bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic components of alkaline proteases of Bacillus strains. Zymograms and electrophoretic isolation
Authors: Zuidweg M. H. J. , Bos C. J. K. , Van Welzen H.
Published: December 2004

Characterization of proteolytic activity in preparations or fermentation broth is accomplished by methods based on a combination of disc electrophoresis and visualization of the activity. The methods permit a direct, comparison with reference preparations. All alkaline proteases studied, including the three subtilisins, were found to consist of several proteolytic components. The zymograms of the subtilisin type preparations show an irregular pattern. At least 14 different components may be observed, belonging to 6 types of mobility pattern, 3 of which could be assigned to the subtilisins. None of the components belongs to the group of metalloproteases. A quite regular pattern is shown by the zymogram of protease preparations produced by the alkalophilic Bacillus strains.

A few of the components of the subtilisin preparations Maxatase and protease A were isolated by preparative disc electrophoresis and by disc electrofococusing, allowing a further characterization. Special attention was directed to the determination of properties specific for the application of subtilisins as additives in household detergents. Thermostability in sodium tripolyhosphate solution was found to range from about 10% for one of the minor components to 80% for the main component of Maxatase. Three types of curves representing the effect of pH on the activity were observed. The curve of the main component of Maxatase shows a characteristic shape with a maximum at pH 10.3: with other components lower pH optima were observed. Isoelectric points of the component were found to range from pH 7 to 10.
Pilot plant conversion of steroid using resting cell suspension as biocatalyst
Authors: McGregor W. C. , Tabenkin B. , Jenkins E. , Epps R.
Published: December 2004

Resting cell suspensions of Sepedonium ampullosporum have been used successfully in the pilot plant, transformation of 9β 10α‐pregna‐4, 6‐dien‐3, 20‐dione to its 16α‐hydroxy derivative. The resting cell enzyme system acted as a stable respiratory unit up to 150 hr after resuspension in water. Semicontinuous addition of substrate to the same cell suspension reduced overall conversion lime by 67%.

Aeration and agitation were important factors affecting conversion rates. The hydroxylating system had a critical oxygen concentration above 90% saturation of air in water. Hydroxylase activity was inhibited by cyanide an d totally inhibited by the respiratory inhibitor antimycin A at concentrations much less that that concentration required to block normal respiration.
Open tubular heterogeneous enzyme reactors: Preparation and kinetic behavior
Authors: Horvath Csaba , Solomon Barry A.
Published: December 2004

Tubes with immobilized enzymes on the inner wall, called open tubular heterogeneous enzyme reactors, were prepared by binding enzymes either directly to the tube inside surface or to a layer of a porous matrix attached to the inner wall. Kinetic studies of the hydrolysis of N‐benzoyl‐L‐arginine ethylester as a model reaction indicated that the reaction was kinetically controlled in reactors with surface bound trypsin and the kinetic parameters were evaluated by conventional methods. On the other hand, substrate diffusion in both the porous matrix and the bulk substrate solution strongly affected the rate of reaction in porous layer trypsin reactors. The highest overall rates of reaction were obtained when the reaction was bulk diffusion controlled and the measured rates were in agreement with those calculated from expressions derived from heat transfer theory. The design of reactors for the limiting cases of kinetic and bulk diffusion controlled reaction as well as a method for the determination of substrate diffusivity are outlined.
Immobilization of glucose oxidate on nickel‐silica alumina
Authors: Herring W. M. , Laurence R. L. , Kittrell J. R.
Published: December 2004

The preparation of a water‐insoluble derivative of glucose oxidase by covalent coupling to nickel on silica alumina is described. The catalyst has superior physical characteristics to conventional immobilized enzymes; although its activity and stability are now comparable to conventional catalysts, potential for significant further improvements has been demonstrated.
Film and pore diffusion studies with immobilized glucose oxidase
Authors: Rovito B. J. , Kittrell J. R.
Published: December 2004

Quantitative studies are reported of external film and internal pore diffusion effects for glucose oxidase immobilized on porous glass in a continuous, tubular, packed bed reactor. The McCune and Wilhelm model for external film diffusion is shown to describe these data over a particle Reynolds number range from 0.2 to 25. The Thiele analysis provides a good description of internal pore diffusion resistances. It appears that immobilized enzymes free of internal diffusional restrictions generally require the use of supports with pore sizes larger than those currently available in porous glass.
Enzymatic solubilization of an insoluble substrate, fish protein concentrate: Process and kinetic considerations
Authors: Archer Michael C. , Ragnarsson Jon O. , Tannenbaum Steven R. , Wang Daniel I. C.
Published: December 2004

The solubilization of fish protein concentrate (FPC) by Bacillus subtilis protease (Monzyme) has been investigated. Conditions have been defined which eliminate the problems of microbial contamination and salt accumulation. A Kinetic treatment revealed that enzyme is adsorbed to the surface of the substrate, exposed to aqueous phase. The overall kinetics were described by a sequence of two first‐order processes—an initial, fast reaction in which loosely bound polypeptide chains are cleaved from an insoluble protein particle, and a second, slower reaction in which more compacted core protein is digested.
Selective substrate utilization by marine hydrocarbonoclastic bacteria
Authors: Soli Giorgio , Bens E. M.
Published: December 2004

Several strains of bacteria, isolated from marine environments, were characterized for their hydrocarbon oxidizing abilities using a complex synthetic mixture of hydrocarbons. Attempts were made at a broad classification of these organisms on the basis of their behavior towards four major groups of hydrocarbons, normal paraffins, iso‐paraffins, cyclo‐paraffins, and aromatics, known to be present in crude oils. Although bacteria appear to be able to oxidize hydrocarbons at random, this study has shown that it may be possible to recognize a rudimental pattern if we view their oxidative abilities in terms of groups of hydrocarbons rather than individual compounds. A study of the action of combined strains on the synthetic hydrocarbon mixture was performed. It was found that no particular benefit could be derived as compared to the use of single strains.
Development of a recovery process for novobiocin
Authors: Belter P. A. , Cunningham F. L. , Chen J. W.
Published: December 2004

A periodic countercurrent whole beer process was developed for the recovery of novobiocin to eliminate the high cost of mycelium filtration and accompanying antibiotic losses in the filter cake. In such a process screened, but unfiltered, novobiocin fermentation beer is contacted with a special grade of an anionic‐exchange resin in a series of specially designed, well mixed columns. Each column is fitted with a screen sized to retain resin within the column but allow the cells to press. Periodically the lead column is isolated from the column train, washed free of beer solids, and eluted. A freshly eluted column is placed in the trail position to allow countercurrent operation. The eluate is then processed to crystalline product.

A mathematical model for the sorption of novobiocin was developed based on a suitable continuity equation and mass transfer and equilibrium relationships determined in the laboratory. Digital computations of this model correlated well with laboratory and pilot plant data, and predicted well the performance of the production units. This simulation has been in continued use to predict and reoptimize plant operation as process changes (such as improved beer titers, increased production rate, and the evaluation of superior ion‐exchange resins) occurred or were anticipated.
Methionine control of cephalosporin C formation
Authors: Drew Stephen W. , Demain Arnold L.
Published: December 2004

DL‐Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.
Accumulation of 5′ guanine nucleotides by mutants of Brevibacterium ammoniagenes
Authors: Furuya Akira , Okachi Ryo , Takayama Kenichiro , Abe Shigeo
Published: December 2004

5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture.
Mixed culture biooxidation of phenol. III. Existence of multiple steady states in continuous culture with wall growth
Authors: Pawlowsky U. , Howell J. A. , Chi C. T.
Published: December 2004

It is shown that two steady states exist in certain regions of operation of a 2‐liter continuous stirred tank biological reactor. Transition was made from one steady state to another by applying shock loads of either phenol substrate which is inhibitory to the culture at high concentrations or by adding large additional amounts of concentrated organisms. The existence of the multiple steady states is ascribed to the existence of wall growth, and their position is determined by the amount of wall growth. Transient behavior of the system did not follow the predictions of the simple wall growth model but the culture appeared to undergo a lag period immediately after applying the shock load to the system. It is concluded that the stability of a continuous culture utilizing an inhibitory substrate is improved by increasing the degree of wall growth and decreasing the substrate feed concentration. It is also concluded that small scale experiments can usually not be interpreted correctly unless the effect of wall growth is taken into account.
Large scale production of bacteriophage Φ × 174
Authors: Siquet‐Descans F. , Calberg‐Bacq C. M. , Delcambe L.
Published: December 2004

Bacteriophage Φ × 174 was produced in 20 and 200 liter fermentors. The phage was concentrated without any loss of infectivity by precipitation with 10% (w/v) Poly(ethylene glycol) 6000 in 0.5M NaCl. The total yield of a 100 liter culture is around 1016 infectious particles. The final purification step in a CsCl gradient results in a 60 to 80% inactivation.
Activity and specificity of covalently immobolized wheat germ agglutinin toward cell surfaces
Authors: Zabriskie Dane , Ollis David F. , Burger Max M.
Published: December 2004

Wheat germ agglutinin protein, which is able to agglutinate tumor cells better than normal cells, was covalently bound to polyacrylamide gel beads. The specific binding activity of the protein was preserved on these beads and was expressed heterogeneously by the binding of mouse leukemia cells (L1210) to the protein coupled gels. The selective activity of the immobilized protein was maximal when the number of sites available to covalently couple the protein was lowest. The application of this observation to the general field of covalent immobilization of proteins and enzymes may be of considerable utility.
Biological wastewater treatment model building fits and misfits
Authors: Boyle W. C. , Berthouex P. M.
Published: December 2004

The modeling of biological wastewater treatment processes has received much attention over the past ten years. Efforts are underway to develop a unified model for these processes which will greatly aid in the design and operation of wastewater treatment facilities. This paper presents a philosophical discussion of model building strategies augmented by a discussion of statistical problems associated with model parameter estimation and model discrimination. This discussion further illustrates with actual data, that goodness of fit is not a sufficient condition for model acceptance. Numerous rival models are examined to illustrate this point. In order to verify a model or to discriminate between rival models, they must be “Put in jeopardy.” If this attitude is not employed in model building efforts, important discrepancies in the proposed model may go undetected.
Development and application of the thin channel rectangular air lift mass transfer reactor to fermentation and waste‐water treatment systems
Authors: Gasner Larry L.
Published: December 2004

The consideration of fermentation principles in the treatment of industrial and sanitary waste waters leads to substantial process improvements. In particular, the rate of reaction can be improved by several fold by establishing the proper environmental conditions for microbial growth in a fermentation system. Recent work on the concentric cylinder air lift has shown it to be an economical fermentor with many advantages over conventional fermentors. An attempt to improve the economical performance of this system led to the development of the thin channel rectangular air lift fermentor. This was based upon a theoretical analysis of performance parameters. The analysis indicates decreased bubble coalescence, increased bubble entrainment, decreased power costs, increased mass transfer coefficients, and decrease capital costs. Experience using a prototype thin channel rectangular air lift system with sanitary and industrial waste‐water treatment systems has demonstrated exceptionally high rates of BOD removal at low operating and capital costs.
Effect of aeration intensity on the biochemical composition of baker's yeast. II. Activities of the oxidative enzymes
Authors: Oura Erkki
Published: December 2004

When the effect of catabolite repression is eliminated Saccharomyces cerevisiae prefers an aerobic metabolism. The potential for completely aerobic catabolism exists even in circumstances where its action is limited by the oxygen available. When the oxygen absorption in the medium is adequate, yeast uses a solely oxidative metabolism for energy‐yielding reactions. The changes observed in the activity of malate dehydrogenase can be described as a function of two isoenzymes, both of which are affected by oxygen; the isoenzyme participating in the glyoxylate cycle shows variations in activity similar to that observed in isocitrate lyase. NAD‐linked glutamate dehydrogenase activity roughly follows that of malate dehydrogenase and isocitrate lyase; in cultivations with the same growth rate the NADP‐linked dehydrogenase is insensitive to the oxygen level. The cytochromes aa3, b, and c have a clear maximum at low oxygen tension, the most sensitive being cytochrome aa3. The imbalance between cytochrome c:oxygen oxidoreductase activity and the amount of cytochrome aa3, and the correlation observed between respiration rate and the activities of cytochrome c oxidase and NADH2:cytochroine c oxidoreductase are discussed. Methods used for estimation of cytochromes are compared.
Production of a new thermostable neutral α‐galactosidase from a strain of Bacillus stearothermophilus
Authors: Delente J. , Johnson J. H. , Kuo M. J. , O'Connor R. J. , Weeks L. E.
Published: December 2004

An intracellular, thermostable, neutral α‐galactosidase (α‐D‐galactoside galactohydrolase EC 3.2.1.32) was produced in pilot plant quantities from a strain of Bacillus stearothermophilus. The organism was cultured at 50°C in a soluble neutral medium containing water extract of soybean meal (3%) and 0.5% yeast extract. The enzyme biosynthesis was inducible and sensitive to catabolite repression. After autolysis of the cells, the α‐galactosidase was selectively and quantitatively complexed from clarified beer directly onto DEAE Sephadex; and enzyme‐rich fractions were batchwise eluted with an increasing gradient of NaCl solutions. The eluates were given two consecutive isopropyl alcohol precipitations, and the aqueous solutions of the second precipitate were dialyzed and lyophilized. Final product activity recovery was 72% based on the crude fermentation beer. Best specific activity was 5.2 u/mg protein. Further laboratory purification (DEAE Sephadex and Bio‐Gel P200) yielded a product with 14.2 u/mg protein.
Death kinetics of yeast in spray drying
Authors: Elizondo Hector , Labuza T. P.
Published: December 2004

The death kinetics of a strain of Saccharomyces cerevisiae were studied in an industrial scale spray drier. In solution studies, the death kinetics of yeast was found to be comparable to pathogen destruction. From the studies in drying of yeast a prediction of a 4 log cycle decrease in viable cells of pathogens could be made for normal processing conditions. This should insure the safety of spray‐dried foods unless after contamination occurs. It was found that during drying, although the rate of death is high, the activation energy is greatly decreased over that of death in aqueous solution (reduction from 130 kcal/mole to 5 kcal/mole). The reduction in Ea may be attributed to the thermodynamic compensation phenomenon in which the resulting negative entropy of reaction acts to protect the cells through a water–protein interaction. However, the possibility of a change in death mechanism cannot be precluded. Overall, these results suggest the danger in extrapolating death kinetics to high temperature.
Continuous culture of Thiobacillus ferrooxidans on a zinc sulfide concentrate
Authors: Gormely L. S. , Duncan D. W. , Branion R. M. R. , Pinder K. L.
Published: December 2004

A zinc sulfide concentrate was leached microbiologically by Thiobacillus ferrooxidans in a continuous stirred tank reactor. A model was developed to predict, the leaching kinetics when the bacterial growth rate was not limited by any substrate other than the zinc concentrate, and it was modified to explain the observed results.

Stable steady sates were obtained over a range of dilution rates from 0.0171 to 0.1038 hr−1. Because a solid substrate was used, the specific growth rate of the bacaeria was not a unique function of the subastrate concentration, and conventional contnuous culture theory based on the Monod equation did not apply to this system. The leaching rates and bacterial growth rates were first order in mineral surface area cocentration.
A rational approach for predicting the dissolved oxygen profile in receiving waters
Authors: Peil K. M. , Gaudy A. F.
Published: December 2004

A practical method for predicting the dissolved oxygen (DO) profile in a stream receiving biodegradable waste is presented. In this method the BOD (accumulated O2 uptake) curve is obtained using an open jug technique. The accumulated O2 uptake curve thus developed is employed in numerical integration with the physical reaeration data for the receiving stream to predict the DO profile in the stream. In the present study, the method was examined using 10‐liter open jar reactors to obtain the O2 uptake curves, and the receiving stream was a 670‐liter simulated stream apparatus which has been employed in previous studies on stream reaeration. The method was found to provide a fairly good prediction of the actual dissolved oxygen profile observed in the receiving stream. The effect of the reaeration constant, K2 )agitation effect(, on the kinetics of O2 uptake was also investigated and it was found that increased agitation (higher K2 value) caused some increase in the accumulated oxygen uptake (BOD) curve with most of the increase coming after the so‐called “plateau” area in the O2 uptake curve, i.e., in the general case, after the low point along the DO sag curve.
Some experiments and analysis of a predator‐prey model: Interaction between Colpidium campylum and Alcaligenes faecalis in continuous and mixed culture
Authors: Sudo Ryuichi , Kobayashi Kobee , Aiba Shuichi
Published: December 2004

Following previous work in which a mass and monoxenous culture of Vorticella microstoma had been successfully established (Water Res., 7, 615 lpar;1973) another species of Ciliata, Colpidium campylum was subjected to continuous cultivation using Alcaligenes faecalis as the sole bacterial food and asparagine as the limiting substrate. This work was primarily undertaken to reveal the interaction and biological oscillation between these two types of organisms which simulate theecological behavior of activated sludge.

The fact that the bacteria tended to flocculate and/ or deflocculate depending on the protozoan populastion density was incorporated into the rate equations to account for the oscillation in individual population density of the predator‐prey system The mathematical approach presented earlier by canal and other workers forbiological oscillation used a homogeneously of the bacterial food wasoverelooked in the earlier publication.
Electrochemical evaluation of glucose oxidase immobilized by different methods
Authors: Lahoda E. J. , Liu C. C. , Wingard L. B.
Published: December 2004

Glucose oxidase electrodes were constructed on a platinum screen using polyacrylamide gel, glutaraldehyde crosslinking, and glutaraldehyde crosslinking with +0.04 volts dc on the platinum screen as the methods of enzyme immobilization. The electrodes were evaluated in an electrochemical cell for the oxidation of glucose at the enzyme electrode and the reduction of oxygen at a platinum auxiliary electrode, using constant current voltametry or under external load operation. The method of immobilization affected the extrapolated opencircuit potential as well as the half‐cell potential and the steady current under external load operation. The charged glutaraldehyde electrode gave the best current performance; however, the small output (microamps) indicated that major problems in electron transfer from an enzyme catalyst to an external circuit must be resolved before such electrodes can be used in practical application.
Sucrose inversion by immobilized yeast cells in a complete mixing reactor
Authors: Toda K. , Shoda M.
Published: December 2004

Using whole cell invertase of Saccharomyces pastorianus, entrapped in spherical agar pellets, sucrose hydrolysis was carried out in a continuously fed fluidized bed reactor. The effective rate of reaction determined experimentally for the catalytic pellet was correlated with particle radius (R), intraparticle concentration of enzyme (Ep) and external concentration of substrate (SR).

The results were elucidated by theoretical analysis incorporating internal mass transfer resistance. At high degrees of diffusional resistance, the effectiveness factor was successfully estimted from Bischoff's equation. A dimensionless number, mA  R(k2Ep/KmD)0.5(Km/(Km + SR)), was used conveniently to predict the effectiveness factor in those cases wher the intraparticle diffusional effect was less significant. This number was employed to determine critical pellet size for an optimal reaction.

The relationship between the properties of the pellet (size and intraparticle enzyme activity) and its apparent kinetic constants (k′2 and K′m), estimated according to Lineweaver‐Burk, are discussed.
Dynamic response of a polarographic oxygen probe
Authors: Kok R. , Zajic J. E
Published: December 2004

The dynamic characteristic of dissolved oxygen probes is usually modeled as being equivalent to a single diffusion layer. Other workers have shown that in response to a downstep in oxygen tension a polarographic probe initially follows single diffusion layer dynamics but that during the last 10% of response the probe deviates significantly from this behavior.

Probe response to a series of downsteps of various magnitudes after exposure to calibration gases for 1, 2, and 3 min was recorded. When the probe membrane was new the response behavior was found to be largely independent of the step size as well as the exposure duration.

The deviation fro the single diffusion layer model was explained in terms of lateral diffusion of oxygen from the anodic compartment to the cathode. By use of a model incorporating the lateral diffusion, probe response to a general oxygen tension‐time function was calculated.
Using multiresponse data to estimate biokinetic parameters
Authors: Johnson David B. , Berthouex Paul M.
Published: December 2004

Biokinetic parameters are usually calculated from slopes and intercepts taken from plots of experimental data. One response at an item is plotted and used for parameter estimation. Aside from problems that may be caused by transformations made when the data are plotted, this approach has the weakness of not using all the data simultaneously when there is more than one response. This paper shows how multiresponse biological data can be handled to get parameter estimates that are much more precise than those obtained using conventional methods.
Continuous coagulation of milk using immobilized enzymes in a fluidized‐bed reactor
Authors: Cheryan M. , Van Wyk P. J. , Olson N. F. , Richardson T.
Published: December 2004

Milk‐clotting enzymes such as pepsin, chymosin, chymotrypsin, and M. miehei proteases were immobilized on porous, alkylamine glass and incorporated into a fluidized‐bed continuous coagulation scheme. Only pepsin and calf rennet retained sufficient activity towards skim milk to warrant further studies. Comparison of kinetic data with fixed‐bed reactors revealed the overall superior performance of fluidized beds; higher clotting activities were possible while avoiding plugging problems and high pressure drops common to fixed‐bed reactors. Film diffusion and catalyst back‐mixing appear to be significant factors in the overall kinetics.

All enzymes lost activity on exposure to skim milk. The inactivation rates were lower at high substrate pH and insignificantly affected by reactor temperature. Nitrogen and sialic acid accumulation on the porous glass paralleled the loss in activity in the initial stages. Attempts to regenerate the immobilized enzymes were partially successful.
Temporal structure of S phase analyzed using an automated synchrony system
Authors: Klevecz Robert R.
Published: December 2004

An automated system for cell cycle analysis is described in which synchronous populations of greater than 95% mitotic cells can be selected and subcultured without resorting to inhibitors or altered media. Selection of mitotic cells from roller bottle cultures generates synchronous populations of up to 5 × 107 cells, as often as every 30 min, under conditions of constant temperature. A detailed analysis of the temporal pattern of DNA synthesis in the S phase of several Chinese hamster cells is presented. Several potential artefacts and uncertainties regarding the cell cycle blockage point in S phase synchronization are discussed.
Primary suspension culture of calf anterior pituitary cells on a microcarrier surface
Authors: Horng Chi‐Byi , McLimans William
Published: December 2004

In order to achieve a steady‐state primary culture system for mammalian cells, with the potential to eventually correlate and control cell function and growth, a serious evaluation of various suspension systems was made. Calf anterior pituitary cells were employed as a differentiated cell type and successfully cultivated in a microcarrier suspension culture system. DEAE‐Sephadex was demonstrated to be a satisfactory type of microcarrier. The cells readily attached to the bead and, after a short lag period, they actively proliferated on the bead surface to yield growth of a predominantly epithelial cell type. Under specific conditions the microcarrier supported primary cell growth up to levels of 2 × 106 cells per ml. High bead concentrations inhibited cell growth. The inhibition could be overcome by using proportionately higher cell inoculum so that a concentrated culture with 5 × 106 cells per ml was achieved. The inhibitory effect of high bead concentration was found to be due to the absorption of serum protein and certain growth enhancing factors. The fact that the growth enhancing factors were released from cells during the period of trypsinization and were both thermostable and nondialyzable, seems to suggest one approach to a dialysis culture system. In addition, relatively trauma‐free primary cell cultures can be achieved by using explant culture without prior trypsinization. In microcarrier suspensions direct growth of primary rat mammary tumor cells was also demonstrated.
Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain
Authors: Lasfargues Etienne Y. , Lasfargues Jennie C.
Published: December 2004

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one‐year‐old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.
Growth and immunogenicity of foot‐and‐mouth disease virus in baby hamster kidney cells adapted to and continuously grown in a serum‐free chemically defined media
Authors: Tomei L. D. , Issel C. J.
Published: December 2004

The production of foot‐and‐mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum‐free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK‐21 cells adapted to and continually passed in a glutamine‐free autoclavable, chemically defined medium (BHK‐S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10−1 to 10−7 plaque‐forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement‐fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 108.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK‐S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.
Effects of growth conditions on the oxygen equivalence of microbial cells
Authors: Grady C. P. L. , Findley P. L. , Muck R. E.
Published: December 2004

A natural microbial population of sewage origin was grown at four temperatures (10, 20, 30, 40°C) and five mean residence times (3, 6, 12, 18, 24 hr) in a continuous stirred tank reactor. Samples of cells were removed during steady‐state operation and analyzed for chemical oxygen demand (COD), ash content, protein, carbohydrate, and nucleic acids. The oxygen equivalence (OE) of the cells was measured directly as g COD/g ash‐free cells and was determined indirectly by calculating the calorific content from the macromolecular composition. The results indicate that OE is a function of growth conditions. Furthermore the COD test is a poor measureof OE because of a high level of variation associated with the test. Additional studies should be performed using bomb calorimetry to establish a quantitative relationship between growth conditions and OE.
An automatic homocontinuous culture apparatus
Authors: Ferrara Romano , Grassi Sergio , Del Carratore Giovanni
Published: December 2004

An apparatus for the cultivation of microorganism under controlled conditions is described. This apparatus is basically a turbidostat improved with automatic devices for sample withdrawal and tube cleaning. The results of experiments, using Euglena gracilis to check the constancy of some physiological parameters (in particular phototactic reactivity), are reported.
Effect of carbon dioxide on the formation of α‐amylase by Bacillus subtilis growing in continuous and batch cultures
Authors: Gandhi A. P. , Kjaergaard L.
Published: December 2004

The influence of CO2 on the formation of α‐amylase by Bacillus subtilis NCIB 8646 growing in continuous and batch cultures was investigated.

Different levels of CO2 examined in the batch cultures stimulated the formation of α‐amylase, with the highest activity being obtained using 6% CO2(V/V). The additions of CO2 inhibit the growth and division ofvegetative cells of B.subtilis when CO2 is present in a concentration of more than 3%(V/V).

In chemostat cultures, air containing 8% CO2 (V/V) increased the specific enzyme productivity almost three times over the control, without affecting the cell growth.

An attempt is made to correlate the obtained results withthe already established theory for the regulation of the α‐amylase synthesis.
Kinetic Behavior of Microencapsulated β‐Galactosidase
Authors: Wadiak D. T. , Carbonell R. G.
Published: December 2004

Escherichia coli β‐D‐galactosidase (E.C. 3.2.1.23) was immobilized in cellulose nitrate membrane microcapsules and the reaction kinetics with o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG), lactose, and whole milk were studied using both continuous stirred tank and packed bedreactor configurations. The results of the experiments gave effectiveness factors of 0.3 for ONPG, 0.6 to 0.7 for lactose in solution, andclose to unity for lactose in milk. Using a coupled mass transfer and kinetic model, it was possible to estimate the permeability of the microcapsule membrane from the reactor data. Membrane permeabilities on the order of 5 × 10−3 and 3 × 10−4 cm/sec were estimated for ONPG and lactose, respectively. It was determined that the membrane was the limiting mass transfer resistance for the overallreaction. The analysis showed that within the microcapsule, the reaction is reaction rate limited for lactose and slightly diffusion limitedfor ONPG.
Submerged cultivation of a thermotolerant basidiomycete on cereal flours and other substrates
Authors: Hofsten Bengt V. , Rydéean Anna‐Lisa
Published: December 2004

The imperfect state, Sporotrichum pulverulentum, of thecellulolytic basidiomycete Phanerochaete chrysosporiumhas been grown on barley flour and other agricultural products in shake cultures, in an air‐lift fermentor, and in stirred fermentors. The growth morphology varied with cultivation conditions, but it was possible to maintain heavy suspensions of loosely associated mycelia in fermentors. The fungus can grow in temperatures up to 40°C and use ammonium salts or organic nitrogen sources to convert sugars, starch, pectin, and various seed residues to a biomass containing 30–40% protein with a favorable amino acid composition. Serial cultures were grown on flours under conditions where the larger part of the culture was withdrawn and replaced with new medium every, or every other, day. The mycelia are easy to harvest by filtration and have several properties which make the product attractive as a potential food ingredient.
Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast
Authors: Peitersen Nicolai
Published: December 2004

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO2 and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.
The total containment of a batch‐type zonal centrifuge
Authors: Webb N. L. , Richards B. M. , Gooders A. P.
Published: December 2004

A batch‐type zonal centrifuge has been modified and totally contained for use with biologically hazardous materials.

A sealed cabinet encloses the centrifuge and the ancilliary equipment. It is operated with a flow of filtered air when the zonal system is on, decontaminated with ethylene oxide, and maintained at a negative pressure throughout. The centrifuge subsystems can be drained, flushed, and decontaminated with ethylene oxide before an engineer services the machine. The sample handling system within the cabinet is remotely controlled.
Studies on the production of bacterial rennet in a pilot plant fermentor
Authors: Rao Lavu Krishna , Mathur D. K.
Published: December 2004

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K‐26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K‐26 using 5% wheat bran medium in a 13 liter fermentor.
Kinetic studies on insoluble cellulose–cellulase system
Authors: Huang Andrew A.
Published: December 2004

Enzymatic hydrolysis of insoluble amorphous cellulose by Trichoderma viride cellulase was investigated in a batch reactor at several substrate concentrations and three enzyme levels. The reactions were carried out at 50°C and pH 4.8. Enzyme was rapidly adsorbed onto solids on contact, then gradually returned to the liquid phase as the reaction proceeded. A kinetic model that considered the fast adsorption which was followed by the slow reaction, and subsequent product inhibition was developed to interpret the experimental observations. The resulting equation successfully correlated the data for up to 70% conversion. The methods for determining the kinetic parameters are discussed.
Preparation and kinetic properties of gel entrapped urate oxidase
Authors: Hinberg I. , O'Driscoll K. F.
Published: December 2004

Urate oxidase from hog liver (urate: oxygen oxidoreductase, EC 1.7.33) has been entrapped in a crosslinked 2‐hydroxyethyl methacrylate gel with a 47% retention of activity. The kinetic behavior of the gel entrapped enzyme has been studied in a slurried tank reactor using uric acid as substrate. Internal diffusion effects were found to be negligible for particle sizes below 128 μm. A threefold increase in Km (app) was observed for the 128 μm particles and attributed to diffusional effects. The pH activity profile of the gel entrapped enzyme was bell‐shaped at high substrate concentration and could be fitted to a titration curve of two ionizable groups, a basic group having a pK of 7.9 and an acidic group with a pK of 11.0. The gel entrapped enzyme showed excellent stability between pH 6.5 and 10.5.
New suspension culture system (Mini‐AR)
Authors: Dynesius Roselynn , Lange Robert D. , Eraslan Arsev H. , Snyder William T.
Published: December 2004

A new suspension culture system (Mini‐AR) based on the Stoke's drag law for suspended particles is described. This apparatus can be utilized for the maintenance of mammalian kidney cells in both short‐ and long‐term cultures.
The response of a bioelectrochemical cell with Saccharomyces cerevisiae metabolizing glucose under various fermentation conditions
Authors: Videla H. A. , Arvía A. J.
Published: December 2004

Working conditions of a biochemical fuel cell formed by an oxygen cathode and a platinum bioanode in a Saccharomyces cerevisiae suspension metabolizing glucose are described. The biocell response in terms of bioanode potential and current drainage under different fermentation conditions is reported. A kinetic equation relating the current, the number of microorganisms, and the substrate concentration is obtained. The bioanode potential corresponds to that of an oxygen concentration polarization cell.
On‐line measurement of gas‐exchange conditions in fermentation processes
Authors: Nyiri Laszlo K. , Toth Gizella M. , Charles Marvin
Published: December 2004

Gas‐exchange conditions of C. ulilis cultures were analyzed by an on‐line, real‐time operating digital computer interfaced with a 250 liter, highly instrumented fermentor.

Information obtained from the computer was correlated with wet chemical analytical data related to nucleic acid, protein, and ethyl alcohol production rates. Once the correlation was established, the computed gas‐exchange data, especially the RQ which functions as an indicator of the cells physiological condition, could be used as a real‐time process status indicator which follows or forecasts events during the fermentation.

It was also found that electrical noises generted on the sensor‐instrument level may handicap the interpretation of the meaning of computed process status indicators. Since “clear” data is considered as a prerequisite to follow the physiological conditions and perform control of environmental variables, a special computer program is necessary to filter the electric noises at the sensor‐instrument level prior to the on‐line, real‐time data analysis for process status identification.
Interparticle mass transfer study with a packed column of immobilized microbes
Authors: Toda K.
Published: December 2004

The kinetics of whole‐cell lactase of Escherichia coli immobilized in spherical agar gels was determined under the influence of interparticle mass transfer in a fixed bed reactor. The dependence of the pseudokinetic parameters (K′m and v′max) fluid conditions was in accordance with the prediction derived from combinedeffects of kinetics and mass transfer. Within the limited conditions of theexperiments, it was observed that the apparent Michaelis constant could be represented by the following simple equation, K′m/Km = 1 + (ϕ/kLa)(vmax/Km).

The mass transfer coeflicient (kL) needed in the theoretical analysis was determined in correlation with particle size (0.12–0.45 cm) and liquid flow rate (0.30–12.0 cm/min) using the more stable enzyme, cell‐bound invertase of Saccharomyces pastorianus, as the material to be entrapped in the gelatinous particles. The relationship was expressed in the following dimensionless equation, (1–ϕ)Sh = 2 + 0.6Re1/2Sc1/3, except that marked deviation resulting in the reduction of kL was noticed with a Reynolds number less than 0.8.
Effectiveness factors for substrate and product inhibition
Authors: Wadiak D. T. , Carbonell R. G.
Published: December 2004

The transformation technique of Na and Na (Math. Biosci., 6, 25, 1970) is extended to convert boundary‐value problems involving the steady‐state diffusion equation for spherical immobilized enzyme particles exhibiting substrate and product inhibition to initial‐value problems. This allows a study of the influence of external mass transfer resistances on the effectiveness factors. It also considerably reduces the number of calculations required to investigate the effect of changes in the kinetic parameters on the overall rate of reaction. The existence of multiple steady states for substrate inhibition kinetics in spherical catalyst particles is illustrated and a criterion for uniqueness of steady states is developed. Effectiveness factors for competitive and noncompetitive product inhibition increase with increasing value of the Sherwood number for the substrate and increasing value of the ratio of substrate to product effective diffusivities within the particle.
Some properties of a protease (subtilisin BPN′) immobilized to porous glass
Authors: Chapman J. Douglas , Hultin Herbert O.
Published: December 2004

Subtilisin BPN′ was immobilized to porous glass via isothiocyanate coupling. The pH optimum of the enzyme was shifted to the alkaline side on binding. This effect was more pronounced with ethyl lactate than with N‐tosyl arginine methyl ester (TAME). Presumably, the shift is a reflection of the negative charge on the surface of the glass. The Michaelis constant and Vmax of soluble subtilisin BPN′ with TAME were two and one orders of magnitude, respectively, lower than with ethyl lactate. Vmax, calculated per g of active enzyme, with TAME as the substrate was not affected by immobilization, while Vmax with ethyl lactate decreased greater than tenfold. The apparent KM decreased on immobilization with ethyl lactate as substrate and increased with TAME. Results are explained in terms of diffusional resistance and a possible attraction of ethyl lactate to the glass surface. Active site titration indicated that about 25%, of the immobilized enzyme was active.
Urocanic acid production using whole cells immobilized in a hollow fiber reactor
Authors: Kan J. K. , Shuler M. L.
Published: December 2004

The feasibility of using hollow fiber membrane dialyzers (C‐DAK) for immobilization of microbial whole cells was investigated. The cells are located on the shell side of the dialyzer, while substrates and products are free to diffuse across the hollow fiber membranes. The biochemical reaction studied was the conversion of L‐histidine to urocanic acid and catalyzed by L‐histidine ammonia‐lyase. C‐DAK dialyzers containing a heat‐treated suspension of Pseudomonas fluorescens ATCC 11299b (with L‐histidine ammonia–lyase activity) were incorporated into constant volume recycle reactor systems for continuous product formation. A simple model successfully correlated the data and predicted performance. It was found that the reaction was not likely to be diffusion limited, and such a cell immobilization scheme is convenient and workable for continuous production of biochemicals.