the enzyme activity being faster than that expected from their half‐life times determined individually in the homogeneous system.
A population balance model of enzymatic lysis of microbial cells
Authors: Hunter J. B. , Asenjo J. A.
Published: December 2004

A model is proposed for enzymatic lysis of microbial cells based on number balances over the distribution of cell‐wall mass in a population of cells. Analytical solutions to the population balance equations were obtained by the method of characteristics for simple reaction kinetics. The model has been used to analyze the following cases of lysis in a nonhomogeneous cell population: wall hydrolysis with cell rupture and product release, the effect of a distribution of lysis rates, and lysis of two‐layer cell walls. Rate expressions for the reactions of lysis can be derived from bulk‐phase experiments; the distributions of cell size and product content can be measured independently by flow cytometric techniques. The population model also provides an explanation for the initial lag seen in lysis kinetics for virtually any initial distribution. The model demonstrates patterns of lysis and product recovery for heterogeneous populations of cells and also applies to the more general problem of soluble‐enzyme reactions with heterogeneous solid substrates.
In situ methane enrichment in anaerobic digestion
Authors: Hayes T. D. , Isaacson H. R. , Pfeffer J. T. , Liu Y. M.
Published: December 2004

A major cost consideration in the use of anaerobic digestion to convert biomass and waste to utility‐grade gas is the expense of separating CO2 from the product gas. Anaerobic digestion has a number of inherent properties that can be exploited to increase the methane content of the gas directly produced by the digester, the most important of which is the high solubility of CO2(40‐60 times that of methane) in water under digestion conditions. The methane enrichment concept examined in this study involved the recirculation of a liquid stream from the digester through a CO2 desorption process and the return of the liquid stream back to the digester for absorption of additional CO2 produced by the conversion of organic materials. A steady‐state equilibrium model predicted that a digester gas methane content exceeding 94% could be achieved with this scheme using modest recirculation rates provided a desorption process could be designed to achieve a 60+% CO2 removal efficiency in the degassing of the liquid recycle stream. Using fixed‐film laboratory digesters operated on synthetic feedstocks, the technique of methane enrichment was tested under pressurized and unpressurized conditions. A 93 + 2% methane gas stream was produced from a volatile‐acid‐fed bench‐scale digester simulating the methanogenic stage of two‐phase digestion under conditions of (1) a pH swing achieved without caustic addition that allowed digestion at pH 7. 5 and air stripping at pH 6. 5‐7. 0, (2) digester pressurization to 30 psig, and (3) a recycle rate of 0. 33 L/L reactor/day. Significant but lower levels of methane enrichment were achieved with the single‐stage digester at the low experimental recycle rate. However, the narrow range among all experiments of CO2 desorption efficiencies achieved in air stripping the recycle stream (35‐60% CO2 removal) suggests that comparable methane enrichment‐may be achieved with unpressurized single‐stage digestion using greater recycle rates. A materials balance analysis of data from an unpressurized, single‐stage digester employing no chemical addition and using laboratory degassing efficiencies indicated that 94% methane could be produced at recycle rates of less than 1. 4 L/L reactor/day with a methane loss of less than 2%.
A model for energy‐sufficient culture growth
Authors: Tsai S. P. , Lee Y. H.
Published: December 2004

Pirt's maintenance model has been widely accepted for the effects of growth rate and maintenance on growth yield. However, the interpretation of parameters in Pirt's model as biological constants is difficult for energy‐sufficient culture growth. In this study, a mechanistic model for the growth energetics of energy‐sufficient chemostat cultures is proposed and verified with literature data. In the model, the overutilization of the energy substrate in energy‐sufficient culture growth is attributed to the defective regulation of the energy substrate metabolism and energy uncoupling. The model also uses an “energy surplus” concept to collectively represent the effects of energy excessiveness. The proposed model provides a better quantitative understanding of the maximum growth yield and maintenance of energy‐sufficient cultures. It also explains the glucose concentration effect reported in the literature.
A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production
Authors: Dalili Mina , Ollis David F.
Published: December 2004

A flow cytometric kinetic study of hybidoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody‐producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourge consideration of structured kinetic studies which recognize the quality of the biophase.
Effect of metal ion concentration on a biological reactor
Authors: Ibbotson P. G. , Spedding P. L. , Otterburn M. S.
Published: December 2004

Saccharomyces cerevisiae (yeast) cells were employed as a source of alcohol dehydrogenase in the NAD+‐to‐NADH reaction. The cells were immobilized in calcium alginate monofilament fibers and used in a biological reactor. The alginate could not be heat sterilized since temperatures above 80 °C caused the polymer chains to degrade. The same proved true for the high pH necessary for the reaction, but the alginate strength was increased by Ba2+ solution treatment. X‐ray probe analysis showed that about 30% of the Ca2+ sites exchanged with the Ba2+ ions. The Ba2+ ions (as well as the Ca2+ ions) permeabilized the cells and increased the reaction rate. Long term trials showed that Ba2+ ions were slowly elutriated from the fiber biocatalyst, causing a drop in reaction rate. The trend certainly was reversible as far as the fiber was concerned. It is assumed that the permeabilization of the cells by the Ba2+ ions was a reversible process.
Hydrodynamic model for liquid‐impelled loop reactors
Authors: van Sonsbeek H. M. , Verdurmen R. E. M. , Verlaan P. , Tramper J.
Published: December 2004

A new type of bioreactor, in which two liquid phases are used, is introduced and a model description of its hydrodynamic behavior is given. The model is based on the two‐phase drift‐flux model of Zuber and Findlay and on a friction coefficient derived from one‐phase flow theory. Drop sizes, necessary for model calculations, were estimated from literature correlations and photographically verified. The model predicts the continuous‐phase circulation velocity and dispersed‐phase hold‐up within an accuracy of 5% for a pilot‐plant reactor, and within 10% for a lab‐scale reactor. Only at very low ranges, especially on lab scale, is this accuracy not attained.
Factors influencing recombinant protein yields in an insect cell–bacuiovirus expression system: Multiplicity of infection and intracellular protein degradation
Authors: Licari P. , Bailey J. E.
Published: December 2004

The insect cell (Sf9)‐baculovirus (AcNPV) expression system was employed for the synthesis of β‐galactosidase, a model heterologous protein. In the recombinant virus studied, the lacZ gene is fused to a portion of the polyhedrin structural gene and is under the control of the polyhedrin promoter. The effect of the multiplicity of infection (MOI) on product titer was determined by infecting cells with MOI values ranging from 0 to 100 and monitoring the production of β‐galactosidase with time. The relationship between final product titer and MOI was dependent on the growth phase of the cells prior to infection. The final product titer from cells infected in the early exponential phase was relatively independent of MOI. For cells infected in late‐exponential phase there was a logarithmic relationship between the final β‐galactosidase titer and the MOI used, with the highest MOI studied resulting in greatest protein synthesis. The synthesis and degradation rates of β‐galactosidase were investigated by a pulse‐chase technique using L‐[35S]‐methionine. At 24 h postinfection, the degradation rate is of the same order of magnitude as the synthesis rate. However, the synthesis rate of β‐galactosidase increases dramatically at 96 h postinfection. During this later period, the degradation rate is negligible. Although degradation of recombinant protein occurs in this system, degradation activity declines as infection proceeds and is insignificant late in intention when recombinant protein expression is intense.
Regulation and utilization of microsin promoter turn on in a chromosomal fusion
Authors: Meehan A. J. , Minkley E. G. , Domach M. M.
Published: December 2004

Prior work has demonstrated that the microsin antibiotics are produced by enteric bacteria when the growth medium is depleted of nutrients. Because the control loci could have biotechnical potential, and general stress‐response phenomena are of importance to understanding how bacteria survive in natural and bioreactor environments, we examined further the growth rate dependence of gene expression under the control of the microsin B17 promoter. This work entailed performing batch and chemostat growth experiments with a strain of E. coli K‐12 containing a mcbA‐lacZ gene fusion in the chromosome. Our results indicate that when a culture is presented with excess respiratory substrate, a well defined growth rate exists, below which a significant induction event occurs. However, cultures that are fermenting or highly glycolytic tend to express poorly. Additionally, the utility of the fusion strain was examined by performing fed‐batch cultivation experiments. We found that sustained production in a fed‐batch reactor can be accomplished by using a straightforward, exponential nutrient feeding profile.
A physical characterization of GAP A3 hybridoma cells: Morphology, geometry, and mechanical properties
Authors: Needham D. , Ting‐Beall H. P. , Tran‐Son‐Tay R.
Published: December 2004

Morphological, geometrical, and rheological properties of the GAP A3 hybridoma cell line have been evaluated as a function of the cell cycle. Interference contrast video microscopy and scanning electron microscopy (SEM) showed that a sample of cells taken from the middle of the exponential growht phase displayed a range of cell morphologies, consistent with a heterogeneous growing culture. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (cortical tension and apparent cell viscosity) properties of single cells selected at random from a sample in the middle of the exponential growth phase. Consistent with the range of morphologies, cell volumes (1400 to 5700 μm3) and apparent viscosities (430 to 1.2 × 104 P) showed a wide range of values at 37°C, demonstrating that a hybridoma cell line cannot be characterized by a single value for any one property, and that properties must be related to their cycle dependence when considering proliferating cells. Direct, video‐microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle, allowed us to correlated distinct morphologies with phases of the cell cycle. As the cell cycle progresses, an increase in cell volume by a factor of 3 to 4is accompanied by an overall increase in apparent cell viscosity by approximately the same ratio, consistent with an accumulation of more cytoplasmic material in the older cells. Also, a decrease in average apparent viscosity by a factor of 10. These results are important in order to evaluate the possible role of certain structural, cell‐cycle dependent features in shear and abrasion sensitivity. This is a problem of current concern in the bioreactor culture of mammalian cells.
On the calculation of the free energy change accompanying the growth of escherichia coli K‐12 on succinic acid
Authors: Battley Edwin H.
Published: December 2004

Determinations of the ΔG0′ accompanying the growth of Escherichia coli K‐12 on succinic acid are made using 2 different methods. The ΔG0′ accompanying catabolism could be calculated directly because the thermodynamic properties of the reactants and products are known. The ΔG′accompanying anabolism could not be calculated directly because the ΔGf value for a unit mass of cells was not known. A description is given of a deduction that the ΔG′ accompanying anabolism is zero, or nearly so. This is followed by a description of 2 methods, whereby the free energy of formation of a unit quantity of cellular substance can be calculated. The 2 values obtained by these methods are used to calculate the free‐energy change accompanying anabolism, the resultant values being 1.72 and ‐11.68 kJ, respectively, with an average of −4.98 kJ (−1.19 kcal). This value is sufficiently close to zero that it can be considered to be so, indicating that the ΔG′ accompanying metabolism is that of catabolism alone.
A structured model for simulation of cultures of the cyanobacterium Spirulina platensis in photobioreactors: I. Coupling between light transfer and growth kinetics
Authors: Cornet J. F. , Dussap C. G. , Dubertret G.
Published: December 2004

The study of the interactions between physical limitation by light and biological limitations in photobioreactors leads to very complex partial differential equations. Modeling of light transfer and kinetics and the assessment of radiant energy absorded in photoreactors require an equation including two parameters for light absorption and scattering in the culture medium. In this article, a simple model based on the simplified, monodimensional equation of Schuster for radiative transfer is discussed. This approach provides a simple way to determine a working illuminated volume in which growth occurs, therefore allowing indentification of kinetic parameters. These parameters might then be extended to the analysis of more complex geometries such as cylindrical reactors. Moreover, this model allows the behavior of batch or continuous cultures of cyanobacteria under light and mineral limitations to be predicted. © 1992 John Wiley & Sons, Inc.
Determination of antibody content in live versus dead hybridoma cells: Analysis of antibody production in osmotically stressed cultures
Authors: Reddy Sridhar , Bauer Kenneth D. , Miller William M.
Published: December 2004

Hybridomas are known to exhibit increased specific antibody production rated when subjected to environmental stress. Under these conditions, viability is low so that population‐average measurements do not properly reflect the state of viable cells. Even for flow cytometry, which gives a population distribution, special techniques must be used to discriminate between viable and nonviable cells. We describe the use of the vital stain ethidium monoazide (EMA) for independent measurement of intracellular antibody content in live and dead cells via flow cytometry. EMA is shown to be superior to light scattering techniques in identifying dead cells. We apply this technique to show that, in control batch culture, the specific antibody prodution rate and antibody content in live cells are constant during exponential growth, but decrease as cells enter the stationary phase. Antibody is retained in dead cells, but at a lower level than in live cells. We further show that, under hyperosmotic stress, the specific antibody production rate and antibody content in live both remain high during death phase. © 1992 John Wiley & Sons, Inc.