bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of inorganic phosphorus using immobilized pyruvate oxidase
Authors: Tabata Masayoshi , Murachi Takashi
Published: December 2004

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one‐month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well‐established method.
Isoelectric precipitation of soy protein. II. Kinetics of protein aggregate growth and breakage
Authors: Petenate Angela M. , Glatz Charles E.
Published: December 2004

Protein aggregate growth and breakage in agitated suspensions are modeled. The model includes growth of particles by a turbulent collision mechanism and breakage by a hydrodynamic shear mechanism. In the model, breakage results in the splitting of the particles into several small fragments. The model parameters are a growth rate constant and a breakage rate constant. Aggregate size distributions were measured with a Coulter counter and the data interpreted using a population balance that governs the steady‐state particle size distribution in a continuous stirred tank reactor. Effects of changes in the operating variables pH, concentration, mean residence time, ionic strength, and mixing power input on the model kinetic parameters are investigated.
Collagen strip with immobilized luciferase for ATP bioluminescent determination
Authors: Blum Loïc J. , Coulet Pierre R. , Gautheron Daniéle C.
Published: December 2004

Firefly luciferase from Photinus pyralis has been covalently bound to a collagen strip via an acylazide activation process. Immobilization performed in the presence of both substrates ATP and luciferin allows to increase the activity retained on the strip. The best activity exhibited by immobilized luciferase was obtained in a 0.05M Tris‐acetate buffer, pH 7.75. The pH optimum and the activation energy of luciferase have been found unchanged after immobilization. In the chosen stirring conditions, no diffusional limitations of substrates appear. ATP measurements can be performed with collagen‐bound luciferase in the range 1.10−11 M−3.10−6 M. It was possible to store the strips at 4°C in a dehydrated form; then, the bound enzyme retains 20% of its initial activity after eight months. Human blood ATP was measured with this collagen‐bound luciferase and the results were found in good agreement with those obtained by soluble luciferase.
Alkaline treatment of wheat straw for increasing anaerobic biodegradability
Authors: Pavlostathis Spyros G. , Gossett James M.
Published: December 2004

Wheat straw was treated with NaOH and anaerobically digested for methane production. Alkaline treatment resulted in a greater than 100% increase in biodegradability of wheat straw. The potential of a process flow scheme employing high alkali concentration at ambient temperature with solids separation and recycle of filtrate containing residual alkali was explored. The effect of NaOH on the solubilization of cell wall constituents and potential problems of toxicity are discussed. A solubilization model was developed which is used to predict biodegradability of whole samples based on solids and filtrate biodegradabilities. Energy requirements and chemical costs are also addressed.
Dilute acid hydrolysis of paper birch: Kinetics studies of xylan and acetyl‐group hydrolysis
Authors: Maloney Mark T. , Chapman Thomas W. , Baker Andrew J.
Published: December 2004

Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18M and temperatures of between 100 and 170°C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based on the existence of reactive and resistant xylan portions. The resulting rate equation predicts the experimental xylan concentrations in the residue to within 10%. Hydrolysis of xylan‐associated acetyl groups was found to occur at the same rate as that of xylan, except at 100°C, where acetyl is released preferentially. No effect of acid concentration on the rate of acetyl removal relative to that of xylan was evident.
A microbial culture with oxygen‐sensitive product distribution as a potential tool for characterizing bioreactor oxygen transport
Authors: Moes J. , Griot M. , Keller J. , Heinzle E. , Dunn I. J. , Bourne J. R.
Published: December 2004

The dissolved oxygen (DO) level has been shown to have a profound effect on the product distribution of a Bacillus subtilis culture, with acetoin being excreted with DO above 100 parts per billion (ppb) and butanediol below 100 ppb. The product concentration ratio changed rapidly in the 80–90 ppb range. Switching from one oxygen level to another caused one already accumulated product to be converted to the other in a reversible manner. Rates of change of 0.5–1 g/L h enabled detection within 10 min. Detection sensitivity is enhanced because the ratio of two concentrations can be measured. Remarkably sensitive to mixing rates, the culture responded to changes in stirring speed during experiments in which the dissolved oxygen was controlled at a constant level with a novel control system. Thus, the culture is capable of detecting dead zones in relatively well‐mixed reactors and oxygen gradients in column and tubular reactors. High‐viscosity effects can also be investigated since the culture grows well in xanthan gum solutions. Preliminary kinetic model development indicates that a useful model for simulating reactor mixing and transport effects can be developed to aid in the planning of experiments.
A Multispecies model of biological treatment with sequential pollutant oxidation
Authors: Vasiliev V. B. , Vavilin V. A.
Published: December 2004

The multispecies biological treatment model including the consequent substrates consumption is suggested. The model describes the adaptation in the distribution of species that are included in the composition of activated sludge. It was shown that the effects of adaptation are similar for the consequent as well as simultaneous mechanisms of substrate oxidation.
Purification and characterization of endo‐xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points
Authors: Frederick Mary M. , Kiang Chih‐Hen , Frederick James R. , Reilly Peter J.
Published: December 2004

Two endo‐xylanases (1,4‐β‐D‐xylan xylanohydrolase, EC 3.2.1.8) were purified to homogeneity from a crude Aspergillus niger pentosanase preparation by Ultrogel AcA 54 gel permeation chromatography, SP‐Sephadex C‐25 cation exchange chromatography at pH 4.5, Sephadex G‐50 gel permeation chromatography, and a second SP‐Sephadex C‐25 step, this one at pH 5.8. The two xylanases hydrolyzed soluble xylan more rapidly than insoluble branched xylan, but attacked each substance to an equal extent. Their low activity on a linear xylooligosaccharide mixture and absence of activity on insoluble xylan freed of branches suggest that the xylanases require a branch point nearby for significant attack. No xylose or L‐arabinose was produced, the major products of low molecular weight being tri‐ and pentasaccharides and smaller amounts of di‐, tetra‐, and hexasaccharides. There was low activity on untreated and crystalline cellulose and on carboxymethylcellulose and no activity on other polysaccharides tested. These two xylanases had molecular weights of ca. 1.3 × 104 and similar amino acid profiles, high in acidic and low in sulfur‐containing residues. Isoelectric points were 8.6 for I and 9.0 for II. Optimum pH values for activity were 6.0 and 5.5, respectively. In a 20‐min assay at pH 5.5, each was most active at 45°C, with activation energies up to 40°C of 30.4 and 38.8 kJ/ mol, respectively. Optimum pH levels for stability were 5.0 and 6.0, with half‐lives at 60°C and those pHs of 20 and 75 min, respectively.
Continuous production of ethanol from fructose by immobilized growing cells of Zymomonas mobilis
Authors: Jain W. K. , Toran‐Diaz I. , Baratti J.
Published: December 2004

Immobilized growing cells of Zymomonas mobilis were found to ferment rapidly and efficiently media containing 100 g/L fructose in a continuous reactor. A volumetric ethanol productivity of 94.8 g/L h was achieved at a substrate conversion of 75.5%. With 97% conversion of substrate the productivity was 28.4 g/L h. At fructose concentrations of 150 and 200 g/L substrate and product inhibitions limited the performance of the reactor. Ethanol production was constant over a period of 55 days.
Carboxypeptidase a immobilization on activated styrene–maleic anhydride systems
Authors: Dua R. D. , Kumar Sanjay , Vasudevan Padma
Published: December 2004

The copolymer styrene–maleic anhydride (SMA) was activated to various forms to create enzyme coupling groups. Carboxypeptidase A (CPA) was Immobilized on these supports to enhance their thermal and chemical stability. Immobilized enzyme retained 60–70% of the original activity. When kept at 60°C, while free enzyme was deactivated within 30 min, the immobilized enzyme retained 40% of initial activity at the end of 3 h. The half‐life of free enzyme was only 21 min, while for immobilized enzyme it was enhanced up to 3 h. Also, the immobilized enzyme could be repeatedly used over 50 times retaining almost 50% of original activity.
Effects of temperature and dilution rate on the copy number of recombinant plasmid in continuous culture of Bacillus stearothermophilus (pLP11)
Authors: Koizumi Jun‐ichi , Monden Yoshiaki , Aiba Shuichi
Published: December 2004

The penicillinase yield YP/X (U/g cell) of a transformant Bacillus stearothermophilus CU21 (pLP11) in continuous culture of LGP broth at 44, 47.5, and 50°C, respectively, depended not only on temperature, θ (°C), but also on dilution rate, D (h−1), in a peculiar fashion that could not have been realized if the product were from the gene on the chromosome rather than the plasmid. The gene dosage effect could account for the unusual dependence of YP/X on θ and D, because the mode of YP/X as a function of D at a given temperature resembled that of plasmid content Cp (mg plasmid/g cell) vs. D at the same temperature. In other words, Cp, corresponding to the copy number of plasmid per grams of cell, could be controlled by either θ, D, or both in this instance. However, when the gene expression efficiency, ε (U/ng plasmid), was plotted versus Cp, the effects of θ and D on the expression became indistinct.
Kinetics of chitinase production. II. Relationship between bacterial growth, chitin hydrolysis and enzyme synthesis
Authors: Young Manuel E. , Bell Richard L. , Carroad Paul A.
Published: December 2004

A comprehensive model for chitinase production during growth of Serratia marcescens QMB 1466 on chitin was developed taking into account the rate of chitin hydrolysis in order to estimate the rate of bacterial growth. In relating growth with enzyme synthesis the total enzyme concentration was used as the sum of the enzyme present in the bulk of the fermentation broth and the enzyme adsorbed on the chitin particles. The equations constituting the proposed model were fitted to the experimental results from both continuous and batch fermentation to obtain parameters describing substrate yield, metabolic maintenance, and enzyme yields.
β‐Glucosidase of Penicillium funiculosum. I. Purification
Authors: Kantham Lakshmi , Vartak H. G. , Jagannathan V.
Published: December 2004

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo‐ and exo‐glucanases and β‐glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the β‐glucosidase of this organism, which differs from other β‐glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose‐casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose‐wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of β‐glucosidase and cellulases.
β‐Glucosidase of Penicillium funiculosum. II. Properties and mycelial binding
Authors: Kantham Lakshmi , Jagannathan V.
Published: December 2004

The properties of two isozymes of β‐glucosidase of Penicillium funiculosum (part I of this series) are described. The molecular weights of isozyme 1 was 2.3 × 105 by gel filtration and 1.2 × 105 by SDS gel electrophoresis, indicating two subunits. The molecular weight of isozyme 2 was unusually low for a fungal β‐glucosidase: 1.6 × 104 by gel filtration and 3.7 × 104 in the presence of isopropanol. The two enzymes differed from other fungal β‐glucosidases in their substrate specificities. They showed high activity with pNPG, cellobiose, cellotriose, cellotetraose, cellopentaose, gentiobiose, and laminarin, but were inactive with filter paper, CM cellulose, or derivatives or stabilized by bovine serum albumin and several alcohols such as butanol and propanol. It was inhibited by glucono‐δ‐lactone (Ki = 0.67μM) and glucose (Ki = 0.92mM).

The enzyme was quantitatively adsorbed by P. funiculosum mycelium at pH 4 and the immobilized enzyme was as enzymically active as the free enzyme, but more heat stable. The binding efficiency was very high (5000 IU enzyme/g mycelium). It could be quantitatively eluted with buffers at pH 7 or by 0.02M Ca, Mg, or Al chlorides. The binding was selective, since mycelium grown on lactose could produce and also bind only β‐glucosidase isozyme 1, whereas mycelium grown on cellulose could produce as well as bind both β‐glucosidase isozymes as well as cellulases. Mycelial binding was unaffected by washing with EDTA or trypsinization, but was totally lost by washing with dilute KOH, HCl, or ethylenediamine.
The dependence of protein release from Bacillus amyloliquefaciens on the growth phase in batch culture
Authors: Cumming R. H. , Tuffnell J. , Street G.
Published: December 2004

For the recovery of intracellular material from bacteria it is often necessary to disrupt the cells. Much work has been done on the kinetics of protein release in beadmills,1 homogenizers,2 and by ultrasonication.3 In this paper we report how the growth phase of Bacillus amyloliquefaciens grown in batch culture affects the rate of protein release by ball milling, ultrasonication, and autolysis. We further suggest that autolysis is a feasible method for disrupting Bacillus.
Comparison between immobilized Kluyveromyces fragilis and Saccharomyces cerevisiae coimmobilized with β‐galactosidase, with respect to continuous ethanol production from concentrated whey permeate
Authors: Hahn‐Hägerdal B.
Published: December 2004

Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with β‐galactosidase, for continuous ethanol production from whey permeate in packed‐bed‐type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15, and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than K. fragilis. The study went on for more than 5 weeks. K. fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stableduring the entrire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose‐adapted immobilized S. cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/L ethanol was produced with a productivity of 2.5 g/L h. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/L with a productivity of 4.5 g/L h, whereas immobolized K. fragilis only produced 13 g/L ethanol with a productivity of 1.1 g/L h. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration.
Conversion of cellulose to sugars by resting cells of a mesophilic anaerobe, Bacteriodes cellulosolvens
Authors: Giuliano Christine , Khan A. W.
Published: December 2004

During the growth of Bacteroides cellulosolvens in media containing cellulose, the accumulation of unutilized sugars in the culture broth occurred mainly during the stationary phase of growth. Cells harvested during the stationary phase of growth continued to convert both cellulose and hemicellulose to cellobiose, glucose, and xylose. These three sugars caused feedback inhibition. Continuous removal of these sugars during the incubation of cells with cellulose at pH 5 accumulated ca. 32 g/L of sugars as compared to ca. 17 g/ produced under batch conditions of growth. Sugar formation by resting cells also increased with increasing cell concentration and did not require any nutrient.
Dual aerobic hollow‐fiber bioreactor for cultivation of Streptomyces aureofaciens
Authors: Robertson Channing R. , Kim In Ho
Published: December 2004

Streptomyces aureofaciens (ATCC 12416c) was grown in the interstitial region formed by a parallel arrangement of three hollow silicone tubules contained within a microporous polypropylene hollow fiber. Liquid‐soluble nutrients were supplied by diffusion across the polypropylene fiber to the interstitial cell‐containing region whereas air or oxygen was provided by diffusion from the silicone tubule lumina to the cell mass. In this bioreactor, S. aureofaciens grew to high cell densities (greater than 1011 cells/cm3) and the culture so‐obtained continously synthesized the secondary metabolite tetracycline. The volumetric productivity of tetracycline based on the interstitial volume was 90 μg/ml/h and based on the total reactor volume was 5.5 μg/mL/h. The high surface area‐to‐volume ratio afforded by the cylindrical configuration together with spatially distinct conduits to continuously transport liquids and gases, each of which may be nutrients or products of biosynthesis, to or from a tissuelike cell mass provides an alternative to the conventional air‐ or oxygen‐sparged fermentation vessel. High volumetric reactor productivities may be achieved by virute of the concentrated stationary cell mass and by the appropriate selection of fiber sizes and materials so as to ensure adequate supplies of liquid and gaseous substrates to, as well as removal of metabolites from, most cells in the culture. This reactor topology is quite general and may be adapted to most microbial as well as mammalian and plant cell systems.
Simulation of CFSTR through development of a mathematical model for anaerobic growth of Escherichia coli cell population
Authors: Ataai M. M. , Shuler M. L.
Published: December 2004

A computer model is described that simulates a population of Escherichia coli B/r‐A cells growing under anaerobic conditions. This population model is an ensemble of single‐cell models. The ability of the model of predict the dynamic response of a cell population in a CFSTR to a change in feed flowrates or concentrations was investigated. With glucose as the limiting nutrient the feed concentration of glucose was shifted from 1.0 to 1.88 g/L. With a fixed concentration of glucose (1.0 g/L) step changes in residence time (4.1–1.95 h) were examined. The predicted changes of cell size distribution, substrate concentration, RNA content, and cell dry weight during the transition period compared reasonably well to those observed experiementally. We believe this model is the only model currently available that can make such predictions on an a priori basis.
A kinetic study of Saccharomyces strains: Performance at high sugar concentrations
Authors: Converti Attilio , Perego Patrizia , Lodi Alessandra , Parisi Federico , del Borghi Marco
Published: December 2004

Alcoholic fermentation represents a significant example of production of compounds utilizable as alternative energy sources. High ethanol concentration in the fermented wort is needed in order to reduce the energy consumption in the process of alcohol recovery. A particular Saccharomyces strain, of the oviformis species, obtained from fermented worts exhibiting high ethanol concentrations is studied and compared with a common S. cerevisiae strain in order to show its skill in fermenting very concentrated sugar solutions with an energy saving of ca. 10%.
Fuzzy bioengineering models
Authors: Dohnal Mirko
Published: December 2004

A classical quantitative (analytical and/or statistical) analysis is not appropriate for some ill‐defined and/or very complex bioengineering problems. Therefore, a new form of analysis using fuzzy mathematics has been developed. The fuzzy model can utilize semiqualitative and linguistic data which are to a certain level inconsistent. Bioengineering interpretation of the basic concept of fuzzy mathematics is given together with a numerical algorithm for a fuzzy evaluation of a model. A simple fuzzy model of a fermentor is studied in detail.
Startup of anaerobic downflow stationary fixed film (DSFF) reactors
Authors: Kennedy K. J. , Droste R. L.
Published: December 2004

One of the serious problems limiting the application of full‐scale anaerobic fixed film processes is reactor startup. To better understand startup, studies with downflow stationary fixed film (DSFF) reactors were conducted to characterize the effects of influent concentration, support material, and surface‐to‐volume ratio on biofilm development and overall reactor performance. Materials with roughened surfaces gave the best startup performance and as expected increased surface area in the reactors led to more rapid increases in loading rates and higher ultimate loadings. Soluble influent COD concentrations between 5 × 103 and 2 × 104 mg/L influenced the rate of biofilm development. Lower COD concentrations resulted in faster development of the biofilm, even though ultimate loadings were not necessarily achieved as rapidly as in reactors fed higher strength wastes. No decrease in specific activity of the biofilms in each reactor was observed as the thickness of the biofilms increased to their maximum value at the ultimate loadings. The operation of reactors fed lower strength wastes was more stable than reactors receiving higher strength feeds at comparable loadings. Biofilm yield and activity, COD removals, suspended growth and activity, and other system parameters are discussed.
Noninferior periodic operation of the biological reactor
Authors: Matsubara Masakazu , Hasegawa Sachiyuki , Shimizu Kazuyuki
Published: December 2004

The optimal periodic operation of the biological reactor was studied from the standpoint of the two‐objective programming problem. The noninferior set with respect to the cell productivity and the conversion of the substrate into the biomass was determined by use of the optimization technique due to Miele. It was shown that the noninferior set was composed in general of the repeated batch branch and the repeated fed‐batch branch, which occupy the high‐productivity portion and the high‐conversion portion of the noninferior set, respectively. However, the latter branch disappears in the case of growth kinetics with no substrate inhibition. In addition, the extreme points of the noninferior set yielding the maximal productivity and the maximal conversion represent such operations that are equivalent to the steady‐state operation (chemostat culture) and the batch operation, respectively.
Gas chromatography and gateway sensors for on‐line state estimation of complex fermentations (butanol‐acetone fermentation)
Authors: McLaughlin Joseph K. , Meyer Charles L. , Papoutsakis Eleftherios T.
Published: December 2004

A fermentation system has been designed to demonstrate the use of gas chromatography (GC) for on‐line monitoring of the butanol‐acetone and other complex saccharolytic fermentations. Tangential flow ultrafiltration was used to sterilely and continuously obtain a cell‐free filtrate from the fermentation broth for on‐line GC analysis of butanol, butyrate, acetate, acetone, ethanol, and acetoin. The liquid injection system consists of a phosphoric acid contactor, a slider‐type injection valve, and a heater to address the difficulties (ghosting) encountered in the analysis of carboxylic acids. The fermentation headspace gas was also analyzed by on‐line GC for nitrogen and carbon dioxide, while hydrogen was measured by difference. Raw chromatographic data were analyzed by a chromatography data system. Both raw and processed data were transmitted to a VAX 11/750 computer for further processing (using the fermentation equation) and archiving. The fermentation equation, which has recently been derived and tested on completed fermentation data, was also found to be valid during transient fermentations and thus useful as a gateway sensor for calculating various fermentation parameters on‐line. Such parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as YATP, excess ATP, and NAD reduced by FdH2), which characterize the state of the fermentation.
Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas circulation type fermentor
Authors: Fujio Yusaku , Ogata Masafumi , Ueda Seinosuke
Published: December 2004

Studies have been conducted in a gas circulation type fermentor in order to characterize the ethanol fermentation of uncooked cassava starch with Rhizopus koji. Results showed that ethanol concentration reached 13‐14% (v/v) in 4‐day broth, and the maximum productivity of ethanol was 2.3 g ethanol/L broth h. This productivity was about 50% compared to the productivity of a glucose‐yeast system. Ethanol yield reached 83.5‐72.3% of the theoretical yield for the cassava starch used. The fermentor used in the present work has been proven by experiment to be suitable for ethanol fermentation of the broth with solid substrate.
Influence of glucose on the kinetics of maltodextrin hydrolysis using free and immobilized glucoamylase
Authors: Vallat I. , Monsan P. , Riba J. P.
Published: December 2004

The study of the effect of glucose addition to the kinetics of maltodextrin hydrolysis catalyzed with free and immobilized Aspergillus niger glucoamylase does not show any significant glucose inhibitory effect. This result is in contradiction with data previously reported in the literature. On the contrary, a slight glucose‐activating effect was observed. This effect was greater in the case of the immobilized enzyme. The glucose inhibitory effect may thus not be involved in the case of practical saccharification conditions catalyzed with glucoamylase when maltodextrins are used as substrate.
Regeneration of NAD(P)H by immobilized whole cells of Clostridium butyricum under hydrogen high pressure
Authors: Matsunaga Tadashi , Matsunaga Naoki , Nishimura Shigeo
Published: December 2004

Immobilized whole cells of Clostridium butyricum reduced both NAD+ and NADP+ in the presence of hydrogen at a pressure of 100 atm. The NAD+ and NADP+ reduction activities were 4.45 and 4.30 U/g dry cells, respectively [U = NAD(P)H regenerated, μ mol/min]. The amount of NADH regenerated by immobilized cells increased with increasing hydrogen pressure above 10 atm. Immobilized cells (6 mg dry cells) of Cl. butyricum completely converted NAD+ (6.4 μmole) to NADH for 5 h, whereas only 60% of NAD+ were reduced by free cells. Immobilized cells retained 89% activity after the 5‐h reactions were repeated 4 times. L‐Alanine was continuously produced at the rate of 12.8 μmol/min g dry cells from hydrogen, ammonium, and pyruvate with immobilized Cl. butyricum‐alanine dehydrogenase.
Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose
Authors: Caminal G. , López‐Santín J. , Solà C.
Published: December 2004

The production of sugars by the enzymatic hydrolysis of cellulose is a two‐step process that includes conversion of the intermediate cellobiose to glucose by β‐glucosidase. The hydrolysis was followed by analyzing the two sugar products (cellobiose and glucose). The enzyme showed maximum activity at pH 4.8. Thermal deactivation was significant at temperatures above 45°C. At 50°C (optimum temperature) thermal deactivation was found to follow first‐order kinetics. Several models were tested by modeling the kinetics of the reaction. Their parameter values were determined by numerical optimization, including temperature dependence. The best fitting model was a competitive product inhibition for the two reactions in the operational range.
Solid‐state ethanol fermentation by means of inert gas circulation
Authors: Sato K. , Nakamura K. , Sato S.
Published: December 2004

A new method for solid‐state ethanol fermentation (the SSEF system) was experimented on for the ethanol production from solid starchy materials, where a packedbed‐type fermentor was used. Both cultivation of Aspergillus saitoi and enrichment of a saccharifying enzyme were effective for hydrolysis of the starch. Ethanol production was set in by a form of parallel fermentation using a respiration‐deficient mutant of Saccharomyces cerevisiae. Produced ethanol was simultaneously stripped by circulating inert gas and separated in a condenser. Average ethanol concentration in the condensate was over 200 g/L, and over 90% of produced ethanol was recovered from the packed bed during 15 or 16 days of stripping. The fermentation efficiency was about 80%, which was evaluated much higher than those of conventional solid‐state fermentations. The residue had lesser volume and a higher solids content compared with the distillery wastewaters of conventional liquid‐state fermentations. This means an advantage for the treatment and the effective conversion of the residue into fetilizers or animal feeds.
Fermentation and recovery of the EcoRI restriction enzyme with a genetically modified Escherichia coli strain
Authors: Botterman J. H. , De Buyser D. R. , Spriet J. A. , Zabeau M. , Vansteenkiste G. C.
Published: December 2004

The fermentation and recovery of the EcoRl restriction endonuclease with a genetically modified Escherichia coli strain is investigated. Vast amounts of product could be obtained after cultivation in a 20‐L computer‐coupled pilot fermentor and purification of the recovered wet cells. It was found that in the end the product is at least inhibitory and probably lethal to the cells (the lethality has been proven with genetic experiments) so that optimum yield requires an optimized choice for the time instant of induction. Growth after induction and product formation require substantial amounts of oxygen, which must be supplied if a high population level is to be achieved. pH control may alleviate the burden of high oxygen supply. Quantitative assessment after the different purification stages indicate that approximately 15% active enzyme can be obtained from the total amount produced.
Monitoring of protein product formation during fermentation with fast protein liquid chromatography
Authors: Gustafsson Jan‐Gunnar , Frej Ann‐Kristin , Hedman Per
Published: December 2004

A method is described using fast protein liquid chromatography (FPLC) for the monitoring of protein formation during fermentation. The procedure consists of centrifugation to recover the cells, sonication of the cells, centrifugation to remove cell debris, and analysis of supernatant on a column of Mono Q (a strong anion exchanger). Analysis of peak areas provides quantitative determination of product concentration. Maintenance and life of the Mono Q column is discussed. We find that FPLC is a convenient method for measuring products in cell homogenates because it gives rapid, highly resolved separations.
An automatic and sterilizable sampler for laboratory fermentors: Application to the on‐line control of glucose concentration
Authors: Ghoul Mohamed , Ronat Evelyne , Engasser Jean‐Marc
Published: December 2004

No Absract.
Transient responses of continuously growing yeast cultures to dilution rate shifts: A sensitive means to analyze biology and the performance of equipment
Authors: Kätterer L. , Allemann H. , Käppeli O. , Fiechter A.
Published: December 2004

Transient responses of continuously growing yeast cultures to step changes in dilution rate were influenced by (1) the preshift conditions of the culture, (2) the magnitude of the dilution rate increase, (3) the organism involved, and (4) the type of bioreactor used. The sensitivity of transient responses to seemingly minor differences in the design of experiments indicated that this type of investigation may be a means to analyze kinetics of microbial cultures as well as the effectiveness of bioreactors.
Models for high cell density bioreactors must consider biomass volume fraction: Cell recycle example
Authors: Monbouquette Harold G.
Published: December 2004

Intrinsic models, which take into account biomass volume fraction, must be formulated for adequate simulation of high‐biomass‐density fermentations with cell recycle. Through comparison of corresponding intrinsic and non‐intrinsic models in dimensionless form, constraints for non‐intrinsic model usage in terms of biokinetic and fermenter operating parameters can be identified a priori. Analysis of a simple product‐inhibition model indicates that the non‐intrinsic approach is suitable only when the attainable biomass volume fraction in the fermentation broth is less than about 0.10. Inappropriate application of a non‐intrinsic model can lead to gross errors in calculated substrate and product concentrations, substrate conversion, and volumetric productivity.
Microencapsulation of mammalian cells in a water‐insoluble polyacrylate by coextrustion and interfacial precipitation
Authors: Sefton Michael V. , Dawson Roger M. , Broughton Richard L. , Blysniuk John , Sugamori Mark E.
Published: December 2004

A process for the microencapsulation of mammalian cells in a commercially available water‐insoluble polyacrylate (EUDRAGIT RL) is described, and the effects of process parameters are outlined The polymer dissolved in diethyl phthalate was pumped along the annulus formed from two concentric needles, while the cell suspension was pumped inside the inner needle Droplets of polymer solution containing cells were blown off the end of the needles by a coaxial air stream. The droplets fell into a corn oil–mineral oil curing bath, in which the solvent was removed from the nascent capsule causing the polymer to precipitate around the cell suspension core. Capsules were washed free of oils and solvent in a fractionated plasma that allowed for quantitative transfer of capsules from the oil phase to an aqueous medium. By appropriate adjustment of the coaxial air flow rate, capsule size could be varied from 250–1000 μm, although the most convenient size was found to be 400–700 μm. Adding Ficoll 400 to the cell suspension to match the density of the suspension to the polymer solution resulted in capsules with a well‐centered core but did not affect capsule strength. It appeared that increasing the polymer solution concentration or the polymer to the cell flow rate ratio resulted in an increased capsule strength, although differences in capsule size made unequivocal conclusions difficult. These capsules are of potential use as an artificial pancreas for the treatment of diabetes (with pancreatic islets) or for large‐scale tissue culture and the production of bioactive products (e.g., with fibroblasts).
Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers
Authors: Hu W.‐S. , Wang D. I. C.
Published: December 2004

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS‐4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15‐ to 16‐fold for FS‐4 cells, as opposed to 3‐ to 4‐fold typically obtained in a batch culture.
Dynamics of continuous stirred‐tank biochemical reactor utilizing inhibitory substrate
Authors: Sokół W.
Published: December 2004

The model of a continuous‐stirred tank biochemical reactor was developed in which the instant uptake rate of substrate was used. The solutions of the model found for the oxidation of phenol by Pseudomonas putida fitted the experimental data better than the results obtained from the models cited in the literature. The model enables control of the culture parameters so that the unwanted washout of the biomass from the bioreactor can be avoided. A review of the models cited in the literature is also presented.
Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation
Authors: Lenders J.‐P. , Crichton R. R.
Published: December 2004

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate‐oxidized dextran in the presence of NaBH3CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH3CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water‐soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate‐oxidized dextran T‐70 in the presence of NaBH3CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T‐70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70°C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta‐bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.
Simple nonsingular control approach to fed‐batch fermentation optimization
Authors: Modak J. M. , Lim H. C.
Published: December 2004

Determination of the optimal feed rate for fed‐batch fermentation is normally a problem in singular control with a state inequality constraint and as such is, in general, difficult to solve, especially for those described by a large number of dynamic mass balance equations. In this article we use a new set of state variables and the culture volume as the control variable. In this way the problem is converted to one of nonsingular control with the magnitude and rate constraints on the manipulated variable and can be numerically solved by a gradient‐based technique, thus avoiding the difficulty associated with singular control problems. Examples are given to illustrate the method.
A novel carbonochloridate for activation of supports containing hydroxyl groups
Authors: Büttner W. , Becker M. , Rupprich Ch. , Boeden H.‐F. , Henklein P. , Loth F. , Dautzenberg H.
Published: December 2004

The conditions for the introduction of active carbonate groups into supports containing hydroxyl groups by reaction with 5‐norbornene‐2.3‐dicarboximido carbonochloridate are described. Up to 1.5 mmol carbonate groups/g dry Sepharose 4B could be bound. In the case of glycine the reaction of the activated supports with the amino groups takes place with a 10‐fold higher rate than the hydrolysis of the carbonate groups, and high coupling yields can be reached. It is shown that the activated supports are well suitable for the preparation of carriers for affinity chromatography or the immobilization of enzymes.
Anoxic–oxic activated‐sludge of cyanides and phenols
Authors: Richards Deanna J. , Shieh Wen K.
Published: December 2004

The anoxic–oxic activated‐sludge process has been evaluated in a laboratory investigation as a means for effective treatment of cyanide‐laden wastewaters, with phenols used as the organic carbon sources for denitrification reactions. The performance of the process was evaluated at different levels of feed cyanide concentration and mean cell residence time (MCRT). The results obtained indicate that the phenolic compounds used can be effectively used as the organic carbon sources to promote denitrification reactions. The effects of cyanide inhibition on overall TOC removal can be alleviated at longer MCRTs. Between 1.2 and 2.2 g TOC can be utilized per gram NO2 + NO3− ‐N removed in the anoxic chamber depending on the prevailing MCRT. Microbial oxidation of cyanide and thiocyanate which yields ammonia is the main mechanism responsible for the removal of cyanide and thiocyanate observed in the anoxic–oxic activated‐sludge process. Excellent removal efficiencies have been observed with feed concentrations up to 60 mg CN−/L and 100 mg SCN−/L Frequent exposure of autotrophic and aerobic cyanideutilizing microbes does not impede their activities in the oxic environment. Good nitrification and denitrification efficiencies are attainable in the anoxic–oxic activated‐sludge process in the presence of high feed cyanide and thiocyanate concentrations, provided that MCRT is maintained at a desirable level. As a result, the microbial degradation of cyanide and thiocyanate in conjunction with nitrification and denitrification to produce innocuous nitrogen gas is feasible in the anoxic–oxic activated‐sludge process.
A novel structured kinetic modeling approach for the analysis of plasmid instability in recombinant bacterial cultures
Authors: Bentley William E. , Kompala Dhinakar S.
Published: December 2004

The instantaneous specific growth rate of a recombinant bacterial culture is directly calculated using a simple structured kinetic modeling approach. Foreign plasmid replication and foreign protein expression represent metabolic burdens to the host cell. The individual effects of these plasmid‐mediated activities on the growth rate of plasmid‐bearing cells are estimated separately. The dynamic and steady state simulations of the model equations show remarkable agreement with widely observed experimental trends in plasmid copy number and foreign protein content. The model provides an important tool for understanding and controlling plasmid instability in recombinant bacterial fermentations. The modeling framework employed here is suitable for studying the metabolism and growth of a variety of microbial cultures.
Thermostability of alcohol dehydrogenase: Evidence for distinct subunits with different deactivation properties
Authors: Skerker Paul S. , Clark Douglas S.
Published: December 2004

Several independent experimental techniques, including nondenaturing and denaturing isoelectric focusing, spin labeling, and enzyme immobilization, indicate that four ethanol‐active subunits of horse liver alcohol dehydrogenase (LADH) can be classified as one of two types, designated E1 and E2. Thermal inactivation studies of LADH in solution and immobilized to two different supports demonstrate that the first‐order rate constants of deactivation of E1 and E2 differ by more than an order of magnitude. Furthermore, E1, and E 2 can be distinguished by EPR spectroscopy, with the less stable subunit type, E2, appearing to have the less compactly structured active‐site environment. The less stable enzyme form also loses catalytic activity upon covalent attachment to CNBr‐Sepharose but remains active when adsorbed to Octyl‐Sepharose. Moreover, the immobilization results in conjunction with lysine modification studies suggest that E2 immobilized to CNBr‐Sepharose cannot bind coenyzme. Overall, these results illustrate how EPR measurements in concert with activity assays can pro vide insights into the molecular mechanisms of enzyme stabilization.
Mathematical modeling of growth and production of alcohol oxidase by Hansenula polymorpha grown on methanol–glucose mixtures
Authors: Giuseppin M. L. F.
Published: December 2004

The modeling of growth and production of methanol oxidase (MOX) by Hansenula polymorpha CBS 4732 has been studied to provide a mathematical description of such production processes. Two kinds of mathematical models were constructed for growth on methanol and on mixtures of methanol and glucose. The model for growth on methanol as the sole carbon source consists of kinetics expressions, a limited number of key steps incorporating substrate and production inhibition. This model was used to predict and simulate the culture dynamics at the start‐up, the most critical step in continuous cultivation. The growth on mixtures of methanol and glucose was modeled assuming virtually independent metabolic pathways. The induction and production of MOX could be described by adaptation of various repression equations for various data from the literature. The models describe both experimental data and literature data on growth of H. polymorpha CBS 4732 on glucose–methanol mixtures satisfactorily. All parameters for the induction–repression model for growth of H. polymorpha CBS 4732 on glucose–methanol mixtures yielded evidence that a similar induction–repression pattern is involved in MOX production. Catalase, however, is repressed by a different mechanism.