bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of ethanol by Saccharomyces cerevisiae under high‐pressure conditions
Authors: Thibault Jules , LeDuy Anh , Côté François
Published: December 2004

A research project was initiated to examine the possibility of using supercritical carbon dioxide for in situ recovery of ethanol during its production by yeast Saccharomyces cerevisiae. As a preliminary step, it was necessary to study the behavior of ethanol production under high‐pressure conditions, up to 7 MPa (1000 psi). The results show that pressure has a significant inhibiting effect on the production of ethanol. There is a significant decrease in the initial rate of production as well as in the final ethanol concentration as pressure is increased. This decrease is more significant when carbon dioxide is used to pressurize the fermentor. The pressure affects the ability of the cells to produce ethanol in a reversible way. When the fermentor is returned to atmospheric conditions, the reaction resumes its normal fermentation rate.
The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract
Authors: Bajpai Pratima , Margaritis Argyrios
Published: December 2004

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25–35°C, but dropped sharply beyond 35°C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35°C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25–45°C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40°C. For the pH range between 3 and 7, the free‐cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h−1 and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.
Mixed solvent systems for recovery of ethanol from dilute aqueous solution by liquid–liquid extraction
Authors: Mitchell R. J. , Arrowsmith A. , Ashton N.
Published: December 2004

Distribution coefficients and selectivities of a number of mixed solvent systems have been determined in order to assess their suitability in preferentially extracting ethanol from aqueous solution. The measured values of distribution coefficients and selectivities differ substantially from the values estimated by interpolating between the pure solvents.
Oxygen transfer in animal cell culture medium
Authors: Lavery M. , Nienow A. W.
Published: December 2004

Both kLa and kL measurements were carried out by an unsteady state technique at impeller speeds ranging from 1.6 to 5.8 s−1 in a mechanically agitated animal cell culture vessel of working volume 1.5 L. Checks were made that the time constant of the oxygen electrode was negligible compared to the time for aeration and that the oxygen electrode reading was not a function of agitator speed in the range employed. The kL values by surface aeration of (1.18–3.54) × 10−5 m/s and kLa values by sparged aeration of (2.8–8.5) × 10−4 s−1 were found. The former are in reasonable agreement with published experimental values and the latter in accord with values estimated from published correlations based on agitator power input and aeration rate. The fluids used were water, basal medium, and basal medium supplemented with 5% (v/v) foetal calf serum; for each of these, kL and kLa values were similar. However, the addition of silicone antifoam (6 PPM) reduced the kLa value by ca. 50%.
Continuous‐culture studies of synthesis and regulation of extracellular β(1‐3) glucanase and protease enzymes from Oerskovia xanthineolytica
Authors: Andrews B. A. , Asenjo J. A.
Published: December 2004

This article describes the synthesis and regulation of β(1‐3)glucanase and protease enzymes from the cell lytic system of Oerskovia xanthineolytica LL‐G109 in continuous culture using different concentrations of carbon source (glucose) and inducer (glucan). These two enzyme activities are the main components of a lytic system capable of lysing and disrupting whole yeast cells; it is subject to catabolite repression by glucose and is induced by yeast glucan. Peaks of β(1‐3)glucanase and protease activity are obtained at dilution rates of between 0.05 and 0.15 h−1. The glucanase‐protease ratio is very high compared to other strains. At dilution rates above 0.15 h−1 all activities are similar to those obtained in batch culture. The lytic enzyme system appears to contain several β(1‐3)glucanase enzymes. In continuous culture both productivity and enzyme concentrations are greatly in creased when compared to batch culture, 11‐ and 4.4‐fold, respectively.
Periodic forcing of microbial cultures: A model for induction synchrony.
Authors: Hjortso Martin A.
Published: December 2004

Periodic environmental shifts have been used to induce synchrony in many different microbial populations. In this article, the induction synchrony phenomenon is analyzed using an age distribution model in which the age at which the cells divide is subjected to periodic forcing. It is found that synchrony will occur whenever the period of the forcing lies in the interval between the youngest and the oldest division age that occur in the population during the forcing. The analysis also predicts that under certain conditions it should be possible to obtain a multimodal synchrony in which cells in the population are distributed among a set of discrete, synchronized cell lines. The behavior of the age distribution when the conditions for synchrony are not satisfied is briefly explored. It is found that the age distribution model is able to exhibit a very rich spectrum of possible dynamic behavior. Many of the phenomena observed can be thought of in terms that are familiar from nonlinear analysis, such as stable and unstable limit cycles, period doubling, halving, and chaos. The richness of dynamic behavior opens the possibility that environmental shifts or periodic forcing could be used as a powerful tool in discriminating models of microbial kinetics and cell cycle control.
Preservation of potential fermentables in sweet sorghum by ensiling
Authors: Linden James C. , Henk Linda L. , Murphy Vincent G. , Smith D. H. , Gabrielsen Byron C. , Tengerdy Robert P. , Czako Lajos
Published: December 2004

Pressed and wilted samples of sweet sorghum [Sorghum bicolor (L.) Moench var. Rio] were ensiled for periods up to 155 days. A kinetic study of the biochemical changes which occurred during ensiling showed that in wilted sorghum ensilage invert sugars and mannitol levels collectively were maintained at 65% of the original ferment able sugar content of the sorghum. The acidic environment produced by ensiling also served as a pretreatment that resulted in enhanced yields of reducing sugar when the sorghum was contacted with cellulolytic enzymes. The quantity of sugar obtained from enzymatic hydrolysis more than compensated for carbohydrate used by organisms during the ensiling process. Both Saccharomyces uvarum and Clostridium acetobutylicum were able to ferment a medium constituted from pressed sorghum juice and the solution resulting from enzymatic hydrolysis of sweet sorghum ensilage.
Influence of anions on metal adsorption by Rhizopus arrhizus biomass
Authors: Tobin J. M. , Cooper D. G. , Neufeld R. J.
Published: December 2004

The presence of anions in solution was found to inhibit the uptake of La3+, Cd2+, Pb2+, UO, and Ag+ by Rhizopus arrhizus biomass. The effects ranged from total inhibition of Cd2+ and Pb2+ uptake at equimolar concentrations of EDTA to no change in uptake of La3+ or UO at 12‐fold molar excesses of Cl− or CO. No anion was found to enhance metal uptake levels, and the degree of inhibition generally followed the series:

The chemical equilibrium model REDEQL2 was adapted to treat metal uptake by R. arrhizus biomass and used to predict the effects of anions in solution. Comparisons with the experimental results are made and discussed in light of the assumptions underlying the model.
Separation of dilute aqueous butanol and acetone solutions by pervaporation through liquid membranes
Authors: Matsumura Masatoshi , Kataoka Hiroshi
Published: December 2004

A simultaneous extraction‐stripping process is proposed for separating volatile products from fermentation broths, it is based on pervaporation through a liquid membrane supported with a hydrophobic porous membrane. The liquid membrane prepared with oleyl alcohol was selected as the most suitable for separating volatile products resulting from acetone‐butanol fermentation. The separation performance and stability of the oleyl alcohol liquid membrane were investigated by using dilute aqueous butanol and acetone solutions. The oleyl alcohol liquid membrane was found to be superior by far in both selectivity and permeability of butanol to the better known silicone rubber membrane in its high selectivity for alcohols. Using the oleyl alcohol liquid membrane, the dilute aqueous butanol solutions of around 4 g/L obtained in acetone‐butanol fermentation could be concentrated up to 100 times. The stability of this liquid membrane was also quite good as long as the surface tension of the feed solution was less than the critical surface tension of the support membrane. No change in the separation performance was found after the continuous usage in a long period of 100 h.
Microencapsulation of human fibroblasts in a water‐insoluble polyacrylate
Authors: Boag Alexander H. , Sefton Michael V.
Published: December 2004

Viable human diploid fibroblasts have been micro‐encapsulated in EUDRAGIT RL, a commercially available water‐insoluble polyacrylate, by an interfacial precipitation technique. Cells in medium and polymer solution (in diethyl phthalate) were coextruded and formed into droplets by a coaxial air stream. The droplets fell into a corn‐oil/mineral‐oil mixture to extract the solvent to precipitate the polymer around the cells. Capsules were ca. 500 μm in diameter depending on the air flowrate with a ca. 10‐μm thick wall. When collagen (1 mg/mL) was added to the cell suspension prior to encapsulation and base‐washed corn oil was used, cell growth occurred with one doubling achieved after five to six days as the collagen gel contracted inside the capsule. In the absence of collagen, cells spread on the inner wall of the capsule but did not grow, presumably because the surface charge on the capsule was inadequate. In similar fashion fibroblasts spread but did not grow on films of EUDRAGIT RL but did grow on blends of EUDRAGIT RL and EUDRAGIT E containing 10–30% of the latter more highly aminated polyacrylate. Although not suitable for anchorage‐dependent cell growth by itself, EUDRAGIT RL has been suitable as a model polymer to demonstrate the feasibility of using water insoluble polyacrylates and organic solvents and nonsolvents for the micro‐encapsulation of fibroblasts. Such microcapsules are of potential interest as a mode of large scale tissue culture for the production of novel therapeutic agents.
An enzyme electrode for on‐line determination of ethanol and methanol
Authors: Belghith Hafedh , Romette Jean‐Louis , Thomas Daniel
Published: December 2004

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO2 sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.
New instrument for on‐line viscosity measurement of fermentation media
Authors: Picque D. , Corrieu G.
Published: December 2004

In an attempt to resolve the difficult problem of on‐line determination of the viscosity of non‐Newtonian fermentation media, the authors have used a vibrating rod sensor mounted on a bioreactor. The sensor signal decreases nonlinearly with increased apparent viscosity. Electronic filtering of the signal damps the interfering effect of aeration and mechanical agitation. Sensor drift is very low (0.03% of measured value per hour).

On the rheological level the sensor is primarily an empirical tool that must be specifically calibarated for each fermentation process. Once this is accomplished, it becomes possible to establish linear or second‐degree correlations between the electrical signal from the sensor and the essential parameters of the ferementation process in question (pH of a feremented milk during acidification, concentration of extra cellular polysaccharide). In addition, by applying the power law to describe the rheological behavior of fermentation media, we observe a second‐order polynomial correlation between the sensor signal and the behavior index (n).
Specific and nonspecific glucanases from Trichoderma viride
Authors: Beldman G. , Voragen A. G. J. , Rombouts F. M. , Searle‐Van Leeuwen M. F. , Pilnik W.
Published: December 2004

Six endoglucanases (Endo I, II, III, IV, V, and VI), three exoglucanases (Exo I, II, and III), and a β‐glucosidase (β‐gluc I) isolated from a commercial cellulase preparation of Trichoderma viride origin were examined as to their activities on xylan ex oat spelts. Endo I, II, and III as well as Exo II and III showed no activity toward xylan and were classified as specific glucanases. Less specificity was found for the endoglucanases Endo IV, V, and VI, Exo I, and β‐gluc I, whose enzymes were able to hydrolyze xylan. With respect to product formation these xylanolytic cellulases fit the classification of xylanases generally accepted in the literature. Kinetic experiment with xylan, CM‐cellulose, and p‐nitrophenyl‐β‐D‐glucoside revealed that Endo IV, V, an VI and Exo I prefer to hydrolyze β‐1, 4‐D‐glucosidic linkages. β‐Gluc I showed no clear substrate preference.
Adaptive control of anaerobic digestion processes—a pilot‐scale application
Authors: Renard P. , Dochain D. , Bastin G. , Naveau H. , Nyns E.‐J.
Published: December 2004

A simple adaptive control algorithm, for which theoretical stability and convergence properties had been previously demonstrated, has been successfully implemented on a biomethanation pilot reactor. The methane digester, operated in the CSTR mode was submitted to a shock load, and successfully computer controlled during the subsequent transitory state.
A microfibril‐generating factor from the cellulase of Trichoderma reesei
Authors: Krull L. H. , Dintzis F. R. , Griffin H. L. , Baker F. L.
Published: December 2004

A combination of ionic strength reduction and diafiltration of Trichoderma reesei cellulate complex through a hollow fiber apparatus of 5000 molecular weight (MW) cutoff and subsequent passage of filtrate over a Spherogel‐TSK 3000‐SW column provided extracts that had the ability to generate microfibrils in filter paper and to disrupt filter paper and corn leaf tissue. Milligram quantities of material obtained from these extracts released small amounts of soluble carbohydrate from filter paper, required ferric iron for increased activity, and contained amino acids. Short fiber formation and disruption of filter paper during interaction with these extracts was enhanced by prior acid treatment and eliminated by prior base treatment. The amount of soluble carbohydrate hydrolyzed in 24 h from filter paper by whole cellulase complex was not changed by first disrupting the substrate with the extracts.
High‐Rate two‐phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis
Authors: Gijzen Huub J. , Zwart Kor B. , Verhagen Frank J. M. , Vogels Godfried P.
Published: December 2004

A novel two‐stage anaerobic process for the microbial conversion of cellulose into biogas has been developed. In the first phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermentation of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in a UASB‐type methanogenic reactor.

A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor volume/day (g VS/L/day) did not affect the degradation efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The two‐stage anaerobic process was also tested with a closed fluid circuit. In this instance total methane production was 0.438 L CH4g VS added, which is equivalent to 98% of the theoretical value. The application of rumen microorganisms in combination with a high‐rate methane reactor is proposed as a means of efficient anaerobic degradation of cellulosic residues to methane. Because this newly developed two‐phase system is based on processes and microorganisms from the ruminant, it will be referred to as “Rumen Derived Anaerobic Digestion” (RUDAD‐) process.
Characterization of fractionated polyclonal antibodies for immunoaffinity chromatography
Authors: Sada Eizo , Katoh Shigeo , Kiyokawa Atsuo , Kondo Akihiko
Published: December 2004

Polyclonal anti‐BSA antibodies were ractionated by stepwise elution from an immobilized BSA column by decreasing pH or increasing the concentration of NaSCN. The binding affinities of each fraction and original globulin under physiological conditions and their dependence on pH and ionic environments were compared. Fractions with high association constant under physiological conditions did not necessarily show antigen binding affinity over a wide pH range, but they retained a high affinity at higher ionic strength of NaSCN. Consequently, by combining these two fractionation procedures, a fraction with high affinity and which dissociated at moderate pH was obtained. It is clear that high affinity is not always incompatible with ease of dissociation accompanying a change in conditions.
Activated sludge systems with biomass particle support structures
Authors: Nicol Jean‐Paul , Benefield Larry D. , Wetzel Edward D. , Heidman James A.
Published: December 2004

A mathematical model was developed to simulate the operation of an activated sludge system with biomass support particles. Based on the results of this simulation study, it would appear that a system with biomass particle supports offers several advantages not offered by conventional systems. These include the ability to resist failure from large hydraulic surges; stable nitrification under transient inhibitory conditions resulting from temperature changes, hydraulic surges, and/or toxic chemicals; and the ability to establish stable operating conditions with respect to both carbon oxidation and nitrification at short hydraulic retention times and low sludge ages.
Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods
Authors: Crumbliss A. L. , McLachlan K. L. , O'Daly J. P. , Henkens R. W.
Published: December 2004

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 × 10−5 mmol BCA/m2, and on graphite it was 1.7 × 10−3 mmol BCA/m2 nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4°C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23°C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second‐order rate constant K′Enz for the hydrolysis of p‐nitrophenyl acetate (p‐NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25°C is 2.6 × 102 and 5.6 × 102 M−1s−1 respectively. The corresponding KENZ value for the free enzyme is 9.1 × 102 M−1s−1. Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pKa, are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA‐active site pKa values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.
Increased digestibility of cedar by pretreatment with peracetic acid and steam explosion
Authors: Ando Shingo , Kakimoto Takahiro , Itoh Kazuyoshi , Arai Isamu , Kiyoto Koichi , Hanai Shiro
Published: December 2004

A type of steam explosion method combined with chemical pretreatment was studied. Peracetic acid was an effective reagent to assist the steam explosion reaction and greatly improved the enzymatic saccharification of cedar compared with single steam explosion. The extent of saccharification was directly proportional to the amount of peracetic acid absorbed in the chips, and the function of peracetic acid was revealed as an acid catalyst and as a radical initiator in the steam explosion reaction.
Inhibiting action of chlorophenols on biodegradation of phenol and its correlation with structural properties of inhibitors
Authors: Beltrame Paolo , Beltrame Pier Luigi , Carniti Paolo , Guardione Donatella , Lanzetta Cecilia
Published: December 2004

An activated sludge conditioned to a low concentration of phenol was used for the biodegradation of phenol in the presence of Chlorophenols, measuring the inhibiting action of all the latter compounds and expressing it as p/50. As a structural property to be correlated with the measured values, the lipophilicity (log P) of the Chlorophenols was reevaluated with a new apparatus. Values of p/50p are better correlated with the number of chloro substituents in the inhibitor, provided a specific effect of substituents ortho to the phenolic function is considered. New results are compared with previous measurements effected on a different activated sludge.
Repeated batch production of L‐phenylalanine from phenylpyruvate and NH4Cl by immobilized cells of Nocardia opaca under hydrogen high pressure
Authors: Matsunaga Tadashi , Higashijima Michio , Sulaswatty Anny , Nishimura Shigeo , Kitamura Takanori , Tsuji Masao , Kawaguchi Toshio
Published: December 2004

Among various microbial cells examined under screening conditions, Nocardia opaca showed the highest activity for production of phenylalanine from phenylpyruvate. Here NH4Cl as well as amino acids were used as an amino donor for phenylalanine production. The phenylalanine production rate increased with increasing hydrogen pressure. The specific activity of phenylalanine dehydrogenase was increased by culturing N. opaca cells in nutrient broth containing 0.3% phenylalanine. As a result, the phenylalanine production rate increased from 0.69 to 4.4 μmol/min g dry cells. Immobilized cells were activated in nutrient broth containing ZnCl2 before phenylalanine production. Phenylalanine dehydrogenase activity and cell number in the gel increased with increasing incubation time, and the maximum phenylalanine dehydrogenase activity was obtained at 36 h incubation. Then, phenylalanine was produced from phenylpyruvate, NH4Cl, and 100 atm H2 with the activated immobilized cells. The rate of phenylalanine production was 0.24 μmol/min cm3 gel. The conversion of phenylpyruvate to phenylalanine was 82%. Immobilized cells retained 76% of the initial phenylalanine production rate after 10 h reactions were repeated 11 times with two intervening reactivations.
Protein precipitate recovery using microporous membranes
Authors: Bentham A. C. , Ireton M. J. , Hoare M. , Dunnill P.
Published: December 2004

The use of microporous membranes has been examined for the recovery of precipitated protein suspensions and related soluble protein. Membrane flux rates and soluble protein transmissions are reported for unstirred batch‐cell studies and cross‐flow experiments. The unstirred batch‐cell gave soluble protein transmissions in the range 80–100% for feeds containing either soluble protein or a mix of soluble and isoelectrically precipitated protein. In all cases a sharp decline in flux was observed which was, for example, considerably greater for soluble protein at its isoelectric point, pH 4.6, than at pH 8.8. The presence of precipitated protein led to a further decrease in flux rate. In cross‐flow studies, flux decline was eventually accompanied by a significant decline in soluble protein transmission. The flux protein‐transmission characteristics of microporous membranes are discussed in terms of the interaction of the soluble and precipitated protein with the membrane.
Effects of carbon dioxide on Penicillium chrysogenum: An autoradiographic study
Authors: Edwards Alfred G. , Ho Chester S.
Published: December 2004

Previous research has shown that dissolved carbon dioxide causes significant changes in submerged penicillin fermentations, such as stunted, swollen hyphae, increased branching, lower growth rates, and lower penicillin productivity. Influent carbon dioxide levels of 5 and 10% were shown through the use of autoradiography to cause an increase in chitin synthesis in submerged cultures of Penicillium chrysogenum. At an influent 5% carbon dioxide level, chitin synthesis is ca. 100% greater in the subapical region of P. chrysogenum hyphae than that of the control, in which there was no influent carbon dioxide. Influent carbon dioxide of 10% caused an increase of 200% in chitin synthesis, it is believed that the cell wall must be plasticized before branching can occur and that high amounts of dissolved carbon dioxide cause the cell to lose control of the plasticizing effect, thus the severe morphological changes occur.
Correlation of cell volume fractions with cell concentrations in fermentation media
Authors: Ju Lu‐Kwang , Ho Chester S.
Published: December 2004

Cell volume fractions and cell concentrations were measured in submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Correlations for cell volume fractions with cell concentrations in fermentation media of the microorganisms were established accordingly. Other key properties of microorganisms, such as cell water content, wet cell density, and dry cell density, can also be obtained with the use of the current method. The results are in good agreement with data available in the literature.
Application of fed‐batch culture to citric acid production by Aspergillus niger: The effects of dilution rate and dissolved oxygen tension
Authors: Dawson M. W. , Maddox I. S. , Boag I. F. , Brooks J. D.
Published: December 2004

Studies in conventional batch culture confirmed that the maximum citric acid production rate occurred prior to exhaustion of the growth‐limiting nutrient, i.e., when the growth rate was nonzero. The effects of dilution rate and the culture dissolved oxygen tension (DOT) were studied in chemostat culture. Maximum citric acid yield and production rate were observed at low dilution rate (0.017 h−1) and high DOT value (90% of saturation). These findings were applied to a nitrogen‐limited fed batch culture, and allowed a productivity increase of 100% when compared with conventional batch culture.
Fermentation of corn starch to ethanol with genetically engineered yeast
Authors: Inlow D. , McRae J. , Ben‐Bassat A.
Published: December 2004

Expression of the glucoamylase gene from Aspergillus awamori by laboratory and distiller's strains of Saccharomyces cerevisiae allowed them to ferment soluble starch. Approximately 95% of the carbohydrates in the starch were utilized. Glycerol production was significantly decreased when soluble starch was used instead of glucose. Ethanol yield on soluble starch was higher than that on glucose. The rate of starch fermentation was directly related to the level of glucoamylase activity. Strains with higher levels of glucoamylase expression fermented starch faster. The decline in starch fermentation rates toward the end of the fermentation was associated with accumulation of disaccharides and limit dextrins, poor substrates for glucoamylase. The buildup of these products in continuous fermentations inhibited glucoamylase activity and complete utilization of the starch. Under these conditions maltose‐fermenting strains had a significant advantage over nonfermenting strains. The synthesis and secretion of glucoamylase showed no deleterious effects on cell growth rates, fermetation rates, and fermentation products.
Modeling, optimization, and computer control of the cephalosporin C fermentation process
Authors: Chu Wey‐Bang Z. , Constantinides Alkis
Published: December 2004

In this study, a cephalosporin C producing strain, Cephalosporium acremonium (ATCC 36225), was chosen to determine the optimal conditions that maximize antibiotic production in a mixed substrate of glucose and sucrose. A model for cell growth and cephalosporin C production at different pH and temperature was developed and the associated parameters were evaluated experimentally. Pontryagin's maximum principle, in conjunction with the model, was used to predict the optimal temperature and pH control profiles to maximize the production of antibiotic.
Scaleup of monoclonal antibody purification using radial streaming ion exchange chromatography
Authors: Jungbauer A. , Unterluggauer F. , Uhl K. , Buchacher A. , Steindl F. , Pettauer D. , Wenisch E.
Published: December 2004

A scaleup study of the radial streaming chromatography (ZetaPrep technique) using hybridoma culture supernatant as model protein solution is described in this article. Lab and pilot cartridges were tested. Scaleup factors were calculated from the lab experiments and compared to the data obtained at pilot level. The procedure consists of three different steps: microfiltration, diafiltration, and the ZetaPrep technique using QAE cartridges. Diafiltration was used to condition the clarified culture supernatant. Calculating the elution volumes for the pilot level (ZetaPrep 800) from the smallest lab cartridge (ZetaPrep 15), a difference between calculated and experimental values of 230% was obtained. The difference between calculated and experimental values using results from ZetaPrep 100, a preparative cartridge, was 120%. At pilot level it is possible to purify 10 L culture supernatant within 3 h including regeneration and reequilibration of the cartridge. This procedure is useful for monoclonal antibodies (mAb) with a low isoelectric point (pl). The pl's of the mAb which was used in this work are in the range 5.4–6.1.
Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum
Authors: Martín C. , Negro M. J. , Alfonsel M. , Sáez R.
Published: December 2004

Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55°C and pH ranging from 4.3 to 4.8. Hydrolysis of 4‐nitropnenyl‐β‐D‐glucopyranoside (4‐NPG) and cellobiose by the β‐glucosidase of the complex, showed competitive inhibition by glucose with a Ki value of 0.8 mM for 4‐NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment.
Mathematical descriptions of hybridoma culture kinetics: I. Initial metabolic rates
Authors: Glacken M. W. , Adema E. , Sinskey A. J.
Published: December 2004

The lack of quantitative descriptions of mammalian culture kinetics limits the ability to optimally design and control cell culture bioreactors. This limitation is ad dressed by developing mathematical equations relating the initial growth rate and the antibody productivity of the hybridoma cell line, CRL‐1606, to its environmental state. This initial rate approach, in contrast with steady state analysis of chemostat cultures, approximates steady state behavior, since the rates were measured over brief time intervals and at low cell concentrations (< 50,000 cells/mL). The advantage of this approach is that it is much faster than the chemostat approach.

An equation for the growth rate was developed that superimpoed Monod equations in serum and glutamine with “noncompetitive” ‐type inhibition constants were inversely proportional to the lactate and ammonium concentrations. The Monod constant is critical for relating initial, low cell level culture states to other states.

Lactate was found to be the only environmental parameter that significantly inhibited antifibronectin monoclonal antibody production by the CRL‐1606 hybridimas. Volumetric productivity was strictly related to culture viability, which was observed to decline at growth rates below 0.02h−1. Lactate was also found to significantly inhibit ammonium production.
Synthesis of aspartame precursor: α‐L‐aspartyl‐L‐phenylalanine methyl ester in ethyl acetate using thermolysin entrapped in polyurethane
Authors: Yang Chin‐Pin , Su Chein‐Shyong
Published: December 2004

Cross‐linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z‐L‐aspartic acid (ZA) was reacted with ‐Lphenylalanine methyl ester (L‐PM) in water‐saturated ethyl acetate to give only α‐Z‐L‐aspartylL‐phenylalanine methyl ester (α‐ZAPM). Ninety‐four percent conversion of α‐ZAPM was obtained for 30 h of reaction at 40°C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU‐ll‐G‐3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin‐catalyzed condensation reaction of ZA with L‐PM in water‐saturated ethyl acetate was found to be first order in L‐PM and the Lineweaver‐Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L‐PM with the immobilized enzyme and with the ZA‐immobilized enzyme complex, with the second reaction being the rate determining step.
Separation and concentration of amino acids using liquid emulsion membranes
Authors: Thein M. P. , Hatton T. A. , Wang D. I. C.
Published: December 2004

The separation and concentration of amino acids using liquid emulsion membranes (LEMs) are discussed. Using L‐ phenylalanines as a model solute, it is experimentally shown using a facilitated transport system that separation and concentration can be simultaneously achieved. The rate of separation, final product concentration, and membrane swell are shown to increase with increasing chloride driving forces in the membrane, These effects are shown to be insensitive to the particular salt used as the driving force. Changes in the carrier concentration are shown to result in higher initial fluxes and higher swell rates. Hydrodynamically induced membrane breakage is minimal for the system under consideration. Experiments indicate that osmotically induced water transport (“swelling”) in the LEM system is mediated by both the carrier and the emulsion‐stabilizing surfactant. The data suggest that this swell is a diffusion‐limited process. The specificity of the carrier is examined and is found to be directly related to the hydrophobicity of the solute. Strategies for optimizing LEM formulations are discussed. Emphasis is placed on the hydration characteristics of the surfactant and the specificity of the carrier.
A numerical evaluation of a hollow fiber extractive fermentor process for the production of ethanol
Authors: Naser Samer Fahim , Fournier Ronald L.
Published: December 2004

The economics of a process for the production of ethanol employing a hollow fiber extractive fermentor have been investigated. A computer simulation of the process incorporating a mathematical model of the fermentor was used to calculate the mass and energy balances. The results of the process simulation were read into a computer spreadsheet programmed with the economic calculations from which a final ethanol product cost was obtained. The process was found to be as competitive as conventional fermentation processes even at the currently high cost — $4/sq ft — of hollow fibers. It was determined that the 1986 price of 46.2 cents/L of ethanol produced by the process would be reduced by 1.8 cents/L for every $1/sq foot drop in the price of hollow fibers. A comparison of this process with conventional fermentation processes indicates that its potential savings lie in its ability to use a concentrated sugar feed, and the fermentor's increased productivity and ability to produce a concentrated ethanol stream which is removed by the extracting solvent.
Release of periplasmic enzymes and other physiological effects of β‐lactamase overproduction in Escherichia coli
Authors: Georgiou George , Shuler Michael L. , Wilson David B.
Published: December 2004

When Escherichia coli containing the plasmid ptac11 is induced with 10−4 M isopropyl‐β‐thiogalactopyranoside (IPTG), 90% of the β‐lactamase activity of an overnight culture is present in the medium. The high extracellular activity of β‐lactamase does not result from cell lysis but from an increase in the permeability of the outer membrane. The excreting cells release several other periplasmic enzymes into the extracellular fluid and are more sensitive to lysis by detergents. It was also shown that in these cells the level of two membrane proteins, OmpA and OmpC, is decreased. None of these phenomena were observed with the plasmid pDW17, which has a mutation in the tac promoter that reduces its activity to one fourth of the tac promoter.
Polyelectrolyte precipitation of proteins: II. Models of the particle size distributions
Authors: Fisher Rod R. , Glatz Charles E.
Published: December 2004

A population‐balance model has been used to characterize continuous polyelectrolyte precipitation of egg white proteins. We have modeled the particle size distributions of aggregates formed under a range of mixing conditions. The models, accounting for aggregate growth (by both shear‐driven and Brownian‐like collisions), breakage (by hydrodynamic shear or aggregate‐aggregate collisions), and birth (by the breakage of large aggregates), fit the data well. The kinetic constants show dependencies on shear rate and residence time that have not been previously theoretically predicted; these dependencies are due in part to aging effects on the aggregate. The model constants show a dominance of growth over breakage, supporting qualitative interpretations of the particle size distributions. A mechanism for growth‐rate enhancement, caused by polymer extensions from the particle surfaces, produced improved model performance. A collisional breakage mechanism is supported.
Acyl group transfer by proteases forming acyl–enzyme intermediate: Kinetic model analysis
Authors: Yu. Gololobov M. , Borisov I. L. , Belikov V. M. , Švedas V. K.
Published: December 2004

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl–enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as pmax). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of pmax.

The analysis has demonstrated that at a given ratio of initial S and N concentrations, pmax is affected only by (i) the ratio of the second‐order rate constants for enzymatic hydrolysis of S and P(α) and (ii) the ratio of rate constants for an attack of the acyl–enzyme intermediate by the nucleophile and water (β). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from values of α and β that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.
Diffusion of sucrose and yohimbine in calcium alginate gel beads with or without entrapped plant cells
Authors: Pu H. T. , Yang R. Y. K.
Published: December 2004

The diffusion characteristics of sucrose, a nutrient, and yohimbine, a secondary metabolite, in alginate gel beads, with or without entrapped periwinkle (Catharanthus roseus) or apple (Malus domestica) cells, were investigated. Effective diffusivities of both solutes in the gel beads were determined by two different methods from transient concentration changes in well‐stirred solutions where the beads were suspended. The linear plot method developed in this work is easy to use and requires no data from the initial periods of diffusion experiments. It was found that while the cell‐free beads provided only minor diffusional resistance to both solutes, the effective diffusivities of both solutes decreased significantly with the presence of cells in the beads and the amount of reduction was proportional to the amount of cell loading. Further, the effective diffusivity of sucrose appeared to be slightly larger than that of yohimbine under identical conditions. It was also observed that permeabilization of apple cells with dimethyl sulfoxide (DMSO) led to an increase in effective diffusivity with the effect being more significant for yohimbine.
Optimum fiber spacing in a hollow fiber bioreactor
Authors: Chresand Thomas J. , Gillies Robert J. , Dale Bruce E.
Published: December 2004

A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 × 108 viable cells/mL in the extracapillary space—corresponding to an overall reactor density of 7 × 107 cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.
Uptake rate of phenol by Pseudomonas putida grown in unsteady state
Authors: Sokół W.
Published: December 2004

The uptake rate of phenol by washed cells of Pseudomonas putidagrown on phenol in fermenter in an un steady state, caused by the step increase of dilution rate and/or phenol concentration in the feed, was studied. The Monod‐Haldane type equation was applied to fit the data and the best kinetic parameters were calculated by nonlinear least‐square techniques. It was found that the minimum period of unsteady state required for induction of the phenol metabolic pathway was approximately 30 min. The values of kinetic parameters in an unsteady state varied according to each parameter. The values of um first monotonically increased to reach their highest value after about 120 min and then monotonically decreased to equal the um in new steady state after about five residence times. No regularity in changing of Ks and Ki, in unsteady state was observed. However, the greatest change in the values of Ki, was 45% while the change in values of Ks was as much as two times compared to Ki and Ks in steady state prior to disturbance.
Control of Escherichia coli–Candida utilis continuous, competitive mixed‐culture system using the dynamic matrix control algorithm
Authors: Goochee Charles F. , Hatch Randolph T. , Cadman Theodore W.
Published: December 2004

The control of the continuous, competitive mixed culture of the yeast Candida utilis (ATCC 8205) and the bacterium Escherichia coli B/r (ATCC 12407) was examined. A modified version of the dynamic matrix control (DMC) algorithm was tested in simulation and then was successfully implemented. This represents the first report of the location and stabilization of a competitive mixed culture at a metastable equilibrium point using automatic control.
Substrate‐dependent differences in production of extracellular matrix molecules by squamous carcinoma cells and diploid fibroblasts
Authors: Varani James , Fligiel Suzanne E. G. , Inman Dennis R. , Helmreich David L. , Bendelow Matthew J. , Hillegas William
Published: December 2004

Two human squamous carcinoma cell lines and human diploid fibroblasts were examined for the production of extracellular matrix (ECM) molecules including fibronectin (FN), laminin (LN), and thrombospondin (TSP) when grown on a number of different substrates. The substrates used included glass, plastic, collagen (gelatin), and DEAE‐dextran. Levels of TSP as indicated by enzyme‐linked immunosorbent assay did not vary significantly as a function of substrate. In contrast, LN levels in the culture medium were significantly decreased when the cells were grown on DEAE‐dextran or collagen‐linked dextran as compared to the other substrates. FN levels were slightly lower in the culture medium of the cells grown on DEAE‐dextran. Biosynthetic labeling followed by immunoprecipitation indicated that the reduction in LN was due, in part, to decreased biosynthesis. Previous studies have indicated that LN influences the behavior of epithelial cells in culture and that the cells, themselves, are a major source of the LN. The differences in LN production noted here indicate that the production of this ECM component is influenced by the substratum on which the cells are grown. These differences could contribute to alterations in biological properties that are known to be influenced by the substratum.