bluuebunny/crossref_metadata_2025 huggingface dataset:
Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports
Authors: Flynn A. , Johnson D. B.
Published: December 2004

Glucoamylase from four different companies was studied: three had similar stability (half‐life at 50°C about 140 hr); the fourth was less stable (half‐life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl‐TIO was less stable than enzyme bound to hornblende (metal‐link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half‐life.
Immobilized xanthine oxidase: Kinetics, (in)stability, and stabilization by coimmobilization with superoxide dismutase and catalase
Authors: Tramper Johannes , Müller Franz , Van Der Plas Henk C.
Published: December 2004

Milk xanthine oxidase was immobilized by covalent attachment to CNBr‐activated Sepharose 4B and by adsorption to n‐octylamine‐substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (Ki′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the Ki for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (Km′) for adsorbed xanthine oxidase was also higher than the Km for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O2− and H2O2 side‐products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde‐crosslinked gelatin matrix, considerably enhanced the operational half‐life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).
Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose
Authors: Sternberg David , Dorval Sheila
Published: December 2004

Trichoderma can be cultured in stirred‐tank fermentors on high (8%)cellulose concentrations without increasing the salt concentration of the medium when NH4OH is used to control pH and as a nitrogen source. Approximation 90% of the ammonia consumed by the organism can be added as NH4OH. The advantage of using high concentrations of cellulose is that culture filtrates with greater cellulase activity are obtained. The advantage of a low salts medium is that unwanted solutes in the final enzyme preparation are reduced. The appearance of cellulase in the medium occurs later than net ammonia uptake so that only 20% of the final amount of cellulase has appeared when 80% of the maximum amount of ammonia has been consumed.
Nitrogenase activity of immobilized Azotobacter vinelandii
Authors: Seyhan E. , Kirwan D. J.
Published: December 2004

As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen‐fixing bacterium, Azotobacter vinelandii was undertaken. Immobilization was accomplished by adsorption onto an anionic exchange cellulose (Cellex E)with loadings as high as 1011cells/g resin. Immobilized cell preparations were tested under both batch and continuous‐flow conditions. Nitrogenase activity as high as 4200 nmol/min g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 177 hr in a continuous‐flow reactor. Activity loss appeared to be related to the development of a variant strain.
Time scale in the dynamics of continuous cultivation of microorganisms
Authors: Minkevich I. G. , Utkina L. I.
Published: December 2004

The existence of rapid and slow transients in the continuous culture of microorganisms when limited by one of the nutrients is revealed on the basis of a new method of mathematical analysis that separates these rapid and slow transients. The slow transient occurs along one and the same line of slow movement (LSM) independent of the initial state of the culture. This line is situated in the “substrate concentration S – biomass concentration X” coordinate system and rigidly connects these concentrations. When the initial values of S and X do not lie on this line, the rapid process occurring before the slow one leads the culture to LSM. Arrival to LSM occurs owing to alteration of S at almost constant X. The biomass concentration is always a slow variable but tne substrate concentration is both a rapid and a slow variable. A number of regularities of this time hierarchy are analyzed. A comprehensive description of the dynamics of continuous cultivation of microorganisms including different ways of supplying limiting nutrients is presented. Phaseplane plots of microbial cultures and the families of isochrones (lines making it possible to determine exactlythe transient duration) are given. The isochrone theory is described in the appendix.
Improved nonporous magnetic supports for immobilized enzymes
Authors: Halling P. J. , Dunnill P.
Published: December 2004

Ni powders coated by deposition of TiO2 or controlled oxidation to NiO develop substantial resistance to corrosion. Chymotrypsin immobilized to these coated Ni supports shows very high stability of activity on storage. Chymotrypsin immobilized by adsorption and glutaraldehyde crosslinking was fairly rapidly eluted under operational conditions in the presence of substrate. If 3‐aminopropyltriethoxysilane (APS) was used to produce a covalent linkage, desorption of enzyme still occurred because of relatively unstable bonding of the silane to the oxide surface. A more stable attachment was produced by joining together many silane links with a layer of polyglutaraldehyde. The mechanism of action of APS as a coupling agent under these conditions is discussed. γ‐Fe2O3, and particularly a Mn‐Zn ferrite, are suitable magnetic support materials available with smaller particle sizes. Particles below 1 μm give the expected higher specific activities of immobilized enzymes.
Purification and properties of milk‐clotting enzyme from Bacillus subtilis K‐26
Authors: Rao Lavu Krishna , Mathur D. K.
Published: December 2004

A milk‐clotting enzyme from Bacillus subtilis K‐26 was purified by gel filtration and ion‐exchange chromatography resulting in a 24‐fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a Km of 2.77mg/ml for κ‐casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk‐clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy‐metal ions (Hg2+, Pb2+) inactivated the enzyme.
Process characteristics of cell lysis mutants of Saccharomyces cerevisisae
Authors: Boudrant Joseph , DeAngelo Joseph , Sinskey Anthony J. , Tannenbaum Steven R.
Published: December 2004

Several temperature‐sensitive lysis mutants of Saccharomyces cerevisiae were selected according to their ability to release alkaline phosphatase when incubated at a nonpermissive temperature. For two mutants, cell lysis and release of alkaline phosphatase reached a maximum when cells in the logarithmic growth phase were shifted to the ncnpermissive temperature. Morphological changes, as well as changes in macromolecular composition of the cells, were observed. Growth is necessary and oxygen is important for the expression of cell lysis at the nonpermisseve temperature.
Effect of environmental parameters on the efficiency of biodegradation of basalt rock by fungi
Authors: Mehta A. P. , Torma A. E. , Murr L. E.
Published: December 2004

The biodegradation of an aluminum‐bearing (basalt) rock by Penicillium simplicissimum has been investigated. This organism grows on a sugar substrate and releases organic acid compounds. These acids interact with the mineral matter and cause their partial decomposition. The dissolved metals are then complexed by the excess organic acids. The activity of the fungi was found to be optimum at an initial pH 7 and in the presence of 5% (w/v) substrate concentration. In 30 days of leaching almost 20% of the aluminum in the rock was solubilized and the pH was decreased from 7 to less than 3.5 in the inoculated flasks. The controls showed less than 1% of the aluminum solubilized and the final pH dropped to only 6.8. A surface characterization study performed by scanning electron microscopy indicated that the specific mineralogical phases containing aluminum and iron within this host rock were preferentially corroded. The mineral phases containing olivine and plagioclase were found to be least resistant, while phases containing titanium were most resistant to the acids released by the fungi.
Mathematical model for enzymatic hydrolysis and fermentation of cellulose by Trichoderma
Authors: Peitersen Nicolai , Ross Edward W.
Published: December 2004

This paper describes a mathematical model for the enzymatic hydrolysis and fermentation of cellulose by Trichoderma reesei. The principal features of the model are the assumption of two forms of cellulose (crystalline and amorphous), two sugars (cellobiose and glucose), and two enzymes (cellulase and β‐glucosidase). An inducer–repressor–messenger RNA mechanism is used to predict enzyme formation, and pH effects are included. The model consists of 12 ordinary differential equations for 12 state variables and contains 38 parameters. The parameters were estimated from four sets of experimental data by optimization. The results appear satisfactory, and the computer programs permit simulation of a variety of system changes.
Immobilization of microbial cells containing NAD‐kinase
Authors: Hayashi Toru , Tanaka Yoshikazu , Kawashima Koji
Published: December 2004

Microbial cells having NAD‐kinase activity, Brevibacterium ammoniagenes, were immobilized by the radiation‐copolymerization method under low temperature with the activity recovery of more than 80%. Compared to the native microbial cells the immobilized cells were more stable against heat and pH change. The immobilized cells were subjected to the 5 hr reaction repeatedly 20 times without any activity loss.
Ethanol in brazil: Brief summary of the state of industry in 1977
Authors: Lindeman Louis Robert , Rocchiccioli Carlo
Published: December 2004

This paper presents a summary of present agricultural production of ethanol in Brazil. The highlights of the national program instituted in 1975 to increase production with the aim of extending automotive gasoline are described. Process descriptions are given for present plants for both cane sugar and manioc root fermentation routes.
Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides
Authors: Lawford G. R. , Kligerman A. , Williams T. , Lawford H. G.
Published: December 2004

Leuconostoc mesenteroides NRRL B‐512(F) was grown in continuous culture under conditions of energy‐limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6‐α‐D‐glucan 6‐α‐glucosyltransferase EC 2.4.1.5), was not detected in glucose‐or maltose‐limited cultures. Under Conditions of sucrose‐limited growth, the enzyme activity of the cell‐free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose‐limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell‐free culture supernatant by ammonium sulfate precipitation and DEAE‐cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase‐like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10°C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.
Production of high‐quality edible protein from Candida yeast grown in continuous culture
Authors: Lawford G. R. , Kligerman A. , Williams T. , Lawford H. G.
Published: December 2004

Candida utilis NRRL Y‐900 was grown in aerobic continuous culture with cane molasses as the source of the growth‐limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at Dmax. On the average, the yeast biomass was 50–55% protein. At SR > 2% sugar, the biomass productivity was limited by the oxygen supply. With O2‐supplemented aeration (at SR = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.
Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design
Authors: Ghose T. K. , Tyagi R. D.
Published: December 2004

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL‐Y‐132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μm (1 – P/Pm) and v = vm (1 – P/Pm′), where μm is the maximum specific growth rate at P = 0 (hr−1); Pm is the maximum specific product formation rate at P = 0 (hr−1); Pm is the maximum ethanol concentration above which cells do not grow (g/liter); Pm′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short‐time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.
Method for continuous purification of biological material using immunosorbent
Authors: Hughes James J. , Charm Stanley E.
Published: December 2004

A mechanical device for the continuous purification of biological material using immunosorbent was developed. The system consists of heat‐sealed nylon pouches containing agarose‐bound antibody, attached to an endless 35 mm wide Mylar belt that passes through four chambers sequentially. The biological material is bound and dissociated, and the immobilized antibody is regenerated for repeated isolation and purification of antigen. The belt design incorporates features to minimize carry‐over between chambers and prevent damage to the agarose‐bound antibody in repeated passes through the system. An existing batch method for the purification of human placental alkaline phosphatase using immobilized rabbit antisera was adapted to continuous purification in the device. The belt contained a low affinity immunosorbent and made five complete passes through the system. A decrease in antigen binding capacity between free immunosorbent suspensions and belt immunosorbent in pouches was observed. This was shown to be the result of the diffusion resistance offered by the pouch and the short exposure times of each pouch in the chambers. A decrease in antigen binding capacity between successive belt passes was also observed, and resulted from the inability of the agarose in the pouches to resuspend completely after each pass. The low efficiency of the agitation method and the roller device used to squeeze the pouches were the reasons for this deficiency.
Analysis of pH‐stat continuous cultivation and the stability of the mixed fermentation in continuous yogurt production
Authors: MacBean R. D. , Hall R. J. , Linklater P. M.
Published: December 2004

A pH‐stat fermentor is a continuous cultivator in which the feed rate is controlled to maintain a constant pH, i.e., end‐product acid concentration. This fermentor has application to the continuous cultivation of lactic acid‐producing organisms in milk‐based media. The equations describing the operation of this fermentor are developed. It is shown that, where the limiting substrate is the carbon and energy source, the operation of the fermentor is essentially equivalent to that of a turbidostat. In contrast to this, where the carbon and energy source is in excess and growth is limited by another substrate, pH‐state fermentation is stable both in regions of substrate excess, where D = μmax, comparable with turbidostat operation, and substrate limitation where D < μmax, comparable with chemostat operation. These conditions are met in milk‐based media. An analysis is presented, allowing the prediction of the degree of substrate limitation, cell density, and dilution rate in a pH‐stat fermentor from batch‐growth kinetics. This was confirmed using experimental data for the growth of Streptococcus thermophilus TS2 and Lactobacillus LB1 in skim milk. Stable simultaneous growth of two organisms in continuous culture occurs if their growth rates are determined by separate conditions, so that, at steady state, their growth rqtes are separately madeequal to the dilution rate. It is then predicted, and confirmed by experiment, that a mixed culture of S. thermophilus TS2 and L. bulgaricus LB1 in a pH‐stat continuous fermentor in yogurt mix at pH 5.5 would be stable with the growth of L. bulgaricus being substrate unlimited and the fermentor operting with D = μmax for L. bulgaricus LB1, and the growth of S. thermophilus TS2 being substrate limited so that its growth rate is equal to the existing dilution rate. Finally, it is predicted and confirmed by experiment that if the conditions are altered so that the growth of S. thermophilus TS2 is substrate unlimited the stable association is broken down, the fermentor operates with D approaching μmax for S. thermophilus TS2, and L. bulgaricus LB1 is washed out to the level maintained by wall growth.
Analysis of denitrification with a completely submerged pilot‐scale rotating biological contactor
Authors: Soyupak S. , Murphy K. L. , Jank B. E.
Published: December 2004

The hydraulic characteristics and the denitrifying capacity of a single‐stage 0.5 m diam completely submerged rotating biological contactor were studied. A two compartment model was proposed and fitted the data obtained from pulse dye applications at two different flow rate. Denitrification rates with an influent C:N ratio at 1.5:1 proved to be independent of NO3 + NO2‐N concentration. The pooled denitrification data obtained under the two different flow rates could be fitted by an Arrhenius relationship for temperature over the range of 5 to 25°C. The activation energy was 16500 cal/g−mol. A substantially higher volumetric removal capacity was observed than has previously been reported for either suspended or supported denitrifying systems.
Physical Studies on Cell Immobilization Using Calcium Alginate Gels
Authors: Cheetham Peter S. J. , Blunt Kevin W. , Bocke Christopher
Published: December 2004

Columns of calcium alginate gel pellets have excellent physical properties when used as a cell immobilization support. Columns of pellets were very resistant to compression and abrasion during passage of high concentrations of sucrose at high flow rates, but if the pellets were formed using low alginate and Ca2+ concentrations, compression occurred and flow out of the column was reduced and pressure built up. Transfer of sucrose into the pellets was controlled by internal diffusion, the rate of diffusion being increased by reductions in the alginate and Ca2+ concentrations used for immobilization and by the presence of entrapped active cells. Some leakage of cells occurred during use especially when cell division of the entrapped cells took place, but leakage could be minimized by using more highly polymerized pellets. Therefore, immobilization conditions can be chosen so as to form strong pellets, possessing high substrate transfer rates and low rates of cell leakage.
Convenient method for studying enzyme kinetics
Authors: Hsu Teh‐an , Tsao George T.
Published: December 2004

A convenient method for enzyme kinetic studies is introduced. The method includes identification of reaction mechanism and estimation of the associated kinetic constants with a minimum number of experiments. The application of the method is illustrated by using literature data. Factors limiting the application of this method are also discussed.
Large‐scale preparation of a lectin from sunn hemp seeds (Crotalaria juncea)
Authors: Ersson Bo
Published: December 2004

A procedure for large‐scale preparation of a lectin from Crotalaria juncea seeds is described. The method involve fractionation by pH‐ and ammonium sulfate precipitation followed by biospecific affinity chromatography. The adsorbent used for the affinity chromatography was prepared by coupling galactose to Sepharose 6B activated with divinylsulfone. A comparison of different apparatus and techniques involved in the preparation is discussed. The yield and quality of the lectin prepared at a large scale were comparable with laboratory‐scale preparation. From 50 kg Crotalaria juncea beans, 14.4 g Crotalaria lectin were obtained.
Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells
Authors: Constantinides Alkis
Published: December 2004

The steroid transformation of hydrocortisone to prednisolone, combining the two techniques of immobilized whole cells and high steroid concentrations, was investigated and found to be a feasible process. Freeze‐dried Corynebacterium simplex cells were immobilized in collagen, tanned with glutaraldehyde, and cast into a membrane. The reaction was studied at hydrocortisone concentrations ranging from 5 to 50 mg/ml. The following aspects of the system were examined: (1) the substrate concentration effect upon the reaction; (2) the effect of enzyme concentration; (3) the rate‐concentration relationship; and (4) the product inhibition characteristics of the system. The optimal substrate concentration was found to be 15 mg/ml of a membrane concentration of 80 mg/ml. This reaction attained an 80% conversion in 48 hr. A liner relation was found between the initial reaction rate and membrane concentration. One can thus increase the net production of steroid per unit volume and time by increasing the membrane levels. A physical limit to this increase occurred at membrane concentrations greater than 125 mg/ml. The rate‐concentration relationship was linear when graphed on a Line weaver‐Burk plot: giving a Km′ and Vm′ value of 5.39 mg/ml and 0.556 mg/ml/hr, respectively. When the data were tested for competitive product inhibition, the curves fitted the experimental points fairly well and produced Km′ and Vm′ values of 4.52 mg/ml and 0.566 mg/ml/hr, respectively. Product inhibition experiments showed that the inhibition was not purely competitive. At low substrate concentrations, product inhibited the enzyme; at high substrate concentrations, the enzyme was first stimulated and then depressed by increasing levels of products. This behavior has been analyzed and shown to be possibly a result of the information of a tertiary intermediate produced during the reaction.
High‐yield method for immobilization of enzymes
Authors: Wasserman Bruce P. , Hultin Herbert O. , Jacobson Bruce S.
Published: December 2004

Two types of polyethylenimine‐coated glass microbeads (13–44 μm) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized‐enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl‐activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78–87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.
Growth kinetics under adenine‐limiting conditions of Bacillus subtilis KYA 741, an adenine‐requiring strain
Authors: Tsuchiya Katsumi , Kimura Tetsu , Okazaki Mitsuo , Miura Yoshiharu
Published: December 2004

The growth kinetics of Bacillus subtilis KYA 741, an adenine‐requiring strain, was investigated under adenine‐limiting conditions. The concentration of adenine (the limiting substrate for cell growth) in the culture filtrate remained constant during the stationary phase. In this phase, DNA turnover was active and the DNA content per cell was constant throughout the cultivation period. When cells were transferred to medium without adenine, the cell concentration began to decrease immediately and then reached a constant level due to the supply of adenine from lysing to growing cells. The rates of degradation of cells and DNA were both found to be 0.2 hr−1. An equation for cell growth in this pseudostationary phase was obtained by combining Contois' equation, in which the apparent saturation constant was a function of the cell concentration, with a term for cell degradation. This equation satisfactorily expressed the feature of cell growth and adenine consumption by B. subtilis KYA 741 under adenine‐limiting conditions.
Optimization of the production of progesterone 11α‐hydroxylase by Rhizopus nigricans
Authors: Hanish W. H. , Dunnill P. , Lilly M. D.
Published: December 2004

The progesterone 11α‐hydroxylase of Rhizopus nigricans ATCC 6227b is an inducible enzyme system that is primarily induced by its substrate progesterone. Maximum induction was found at a progesterone concentration of 0.5 g/liter or above. Oxygen is the other substrate for the hydroxylation and this was found to have a major effect on the amounts of hydroxylase synthesized. Optimum induction of the hydroxylase in a fermentation with a 3.1 m/sec impeller tip speed was found to occur at a dissolved oxygen tension (DOT) of 10% of air saturation. The agitation rate also effects the amount of hydroxylase synthesized with an apparent maximum at 3.1 m/sec impeller tip speed. The DOT for a maximum hydroxylation rate was much higher than for enzyme synthesis so that it was preferable to increase the DOT after induction was completed.
Growth of yeast on n‐alkanes. II. Batch experiments
Authors: Verkooyen A. H. M. , Rietema K.
Published: December 2004

In this paper the results of the Monte Carlo simulations as described in an earlier paper are compared with those of batch experiments. A number of batch experiments were carried out at a low inoculation rate so that only a fraction of the oil drops were inoculated. Under these conditions the effect of the segregation of the oil phase is more clearly demonstrated. Special attention is paid to the preparation of actively growing yeast cells with which the cultures is inoculated. Also a method is developed to estimate the amount of actively growing cells with which the culture is inoculated. The other parameters necessary for the Monte Carlo simulation are measured in separate experiments: the maximum growth rate of the cells, oil drop size, and the drop parameters. Finally the growth curves (measured in the batch experiments) are compared with those calculated with the Monte Carlo procedure. A good agreement is found.
Purification and properties of glucose isomerase of Actinoplanes missouriensis
Authors: Gong Cheng‐Shung , Chen Li Fu , Tsao George T.
Published: December 2004

Actinoplanes missouriensis produces an intracellular soluble glucose Isomerase. The soluble enzyme can be purified by a DEAE‐cellulose beads columm with a onestep salt elution. The purified enyzme exhibited a molecular weight of approximately 80,000 daltons, being composed of two identical subunits of about 42,000 daltons each. The Km for glucose is 1.33M, the Km for frucotse is 1.67M. The enzyme has an optimal pH of 7.0. The presence of the cobalt ion is not required to produce optimal activity of the enzyme if the proper amount of magnesium is present.
Chelating, film‐forming, and coagulating ability of the chitosan–glucan complex from Aspergillus niger industrial wastes
Authors: Muzzarelli Riccardo A. A. , Tanfani Fabio , Scarpini Gianfranco
Published: December 2004

Waste mycelia of Aspergillus niger from a citric acid production plant are simply treated with boiling 30–40% NaOH aqueous solutions for 4–6 hr to obtain the insoluble chitosan‐glucan complex whose infrared, ESR, and x‐ray diffraction spectra are reported. A number of transition‐ and post‐transition‐metal are chelated and collected by chitosan‐glucan with higher yields than by animal chitosan. Immediate flocculation occur upon mixing chitosan‐glucan dispersions with alginate and polymolybdate solutions. Membranes are also obtained from chitosan–glucan dispersions in acetic acid or in chloral and dimethyl formamide mixtures.
Staining method for determination of the penetration of immobilized enzyme into a porous support
Authors: Carleysmith S. W. , Eames M. B. L. , Lilly M. D.
Published: December 2004

The penetration of bovine serum albumin and penicillin acylase into Amberlite XAD7 beads was determined by staining split beads. The rates of penetration were measured and correlated with a theoretically derived equation.
Purification and immobilization of Aspergillus niger β‐xylosidase
Authors: Oguntimein Gbekeloluwa B. , Reilly Peter J.
Published: December 2004

β‐Xylosidase from a commercial Aspergillus niger preparation was purified by differential ammonium sulfate precipitation and either gel permeation or cation exchange chromatography, giving 16‐fold purification in 32% yield for the first technique or 27‐fold purification in 19% yield for the second. The second method in addition almost completely removed interfering β‐glucosidase activity. Enzymes prepared by this method was immobilized to 10 different carriers, but only when it was bound to alumina with TiCl4 and to alkylamine porous silica with glutaraldehyde were substantial efficiencies and stabilities achieved. With alumina, the variation of activation procedure, amount of β‐xylosidase offered, and activation solution composition yielded maximum activities of over 40 U/g with approximately 70% immobilization efficiency. Variation of binding pH and incubation time led to a maximum immobilized activity of 1.3 U/g with 78% immobilization efficiency on silica.
Batch and multistage continuous ethanol fermentation of cellulose hydrolysate and optimum design of fermentor by graphical analysis
Authors: Tyagi R. D. , Ghose T. K.
Published: December 2004

Batch and single‐flow four‐stage continuous ethanol fermentations of bagasse hydrolysate have been investigated at pH 4.0 and 30°C with a strain of the yeast Saccharomyces cerevisiae. The studies were carried out in the laboratory four‐stage cascade continuous stirred‐tank fermentors at varying feed glucose concentrations (10, 14, 18, and 22%). The range of dilution rates employed varied from 0.05 to 0.2 hr−1. The hydrolysate was supplemented with a cheap nitrogen source (CNS), CaCl2·H2O and MgSO4·7H2O. A 2% (v/v) CNS concentration was found to be sufficient to avoid growth limitation at a glucose concentration of 116 g/liter. The conditions of continuous culture in a multistage system are predicted by a graphical method based on batch‐culture data. The results thus obtained are compared with those predicted by kinetic models and with the experimental results. The variations between the results obtained experimentally and those computed either by a kinetic model or by graphical analyses were found to be within the limits of experimental error. The solutions based on the concept of minimum residence time necessary to achieve the desired biomass or product concentrations are also discussed.
The Brazilian ethanol program
Authors: Pimentel Luiz Straunard
Published: December 2004

Brazilian needs for petroleum as a primary energy source grew from 13.2% in 1940 to 41.7% in 1977. This resulted in a much greater dependence on foreign sources and prompted the Government to initiate a detailed study of alternatives. The National Alcohol Program established in 1975 is just one of the options being examined. The National Energy Balance forecast shows that annual anhydrous alcohol consumption for automotive purposes should increase from 1.74 × 106m3 to 4.7 × 106 m3 in the period from 1978 to 1987. This paper presents the main objectives of the National Alcohol Program in the context of the overall Energy Program, points out the problems connected with alcohol production and utilization, and reviews the serious problems related to its distribution to the consuming centers. Finally, the indirect benefits resulting from the implementation of the National Alcohol Program are shown, underlining the saving of foreign currency, the substantial increase in employment opportunities, the reduction in regional and individual income discrepancies, and the expansion of capital goods production, together with the improvement of national technology in the agricultural and industrial sectors.
Characteristics of immobilized invertase
Authors: Ooshima Hiroshi , Sakimoto Masuo , Harano Yoshio
Published: December 2004

Five kinds of immobilized invertases (IMI)—covalently of porous glass and ion‐exchange resins and ionically on ion‐exchange resins—have been prepared and their kinetic characteristics for sucrose hydrolysis, such as Km, K, pH profile, and thermal stability were studied. Comparing the values of Km and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.
Immobilized catalase‐containing yeast cells: Preparation and enzymatic properties
Authors: D'Souza S. F. , Nadkarni G. B.
Published: December 2004

The cell of Saccharomyces cerevisiae previously induced for catalase (EC 1.11.1.6) activity were immobilized by entrapment of intact cells in acrylamide polymerized by γ irradiation (100 kR). Yeast cells showed an enhancement in catalase activity on entrapment, an effect similar to that observed on treatment with organic solvents like toluene. The cells pretreated with toluene, however, showed complete loss of catalase activity on entrapment. The entrapped enzyme exhibited a narrow pH optimum, reduced Km for H2O2, and a decrease in thermostability. The temperature optimum of catalase was also decreased from 60 to 40°C on immobilization. A tenfold decrease in the activation energy was also observed. The enzyme in the entrapped cells was, however, stable toward inactivation by γ irradiation. Unlike the intact cells, the entrapped yeast cells did not have the ability to induce catalase.
Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling
Authors: Mukhopadhyay S. N. , Malik R. K.
Published: December 2004

Cultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio = 8.5) produced 4.5 IU/ml celulase 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr). In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml, and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.
Modified kit procedure for the kinetic assay of ethanol
Authors: Bostick William D. , Overton James B.
Published: December 2004

We have examined the utility of a commercial kit procedure for the determination of ethanol (EtOH), based upon its enzymatic oxidation and the concurrent production of NADH, monitored by photometry at 340 nm. We found that the equilibrium production of NADH is not stoichiometric with respect to initial ethanol concentration, and that with this procedure, the calibration curve for end‐point assay of ethanol is linear only for very dilute solutions. Likewise, the kinetic assay of ethanol using the kit procedure is limited to very dilute samples (i.e., concentration in the reaction mixture of ⩽2.3 mg EtOH/liter). We describe a simple modification of the kit procedure which makes it amenable to the precise kinetic assay of up to 150 mg EtOH/liter in the reaction mixture. This increase in the dynamic range for kinetic assay of ethanol results form the use of hydrazine both as a trapping agent for the acetaldehyde reaction product and as a competitive inhibitor of alcohol dehydrogenase enzyme.
Growth yield from electrons available from substrates in aerobic‐ and photoheterotrophic cultures of Rhodopseudomonas sphaeroides S
Authors: Sasaki Ken , Nishizawa Yoshinori , Nagai Shiro
Published: December 2004

A balance of electrons available from acetic acid consumed for growth and oxygen uptake in the aerobic‐ and photoheterotrophic growth of Rhodopseudomonas sphaeroides S on acetate‐minimal medium could be realized the same as in the carbon balance. The unmeasured amounts of yeast extract consumed by the cells grown on propionate–yeast extract media were indirectly estimated from the balance equation of electrons available from carbon substrates. The specific consumption rate of the yeast extract increased with an increase in propionate consumption rate in aerobic and photoheterotrophic cultures. Growth yields from acetic acid and from propionic acid plus yeast extract were calculated on the electron level, i.e., YX/ave g cell produced/equivalent electrons available from substrate consumed. YX/ave values were 5.0 to 5.8 g cell/ave in photoheterotrophic cultures and 2.7 to 3.6 in aerobic–heterotrophic cultures regardless of different medium compositions.
Design of a fast flow reactor for aerobic treatment of dilute waste streams
Authors: Kristiansen B. , Bu'Lock J. D.
Published: December 2004

The design of a 125 liter aerated recirculating tower fermentor is presented. The tower has an external recirculation loop and a broth take‐off point designed to give selective retention of biomass in the fermentor. This allows operation with high throughput rates using a low conentration feed. The aspect ratio in the main tower is approximately 14:1, but good mixing is promoted by the rapid recirculation of the broth. The construction of the fermentor and costs are given in details, illustrating that the fermentor may offer a cheap alternative to conventional systems.
Some observations on immobilized hydrogen‐producing bacteria: Behavior of hydrogen in gel membranes
Authors: Matsunaga Tadashi , Karube Isao , Suzuki Shuichi
Published: December 2004

A hydrogen‐producing bacterium, Clostridium butyricum, was immobilized in polyacrylamide gel membrane, agar gel membrane, and collagen membrane. The apparatus consisted of a compartment(I or II) and a whole‐cell‐entrapped gel membrane which was placed in the center of the two compartments. The behavior of hydrogen through whole‐cell‐entrapped gel membrane was examined for improving the sensitivity of a microbial BOD sensor and the efficiency of biochemical fuel cells. The bacteria‐polyacrylamide gel membrane produced a higher amount of hydrogen in compartment II than the other bacteria‐entrapped gel membranes. The apparent diffusion constant decreased with increasing bacteria contents in the gel membrane. As a result, 100 mg wet cells/g gel was the maximal bacteria content in the membrane for the production of hydrogen. Twenty percent of the hydrogen evolved by the whole cells diffused to compartment II, and there was a linear relationship between the glucose concentration in a bulk solution (compartment I) and the rate of hydrogen diffused to compartment II.
Kinetic study of a membrane enzyme reactor
Authors: Kitano H. , Yoshijima S. , Hotogi S. , Ise N.
Published: December 2004

A flat‐membrane dialyzer was used as enzyme reactor by introducing enzyme solution into one of the membrane‐separated chambers. The apparent Michaelis constant Km(app) of urease was always larger (ten times at [urease] = 1 mg/ml) than that of free enzyme because the permeation of substrate through the membrane was rate determining. Km(app) for urease decreased from 125 to 20mM with increasing flow rate of the substrate solution because of the turbulent flow near the membrane. In the case of glucose oxidase or creatine kinase, the reaction rate was limited by the permeation of less permeable substrates such as oxygen or ATP. Therefore, Km(app) of more permeable substrates such as glucose or creatine became smaller than that of free enzyme. The reaction amount calculated from the permeation data agreed well with experimental results. By designing spacers for the reactor to give turbulence to the solution, the effectiveness of the reactor was improved fivefold.
Photochemical system for regenerating NADPH from NADP with use of immobilized chloroplasts
Authors: Karube Isao , Otsuka Toshiaki , Kayano Hiromichi , Matsunaga Tadashi , Suzuki Shuichi
Published: December 2004

Spinach chloroplasts were immobilized in 2% agar gel. Crude ferredoxin and NADP–ferredoxin oxidoreductase isolated from spinach were used as electron carriers. The activity of the NADP reduction by immobilized chloroplasts increased with increasing ferredoxin concentration and the maximum activity was obtained at 8μM ferredoxin. The saturation of NADP reduction was observed at a light intensity of over 1000 lx. The optimum pH and temperature of NADP reduction were 8 and 25°C, respectively. The reduced NADP in a reaction medium increased linearly with increasing reaction time under illumination. NADP was continuously reduced for 2 hr with a hollow‐fiber reactor containing immobilized chloroplasts. NADPH and NADP were separated with a hollow‐fiber dialyzer from ferredoxin and NADP–ferredoxin oxidoreductase, which were reused. The conversion ratio of NADP to NADPH was from 40 to 80%.
Enzyme activity maintenance in packed‐bed reactors via continuous enzyme addition
Authors: Verhoff F. H. , Schlager S. T.
Published: December 2004

An operational scheme for using immobilized enzymes in packed‐bed reactors that permits operation at a constant throughput rate and constant product quality is described. The scheme used columns operated in series with continuous enzyme addition to compensate for enzyme decay. A mathematical technique was developed to determine the enzyme addition rate, enzyme usage, and enzyme volume in the column system. Operation of columns in series is compared to operation where the flow rate is decreased to compensate for a loss of enzyme activity for both zero‐and first‐order decay. The analyses indicated that columns in series resulted in better enzyme utilization but larger reactor volumes than parallel reactors with decreasing flow rate.
Methods for quantitative representation of the morphology of molds
Authors: Metz B. , de Bruijn E. W. , van Suijdam J. C.
Published: December 2004

A semiautomatic method for the quantitative representation of mold morphology is described.
Estimation of intrinsic kinetic constants for pore diffusion‐limited immobilized enzyme reactions
Authors: Lee Gene K. , Lesch Robert A. , Reilly Peter J.
Published: December 2004

A simple method is presented that establishes intrinsic rate parameters when slow pore diffusion of substrate limits immobilized enzyme reactions that obey Michaelis‐Menten kinetics. The Aris‐Bischoff modulus is employed. Data at high substrate concentrations, where the enzyme would be saturated in the absence of diffusion limitation, and at low substrate concentrations, where effectiveness factors are inversely proportional to reaction modulus, are used to determine maximum rate and Michaelis constant, respectively. Because Michaelis‐Menten and Langmuir‐Hinshelwood kinetics are formally identical, this method may be used to estimate intrinsic rate parameters of many heterogeneous catalysts. The technique is demonstrated using experimental data from the hydrolysis of maize dextrin with diffusion‐limited immobilized glucoamylase. This system yields a Michaelis constant of 0.14%, compared to 0.11% for soluble glucoamylase and 0.24% for immobilized glucoamylase free of diffusional effects.
Application of microbial cells as multistep catalysts in potentiometric biosensing electrodes
Authors: Kobos R. K. , Pyon H. Y.
Published: December 2004

The advantages of using intact microbial cells to mediate complex reaction sequences in the construction of biosensing electrodes are domonstrated. A potentiometric sensor was developed for nitrilotriacetic acid (NTA) in which a strain of Pseudomonas sp. was coupled to an ammonia gas‐sensing electrode. the bacterial cells carry out a four‐step reaction sequence to produce the measured species, ammonia, from NTA. The resulting microbial electrode responded to NTA in the concentration range of 1 × 10−4 M to 7 × 10−4M, with a slope of 35‐40 mV/decade. The electrode was stable for up to one month.
Comparison of measured and calculated oxygen solubilities in fermentation media
Authors: Quicker G. , Schumpe A. , König B. , Deckwer W.‐D.
Published: December 2004

A fast physical method is used to measure oxygen solubilities in various solutions of sugars and salts, in nutrition media, and in fermentation broths of Penicillium chrysogenum. The findings were compared with the predictions of a solubility model which assumes that the solubility reduction is log‐additive with respect to various compounds (mainly sugars and electrolytes). A striking agreement between experimental results and predictions was found.