bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde
Authors: Ohmiya Kunio , Tanimura Shuya , Kobayashi Takeshi , Shimizu Shoichi
Published: December 2004

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA‐1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the Km values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β‐galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose‐6‐phosphate dehydrogenase, and xanthine oxidase became inactive.
Continuous dehydrogenation of a steroid with immobilized microbial cells: Effect of an exogenous electron acceptor
Authors: Yang Ho Seung , Studebaker Joel F.
Published: December 2004

Whole cells of Pseudomonas testosteroni, induced to synthesize steroid‐transforming enzymes beforehand, have been immobilized by entrapment in polyacrylamide gel. The immobilized cells have then been used to catalyze the continuous Δ1‐dehydrogenation of Reichstein's substance S under various conditions in the presence of phenazine methosulfate (PMS), an electron acceptor for the cell‐free Δ1‐dehydrogenase. The presence of PMS substantially increases the rate of reaction when fed with the steroid substrate to a continuous stirred tank reactor containing the immobilized cells. The operational half‐life of the Δ1‐dehydrogenase activity of the cells, about 103 hr under the best operating conditions, is essentially unaffected by the presence of PMS. Though the acceleration of the reaction may be due to PMS‐mediated passage of electrons from some component in the electron transport chain to molecular oxygen, the lack of a similar effect with methylene blue is consistent with the conclusion that PMS functions directly as the electron acceptor for the Δ1‐dehydrogenase.
Determination of redox potential levels critical for cell respiration and suitable for L‐leucine production
Authors: Akashi Kunihiko , Ikeda Shigeho , Shibai Hiroshiro , Kobayashi Kaetsu , Hirose Yoshio
Published: December 2004

The effect of oxygen supply on L‐leucine fermentation was investigated employing a leucine‐producing mutant of Brevibacterium lactofermentum. Since it was not possible to measure oxygen tension below 0.01 atm by a Teflon‐coated oxygen electrode, the degree of satisfaction of the cells' oxygen demand (cells' respiration rate/maximum oxygen demand of cells, rab/KrM) and the redox potential of the culture medium (E. mV) were used as indices to oxygen supply in cultures under low oxygen tension. When the oxygen demand of the cells was satisfied (rab/KrM = 1.0) and the E value was between −90 and −110 mV, L‐leucine formation was 26.5 mg‐ml. When the oxygen demand of the cells was not satisfied (rab/KrM = 0.85) and the E value was between −200 and −220mV, L‐leucine accumulation was 29.7 mg/ml. When the oxygen supply was extremely limited (rab/KrM = 0.27) and the E value was −280 mV. L‐leucine formation was 12.9 mg/ml. A new method which simultaneously measures the redox potential and dissolved oxygen was applied to the determination of the critical dissolved oxygen level for cell respiration (PL crit), which was too small to be detected by conventional oxygen electrodes. The value of PL crit of the leucine producer was estimated as 0.0002 atm.
Disintegration of dried yeast cells and its effect on protein extractability, sedimentation property, and viscosity of the cell suspension
Authors: Lee Cherl‐Ho , Tsang Shek Kwan , Urakabe Rintaro , Rha Cho Kyun
Published: December 2004

The morphology of dried Candida lipolytica yeast suspended in aqueous solutions (H2O, 0.4% NaOH, 2N HCl, and 6N HCl) and organic solvents (95% alcohol and acetone) was studied using a scanning electron microscope (SEM) and an optical microscope. The effect of high‐pressure homogenization on cell‐wall structure and cell clumps was also determined. The protein extractability, sedimentation property, and viscosity of cells subjected to different mechanical and chemical treatments were also investigaged. The dried yeast cells were in a spherical agglomeration consisting of 100s of closely bound cells. The clump was resistant to water, aqueous 2N HCl solution at 25°C, 95% alcohol and acetone, but vulnerable to 6N HCl, aqueous 0.4% NaOH solution, and homogenization. The homogenization of the cell suspension not only broke the clump but also cracked the cell‐wall structure. The aqueous alkaline solution could have weakened the cell wall and increased the solubility of the protein released through the cracks in the cell wall. The destruction of the agglomeration and the cell‐wall structure increased the hydration of the cell and thereby increased the stability of the suspension. The sedimentation and the viscosity of the cell suspension corresponded to the morphological changes and the extractability of protein in the cell suspensions with different treatments.
Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water–organic solvent systems
Authors: Carrea G. , Colombi F. , Mazzola G. , Cremonesi P. , Antonini E.
Published: December 2004

The hydroxysteroid dehydrogenases: β‐HSDH, 20β‐HSDH, and 3α‐HSDH, were immobilized on CNBr‐activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40–60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, β‐HSDH maintained 60% of its original activity two months after continuous use in the water–ethyl‐acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki > 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water–organic solvent systems.
Potentiometric method for subatrate analysis using immobilized NAD+‐dependent oxidoreductase enzymes
Authors: Chen A. K. , Liu C. C. , Schiller J. G.
Published: December 2004

Two coenzyme‐dependent oxidoreductases, glucose dehydrogenase and alcohol dehydrogenase, were immobilized in polyacrylamide gel over a platinum grid matrix and used as enzyme electrodes to measure their substrate concentrations in buffered aqueous solutions. The immobilized enzymes were used to oxidize their substrates in the presence of NAD+. Ferricyanide was used as the redox mediator and electroactive specific. The determinations of glucose and ethenol were utilized to demonstrate and evaluate the performance of the system. The described methodology should be readily applicable to the analysis of numerous other substrates of coenzyme‐dependent oxidoreductases.
Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis
Authors: Gierhart Dennis L. , Potter Norman N.
Published: December 2004

Candida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en‐zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu‐bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS‐PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub‐units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.
Influence of substrate on the amino acid profile and macromolecular composition of a Brevibacterium species
Authors: Miller Leslie T. , Thayer D. W.
Published: December 2004

Brevibacterium JM98A (ATCC 29895) was grown aerobically in carbon‐limited continuous culture on the following substrates: gluconic acid, galactose, fumaric acid, glutamic acid, and aspapagine. Both whole and trichloacetic acid(TCA)‐ex‐tracted cells were analyzed for their amino acid compositions. No significant variations of amino acid profile were induced by change of substrate. Only the valine content varied significantly with growth rate. Some significant variations were observed between whole and extracted cell samples, primarily in the levels of the essential amino acids threonine, cystine, and valine.
Effects of external diffusion and design geometry on the performance of immobilized glucose isomerase reactor system
Authors: Lee S. B. , Kim S. M. , Ryu Dewey D. Y.
Published: December 2004

Using immobilized glucose isomerase, the effects of superficial velocity of the reaction solution flowing through a packed‐bed reactor on the apparent kinetic constants of reversible reaction system were studied. The results showed that the apparent kinetic constants, both Vm″ and Km″, of the forward reaction varied significantly as the superficial velocity is changed, whereas those of the reverse reaction varied only slightly. Using the kinetic data determined experimentally, computer simulation of the enzyme reactor performance was carried out, and the importance of the external mass transfer in the proximity of immobilized‐enzyme particles was recognized. The reactor performance, expressed in terms of productivity, was examined as a function of the reactor height‐to‐diameter ratio, H/D. The productivity of the reactor system goes through a maximum value at a H/D ratio of about 1.6. and decreases as the H/D ratio increases. Theoretical analysis of the reaction kinetics of immobilized‐enzyme system that has reversible reaction kinetics is also presented. The experimental results showed good agreement with the results found from the theoretical analysis and the computer simulation studies. Based on the principles of the methods and the results presented in this paper, it is anticipated that one can predict the optimal design and operating conditions for the glucose isomerase reactor system and that application of the results could be extended to other enzyme systems with reversible reaction kinetics.
Separation of gentamicin by foaming
Authors: Lee S. B. , Ryu Dewey D. Y.
Published: December 2004

In order to develop a more economical separation method and to determine the feasibility of such a method for the recovery of gentamicin from fermentation broth, a foam separation process was evaluated using a batch system. The effects of such process variable as surface‐active agent, pH, collector–colligent ratio, and the gas flow rate on the separation efficiency of gentamicin were studied. Form the experimental results, the optimal operating conditions selected were: pH below 9.0, collector‐to‐colligent ratio of 3, and airflow rate at 0.5 vol air/vol liquid/min. Under these operation conditions, the average recovery efficiency of gentamicin was 73% when sodium dodecyl sylfate was used as a collector. A mathematical expression for the foam separation rate was derived. The theoretically predicted values of separation efficiencies agreed reasonably well with the experimental results.
Analysis of the coupled transport, reaction, and deactivation phenomena in the immobilized glucose oxidase and catalase system
Authors: Reuss M. , Buchholz K.
Published: December 2004

In a previous paper, the overall or macrokinetics of the immobilized glucose oxidase–catalase system has been presented. In this paper a detailed analysis of the interaction of diffusion and reaction in this system will be presented. The mathematical treatment includes two consecutive reactions with two‐substrate kinetics. Furthermore, the deactivation of both enzymes due to the intermediate product peroxide is taken into account. The predicted results suggest that the efficiency of the glucose oxidase reaction depends on the concentration ranges of the two substrates. Furthermore, the external mass‐transfer rate may cause a shift from glucose limitation to oxygen limitation. The efficiency of the coupled system is always higher than that predicted for the uncoupled reaction path. The calculations show that the economics of the coupled system depend a great deal on the deactivation of the enzymes.
Hydrophobic esters of cellulose: Properties and applications in biochemical technology
Authors: Dixon James , Andrews Philip , Butler Larry G.
Published: December 2004

We have investigated the utility for enzyme immobilization of several hydrophobic cellulose esters, as a function of solvent composition, extent of esterification, and enzyme. Phenoxyacetyl cellulose was also used for immobilization of rat liver microsomes, hydrophobic chromatography of proteins, and removal of Triton X‐100 from protein solutions. Phenoxyacetyl groups esterified to cellulose were much less subject to enzymatic hydrolysis than soluble phenoxyacetyl esters.
Temperature effects on ethanol and isopropanol utilization by Candida krusei
Authors: Kilian S. G. , Prior B. A. , Lategan P. M. , Kruger W. C. J.
Published: December 2004

Candida Krusei has a optimum growth temperature of 37°C on SASOL ethanol‐isopropanol mixture. The organism was unable to grow on isopropanol, but oxidized it partially to acetone in the presence and absence of ethanol. Growth at 40°C in the alcohol mixture was slightly faster than at 30°C over an ethanol concentration range of 0.43 to 3.6% (v/v), although at both temperatures the growth rate declined continuously with increasing concentration. At an ethanol concentration greater than 3.6% (v/v), the mixture was much more inhibitory to growth at 40 and 30°C. The inhibitory effect was due to the ethanol rather than the isopropanol. Metabolites such as acetate, acetaldehyde, and ethyl acetate accumulated in the medium, but the degree of accumulation depended upon the temperature and alcohol mixture concentration. At 40°C, acetaldehyde and acetate accumulated to a greater extent than 30°C on a 4.0% (v/v) synthetic alcohol mixture and this may also cause the greater inhibition at this temperature. The alcohol mixture is unsuitable for single cell protein (SCP) production in batch culture because of the low cell densities observed at all alcohol concentrations.
Enhanced product formation in continuous fermentations with microbial cell recycle*
Authors: Bull Daniel N. , Young Mark D.
Published: December 2004

The effect of partial recycle of microbial cells on the operation of a chemostat has been investigated for two fermentations. Stable steady states without partial cell recycle were obtained for the conversion of D‐sorbitol to L‐sorbose by Gluconobacter oxydans subsp. suboxydans 1916B and for the conversion of glucose to 2‐ketogluconic acid by Serratia marcescens NRRL B‐486. The employment of partial cell recycle dramatically increased product formation rates for both fermentations.
Automated fermentation equipment I. program‐controlled fermentor
Authors: Veres A. , Nyeste L. , Kurucz I. , Kirchknopf L. , Szigeti L. , Holló J.
Published: December 2004

A fermentation system with a plug scheme unit has been developed, offering a variety of solutions to measurement, control, and operational problems. By means of the program unit, e.g., automatic pH control assigned to the dynamic of batch cultures and the feed of different ingredients controlled by a time program or a given variable have been solved. The continuous culture volume was controlled by a level controlled by a level controller equipped with a photosensor. A method was developed for variable control that provide information on the activity of the culture, and allows direct measurement of the different rate values, e.g., generation time or specific product formation rate. Applicability of the direct measurement of generation time is presented in the qualification of molasses and in a static off‐line optimization process.
Automated fermentation equipment II. Computer–fermentor system
Authors: Nyeste L. , Szigeti L. , Veres A. , Pungor E. , Kurucz I. , Holló J.
Published: December 2004

An expensive computer‐operated system suitable for data collection and steady‐state optimum control of fermentation processes is presented. With this system, minimum generation time has been determined as a function of temperature and pH in the turbidostat cultivation of a yeast strain. The applicability of the computer‐fermentor system is also presented by the determination of the dynamic KLa value.
Environmental phenotypic variation of Scenedesmus dimorphus in high‐rate algae ponds and its relationship to wastewater treatment and biomass production
Authors: Oron Gideon , Shelef Gedaliah , Levi Anna
Published: December 2004

During the course of operating high‐rate algae ponds (HRAP) for wastewater treatment and protein production, changes were found in the two main algae species. The observed changes were interpreted to be a reflection of the operation regime and loading combined with environmental conditions. To verify that these changes were phenotypic and not genetic, experiments were conducted on Scenedesmus dimorphus growing in miniponds (110 L) as well as in the laboratory. The results showed that the changes in S. dimorphus were external and due to the changes in the loading and operating conditions of the ponds adjusted to changing environmental conditions. It was found that wastewater treatment efficiency and algal yield are also correlated with the S. dimorphus type.
Enzyme stabilization by linear chain polymers in ultrafiltration membrane reactors
Authors: Greco Guido , Gianfreda Liliana
Published: December 2004

The experimental results discussed in this article concern p‐nitrophenylphosphate hydrolysis by acid phosphatase in an Ultrafiltration membrane reactor. The basic conclusions drawn are: (1) Linking the enzyme to a soluble support does not give rise to an increase in its stability while the chemical manipulations involved result in marked reductions in enzymic activity. (2) Enzyme entrapment within a proteic gel produces a considerable increase in its thermal stability as compared to the diluted native enzyme; this presumably stems from drastic reductions in enzyme mobility. (3) Correspondingly, considerable reductions occur in enzyme activity that depend on substrate mass transfer resistances within the gel layer. (4) Small amounts of linear chain water‐soluble synthetic polymers (polyacrylamides) give rise to high macromolecular concentration levels in the reactor region where the enzyme is dynamically immobilized and produce the same enzyme stabilization as gel entrapment. (5) Only minor substrate mass transfer limitations take place in this region and hence enzyme activity is virtually unaffected. (6) Both effects (stabilization and slight activity reduction) seem not to depend strongly on the characteristics of the soluble polymer (molecular weight and ionic character).
The activity of immobilized enzymes on different krill chitin preparations
Authors: Synowiecki Józef , Sikorski Zdzisław E. , Naczk Marian
Published: December 2004

The suitability of krill chitin, prepared by using different concentrations of KOH and HCI for deproteinization and demineralization, respectively, was investigated. The activity of enzymes immobilized on such supports depends on the degree of deproteinization of chitin, availability of amino groups, content of minerals, mesh size, structure of the surface, and conformation of the chitin molecules. It was found that invertase and amyloglucosidase achieved high activity after immobilization on chitin obtained at not too rigorous conditions of deproteinization. However, the activity of immobilized α‐amylase and diastase increased significantly with the increase in concentration of KOH used for deproteinization. High content of minerals and proteins in chitin preparation causes a loss of immobilized enzyme activity.
Effect of oxygen transfer rate on the composition of the pectolytic enzyme complex of Aspergillus niger
Authors: Zetelaki‐Horváth K. , Vas K.
Published: December 2004

Optimal agitation and aeration conditions [assuring O2 transfer rates (OTR) from 12 to 179 mmol L−1 h−1] were determined for pectin lyase (PL) synthesis of an Aspergillus niger strain. Components of the pectolytic enzyme complex were also investigated in order to determine whether their O2 demand is identical with or different from that of pectin lyase. Should the latter be the case, a possibility would be given to produce enzyme complexes of different agitation and aeration conditions. According to our results, mycelium yield of Aspergillus niger attained a maximum at an OTR of 100 mmol L−1 h−1. The yields of the various pectolytic enzymes reached maxima at different OTRs. Pectin lyase production was the highest (0.555 µmol min−1 mL−1) at an OTR of 60 mmol L−1 h−1. Endopolygalacturonase (PG) production showed a maximum at the OTR of 49 mmol L−1 h−1 with a second peak at 100−135 mmol O2 L−1 h−1. Pectin esterase (PE) synthesis showed a maximum at on OTR of 12−14 mmol L−1 h−1, while both apple juice clarifying and macerating activities gave two maxima at 14 and 60 mmol L−1 h−1 due to the optima of PE and endo‐PG. Macerating activity showed a high value at OTR optimal for PL production as well.
A CSTR‐hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics
Authors: Deeslie W. David , Cheryan Munir
Published: December 2004

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR‐hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis‐Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space‐time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.
Preparation and properties of immobilized amyloglucosidase
Authors: Nithianandam V. S. , Srinivasan K. S. V. , Joseph K. Thomas , Santappa M.
Published: December 2004

Amyloglucosidase was immobilized on a copolymer of methyl methacrylate and 2‐dimethylaminoethyl methacrylate. The resulting immobilized amyloglucosidase has 19% of the soluble enzyme specific activity. The pH optimum of immobilized amyloglucosidase is shifted towards acidity by 1.9 units. The temperature optimum of immobilized enzyme is shifted upward by 5°C. The immobilized amyloglucosidase has the maximum stability at pH 4.6, whereas the soluble enzyme has maximum stability at pH 5.5. While soluble amyloglucosidase has a maximum thermal stability at 50°C, the stability of the immobilized amyloglucosidase steadily decreases with the increase in temperature.
The potential of biological methane generation from chicken manure
Authors: Huang James J. H. , Shih Jason C. H.
Published: December 2004

The potential for biological methane generating from the manure of laying hens was investigated in the laboratory. Fresh manure was collected, analyzed, and used to prepare medium for bacterial growth. At 55°C and under anaerobic conditions, methanogenic cultures were initiated by incubating the medium with different inoculations from various natural environments. Since there were no significant differences in gas production among these initiated cultures after 40 days of acclimation, they were mixed to maintain a genetic pool. The mixed culture was then challenged with different retention times (RT) and different volatile solid (VS) concentrations for the selection of optimal conditions and cultures. The conditions were finally selected to be 4‐day RT and 6% VS for the maximal rate of gas production. The optimal pH and temperature were determined to be 7.5 and 50°C, respectively. Under such conditions the selected culture produced total gas at a rate 4.5 L/L day and methane (70% of total gas) 3.2 L/L day. The chicken manure therefore was able to support the methane yield at 270 L/kg of VS, a value comparably higher than other kinds of livestock wastes.
Continuous cultivation of fission yeast: Analysis of single‐cell protein synthesis kinetics
Authors: Agar D. W. , Bailey J. E.
Published: December 2004

A fundamental problem in microbial reactor analysis is identification of the relationship between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecular synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single‐cell protein content in Schizosaccharomyces pombe in balanced exponential growth have been analyzed by two different methods. One approach utilizes the integrated form of the population balance equation known as the Collins‐Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms in order to best fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in small protein content cells as the population specific growth rate increases, leading to parabolic single‐cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is shown in this case to be a far more sensitive method for determination of single‐cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.
Application of data consistency tests and new parameter estimation methods to microbial growth on corn dust in batch culture
Authors: Solomon B. O. , Erickson L. E. , Hess J. L.
Published: December 2004

The microorganism Candida utilis was grown on both filtered and unfiltered substrate obtained from enzymatic hydrolysis of starch in corn dust. For growth on filtered substrate, the average integrated biomass energetic yield value based on biomass‐substrate data was η = 0.55 and for growth on unfiltered substrate an average yield value of η = 0.59 was obtained. Material and energy balances showed that the presence of unfiltered corn residue in the media had no significant effect on the yields. Statistical methods were developed and used to obtain best estimates of the growth parameters. Values of the biomass energetic yield corrected for maintenance (ηmax = 0.619) and the maintenance coefficient (me = 0.043) were obtained for growth on filtered substrate. Values of ηmax = 0.741 and me = 0.142 were obtained for the growth on unfiltered substrate. The consistency of data and parameter estimates was relatively good for filtered substrate; however, parameter estimates for unfiltered substrate were not consistent. Growth experiments without filtration of the products of starch hydrolysis resulted in protein‐enriched products with about 39.73% protein.
Growth of Saccharomycopsis fibuligera in a continuous‐stirred‐tank fermentor
Authors: Admassu Wudneh , Korus Roger A. , Heimsch Richard C. , Lemmel Steven A.
Published: December 2004

Growth rates and amylase production rates were determined for the yeast Saccharomycopsis fibuligera grown on a simulated potato processing waste in a continuous‐stirred‐tank fermentor. S. fibuligera formed large multicellular flocs in the fermentor, and cell growth was reduced at low dilution rates because of mass‐transfer resistance. The average Thiele modulus, which is the measure of extent of substrate diffusion, had a value ranging from ϕav = 2.2 for D = 0.10 to 0.3 for D = 0.40. Growth rates were described by the Monod equation modified to include mass‐transfer effects. This modified Monod equation was used to predict growth rates from measured floc‐size distribution. Experimentally determined growth rates were in close agreement with these predicted values.
Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation
Authors: Yerushalmi L. , Volesky B.
Published: December 2004

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed‐batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO2 evolution rate, O2 uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system.

The comparison of the fractions of substrate energy evolved as heat (δh) with the fraction of available electrons transferred to oxygen (ϵO2) indicated equal values of the two (0.46) in the aerobic respiration (fed‐batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ϵO2 than δh, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.
Evaluation of crude dextran as phase‐forming polymer for the extraction of enzymes in aqueous two‐phase systems in large scale
Authors: Kroner K. H. , Hustedt H. , Kula M.‐R.
Published: December 2004

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T‐500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran‐rich phase and a significant shift in the macroscopic structure of these phases, observed as the “gel‐forming” properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T‐500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T‐500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two‐phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.
Analysis and optimization of methods using water‐soluble carbodiimide for immobilization of biochemicals to porous glass
Authors: Janolino Violeta G. , Swaisgood Harold E.
Published: December 2004

Two methods employing a water‐soluble carbodiimide for carboxyl activation were investigated for the immobilization of biochemicals to succinamidopropyl‐porous glass beads. Immobilization using the simultaneous method (simultaneous addition of carbodiimide and nucleophilic ligand to the beads) and large excess of carbodiimide and a small nucleophile should result in covalent binding to all accessible carboxyl groups. Results obtained with glycine methyl ester indicated that 40% of the total surface carboxyl groups were sterically accessible. Using these reaction conditions with the protein, chymotrypsinogen, suggests that a surface monolayer is immobilized. although far fewer sites are required assuming single point attachment. For ligands containing carboxyl groups and several nucleophilic groups (e. g., enzymes), however, biological inactivation may occur using the simultaneous method. Consequently, a sequential method (activation of the surface with carbodiimide followed by washing and addition of the biochemical to be immobilized) was optimized. Using optimal conditions (20 min activation time at pH 4.75 and room temperature; 2 min wash at pH 7 and 0°C) and 0.1M carbodiimide, nearly half of the accessible surface sites remained in the O‐acylisourea form and reacted with glycine methyl ester upon its addition. The amount of surface loading as a function of activation time was consistent with a model constructed using rate constants for O‐acylisourea formation and hydrolysis previously derived from solution studies with acetic acid [Swaisgood and Natake, J. Biochem 74, 77 (1973)]. Measurement of reaction rates with glycine methyl ester following surface activation suggests that the rate of reaction with amino groups is at least eightfold greater than the hydrolysis rate. Either immobilization procedure gave comparable enzyme loading and specific activities for the case of sulfhydryl oxidase.
Quantitative measurement of oxygen consumption in insect cell culture infected with polyhedrosis virus
Authors: Weiss S. A. , Orr T. , Smith G. C. , Kalter S. S. , Vaughn J. L. , Dougherty E. M.
Published: December 2004

A simple and reproducible quantitative method for measuring dissolved oxygen (DO) in uninfected and baculovirus infected cells in culture is described. To establish this method, an industrial DO measuring system for fermentation was employed. During this process the physical characteristics of the cell culture vessel were taken into account permitting a direct readout of DO in microliters per vessel. During these studies, it was experimentally documented that insect cells, particularly baculovirus infected cells, in culture from 1 to 14 days utilize an appreciable level of DO.
Studies on Scenedesmus acutus growth. The incorporation of 14C‐glucose by Scenedesmus acutus under light and dark periods
Authors: Shamala T. R. , Drawert F. , Leupold G. , Becker W.
Published: December 2004

Qualitative and quantitative estimations of alcohol‐extractable compounds from 14C‐glucose‐incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with 14C‐glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.
The use of continuous cultures for selection and isolation of microorganisms producing extracellular enzymes adapted to extreme environments
Authors: Lelieveld H. L. M.
Published: December 2004

Microorganisms producing extracellular enzymes with special properties can be selected and isolated by growing them in continous culture and using as the growth‐limiting substrate a substance which must be broken down by the enzyme of interest. A diffusion layer around the microbial cell will cause gradients in the concentrations of enzymes and the hydrolyzed substrate. This will cause differences in growth rates between the parent cells and those mutant cells which produce an enzyme better adapted to the selective environment provided.
Modeling enzyme immobilization in porous solid supports
Authors: Do Duong D. , Clark Douglas S. , Bailey James E.
Published: December 2004

Enzymes are often immobilized on the internal surfaces of porous solid by immersing enzyme‐free particles in a well mixed solution of enzyme. The ensuing impregnation process involves coupled transient mass transfer and surface attachment of enzyme. A mathematical model is employed to explore the influences of process parameters on the amount of enzyme loaded and the distribution of immobilized enzyme within the support particles. Nonuniform loading of the support occurs under some conditions. This is significant since the distribution of enzyme within the support particle influences the overall activity and stability of the immobilized enzyme catalyst. The model developed here may also be used to describe removal of reversibly immobilized enzyme during washing or utilization of the immobilized enzyme catalyst.
Design optimization of continuous sterilizers
Authors: Ashley M. H. J. , Mooyman J. G.
Published: December 2004

The development of a design of a continuous sterilization is considered with regard to practical data for temperature/time thermal deactivation of infecting organisms. An example of the optimization of the engineering design for capital and operating costs is given. Systems for continuous sterilization by membrane filtration are also considered.
A Computer‐aided noninterfering on‐line technique for monitoring oxygen‐transfer characteristics during fermentation processes
Authors: Spriet J. A. , Botterman J. , Buyser D. R. De , De Visscher P. L. , Vandamme E. J.
Published: December 2004

The role of computers in the monitoring and control of fermentation processes has increased steadfastly. The ultimate utility of the machines will not depend on the availability of online sensors but also on the availability of techniques that combine direct measurements, leading towards estimates of variable closely related to the microbial process or its control. In this article, a methodology for on‐line and noninterfering evaluation of the volumetric mass‐transfer coefficient Kla is developed. A detailed presentation of the procedure, called “the static method,” is given. Its feasibility is proved through implementation of the method on an antibiotic fermentation process. These experiments indicate that operator actions meant to modify the oxygen‐transfer conditions can be checked on‐line. The quantitative value of the static method is ascertained by comparing the experimental results with Kla estimates obtained with the “gassing‐out” method. A sensitivity analysis was carried out, revealing the need for temperature and pressure corrections and showing that the precision of the oxygen analyzer determines the precision of the static method.
The production of 6‐aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase
Authors: Park Jong M. , Choi Cha Y. , Seong Baik L. , Han Moon H.
Published: December 2004

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two‐stage plug‐flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two‐ and three‐stage PFRs were simulated by computer. In the case of the two‐stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree‐stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.
Use of chemically modified activated carbon as a support for immobilized enzymes
Authors: Osborn James A. , Ianniello Robert M. , Wieck Henry J. , Decker Thomas F. , Gordan Susan L. , Yacynych Alexender M.
Published: December 2004

Loading and activity assays of the enzymes α‐chymotrypsin, α‐chymotrypsinogen, and glucose oxidase covalently bound to an activated carbon support are presented. The activated carbon support material was pretreated using either a radio‐frequency oxygen plasma or an electrochemical oxidation to maximize the enzyme attachment. Cyanuric chloride or water‐soluble carbodiimide linking reactions were used to covalently attach the enzymes to the carbon support. Discussion of the relative merits of each reaction scheme is presented.
Farm‐scale production of fuel ethanol and wet grain from corn in a batch process
Authors: Westby Carl A. , Gibbons William R.
Published: December 2004

The batch production of fuel grade ethanol and distillers' wet grain (wet solids) in a farm‐scale process (1240–15,580 L/batch) is described. The employs yeast fermentation of amylase‐treated corn mash and a two‐stage distillation. Primary emphasis in this study was on the cooking, fermentation, and centrifugation steps. Without recycling, fermentation of the mash yield beers with 10.0–10.5% ethanol. Recycling of stillage supernatant at full, 75, or 50% strengths produced enriched mashes that after 48‐h fermentation yielded beers with 5–;14% more ethanol. Recycling twice with full‐strength supernatant at pH 7.0 increased the ethanol yield in the final beer 16.5%; however, the time to complete the final fermentation was extended form 48 to 72 h and salt buildup occurred. By recycling at pH 5.4, it was possible to avoid rapids salt buildup and obtain beers with 10.3–10.5% ethanol. Recycling resulted in increased levels of glucose, starch, crude protein, and fat in the beer and a reduced moisture content while the wet solids showed an increased starch content. Centrifugation after cooking or fermentation yield in the subsequently produced beer. Fermentation of a volume‐resorted mash supernatant gave a beer with only 9.25% ethanol. Mash wet solids varied somewhat chemically from beer and stillage solids. An economic and energy balance analysis of various modes of plant operation are provided and plant considerations are suggested.
Immobilization of yeast cells containing β‐galactosidase
Authors: Banerjee Mita , Chakrabarty Aloka , Majumdar S. K.
Published: December 2004

Cultural conditions optimum for β‐galactosidase production by Saccharomyces anamensis are pH 4.5, temperature 26 ± 2°C, and 30 h of incubation period. Addition of lactose at 24 h fermentation greatly increase the level of enzyme. Optimum pHl, temperature, pH stability, and thermostability of yeast β‐galactosidase are negligibly affected by immobilization. The Km values of enzyme in the native and immobilized cells are 102mM and 148mM, respectively. Glucose noncompetitively inhibits the enzyme activity. Addition of substances such as dithioerythritol, glutathione, and bovine serum albumin to the native cell during assay procedure and immobilized cell prior to immobilization have stimulatory effects on enzyme activity. Metal ions like Ca2+, Mg2+ enhance the β‐galactosidase activity for both intact and bound cells. Immobilized cells retain 68.6% of the β‐galactosidase activity of intact cells and there is no significant loss of activity on storage at 4°C for 28 days.
Process optimization of a continuous airlift tower‐loop reactor
Authors: Luttmann R. , Thoma M. , Buchholz H. , Lehmann J. , Schügerl K.
Published: December 2004

Based on the experimental investigations with H. polymorpha and Methylomonas M 15 in bench‐scale airlift tower‐loop reactors, a general distributed parameter model was developed and used to simulate to cultivation process in a 40‐m‐high production reactor. This general model was simplified with regard to the gas phase and loop balances and was employed to optimize cell productivity and/or profit in a 20‐m‐high pilot‐plant airlift tower‐loop reactor. Maximum cell productivity always occurs in the oxygen‐transfer‐limited growth range. In case of a high “penalty factor” for nonconsumed substrate, maximum profit is attained at the boundary between substrate and oxygen‐transfer‐limited growth. Oxygen‐transfer limitation exists in the lower half of the tower, whereas in the upper half, substrate limitation prevails. The longitudinal dissolved oxygen concentration passes a minimum in this case as has been determined experimentally in the bench‐scale column. The simulation results agree fairly well with the data measured in the pilot plant.
Dynamic simulator for anaerobic digestion processes
Authors: Kleinstreuer C. , Poweigha T.
Published: December 2004

A transient, two‐culture model simulating methane production from biomass has been developed. The simulator, based partially on the work by Andrews and McCarty, is capable of calculating the hydrolysis products of several common organic materials, accommodating various substrate feeding modes, and simulating the transient physico‐biochemical transport and conversion processes occurring in the biological, liquid, and gaseous phases of a well‐mixed reactor. The mathematical representation of this bioconversion system consists of a set of 11 coupled, nonlinear first‐order rate equations based on the principles of mass conservation and biochemical reaction kinetics. The model can be used in conjunction with laboratory investigations and as a simulator for evaluating process control strategies and cost developments.
Continuous culture of the ciliate Tetrahymena pyriformis on Escherichia coli
Authors: Swift Steven T. , Najita Irene Y. , Ohtaguchi Kazuhisa , Fredrickson A. G.
Published: December 2004

The ciliated protozoan Tetrahymena pyriformis was grown in a chemostat fed with a culture of Escherichia coli overflowing from another chemostat. Densities of the protozoan and bacterial populations, mean volume of protozoan cells, yields of protozoan volumes and numbers, and filtering rates of protozoans per cell and per unit volume of biomaterial were determined at five different dilution rates. The data obtained supplement other data already available for the popular test organism T. pyriformis, and they are also comparable with data available for related ciliates.