bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters
Authors: Mistry Firoz R. , Cooney Charles L. 
Published: December 2004

AbstractThe direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous‐ culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous‐culture cell recycle system showed a two‐fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters—iron concentration in the nutrient medium and the N2 purge rate of the fermentor headspace—allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO4 · 7H2O) and decreased N2 purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h−1 and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.
Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase
Authors: Comfort Ann R. , Albert Elizabeth C. , Langer Robert 
Published: December 2004

AbstractThe immobilization of heparinase to tresyl‐chloride‐activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 ± 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm2 when 4.5 μg protein/cm2 was bound to the fibers.
Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis
Authors: Comfort Ann R. , Albert Elizabeth , Langer Robert 
Published: December 2004

AbstractImmobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic Km value for the system. The intrinsic Km value for heparinase–cellulose is 0.4 ± 0.3 mg/mL (N = 6) and it is the same order of magnitude as the Km value for soluble heparinase.
Separation of L‐valine from fermentation broths using a supported liquid membrane
Authors: Deblay P. , Minier M. , Renon H. 
Published: December 2004

AbstractA carrier‐mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L‐valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.
Chemometric optimization of an asymmetric reduction catalyzed by baker's yeast
Authors: Boccù E. , Ebert C. , Gardossi L. , Gianferrara T. , Linda P. 
Published: December 2004

AbstractThe stereoselective reduction of ethyl acetoacetate to (+)‐(S)‐ethyl 3‐hydroxybutyrate catalyzed by Saccharomyces cerevisiae was optimized by means of chemometric methods. The quantitative effects of temperature, time of incubation, and concentrations of yeast and substrate on the optical purity and on the percent of reduced substrate were investigated using a factorial design at two levels. This approach gave information about the chemical behavior of the catalyst. The variability of the two responses was expressed by means of their corresponding response surfaces. Use of desirability functions allowed the overall optimization of the process, also taking into account the importance of economic factors. The investigation showed that it is possible to reduce the substrate completely obtaining (+)‐(S)‐ethyl 3‐hydroxybutyrate with percent of enantiomeric excess>98% and, at the same time, to operate in more convenient experimental conditions than those previously reported.
Electrochemical regeneration of NAD in a plug‐flow reactor
Authors: Fassouane Aziz , Laval Jean‐Marc , Moiroux Jacques , Bourdillon Christian 
Published: December 2004

AbstractElectrochemical regeneration of NAD was performed at a laboratory preparative scale to illustrate both the efficiency and intrinsic simplicity of the electrochemical method. A powerful plug‐flow reactor was realized with a flow through graphite felt electrode, the ratio of the effective area of electrode/volume of reactor increased to 380 cm2/cm3. This graphite‐felt electrode was able to oxidize NADH coenzyme at a very low overvoltage. On the example of the gluconic acid production catalyzed by glucose dehydrogenase, current as high as 0.1 A was obtained in experience where enzymatic activity was the main limitation. In confirmation of our previous work, the results show that the yield of NADH electrochemical oxidation is better than 99.95%.
Studies on the deactivation of immobilized urease
Authors: Vasudevan P. T. , Ruggiano L. , Weiland R. H. 
Published: December 2004

AbstractThe results of experiments in a fixed‐bed reactor and a CSTR containing urease immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.
Production of recombinant human growth hormone in Escherichia coli: Expression of different precursors and physiological effects of glucose, acetate, and salts
Authors: Jensen E. Bech , Carlsen S. 
Published: December 2004

AbstractThe constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N‐terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three‐fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (μ+/μ−) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met‐ala‐glu‐hGH (MAE‐hGH) was obtained. The fermentation process comprised a glucose‐limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE‐hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE‐hGH—the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells—i.e. with or without formation of acetate—was not critical per se in order to obtain a high specific yield of MAE‐hGH. Therefore it is unlikely that formation of MAE‐hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE‐hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context.From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.
Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels
Authors: Chen Ching‐I , McDonald Karen A. , Bisson Linda 
Published: December 2004

AbstractThe appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h−1, feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30°C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.
Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads
Authors: El‐Sayed Abdel‐Halim M. M. , Mahmoud Wafaa M. , Coughlin Robert W. 
Published: December 2004

AbstractIn fed‐batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 μm and pore size of 0.14 μm) produced the highest total enzymatic activity, followed by Celite R633, alginate‐coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate‐coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme‐enriched, immobilized‐cell cultures was higher than that obtained from free‐cell culture under similar conditions.
Ethanol extraction by supported liquid membrane during fermentation
Authors: Christen P. , Minier M. , Renon H. 
Published: December 2004

AbstractA supported liquid membrane system was developed for the extraction of ethanol during semicontinuous fermentation of Saccharomyces bayanus. it consisted of a porous Teflon sheet as support, soaked with isotridecanol. This assembly permitted combining biocompatibility, permeation efficiency, and stability. The removal of ethanol from the cultures led to decreased inhibition and, thus, to a gain in conversion of 452 g/L glucose versus 293 g/L glucose without extraction. At the same time, the ethanol volumetric productivity was enhanced 2.5 times, due to an improvement of yeast viability, while the substrate conversion yield was maintained above 95% of its theoretical value. Besides these improvements in fermentation performances, the process resulted in ethanol purification, since the separation was selective towards microbial cells and carbon substrate, and likely selective to mineral ions present in the fermentation broth. For pervaporation, a concentration of ethanol four times greater was obtained in the collected permeate.
Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH
Authors: Ikemi Masahisa , Ishimatsu Yoshiaki , Kise Shoichi 
Published: December 2004

AbstractA theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH‐dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell–Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi–Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess‐GDH‐to‐AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to productivity as well as operation ability.
Chemostat‐cultivated Escherichia coli at high dilution rate: Multiple steady states and drift
Authors: Majewski R. A. , Domach M. M. 
Published: December 2004

AbstractThe representation of metabolic network reaction kinetics in a scaled, polynomial form can allow for the prediction of multiple steady states. The polynomial formalism is used to study chemostat‐cultured Escherichia coli which has been observed to exhibit two multiple steady states under ammonium ion‐limited growth conditions: a high cell density–low ammonium ion concentration steady state and a low cell density–high ammonium ion concentration steady state. Additionally, the low‐cell‐density steady state has been observed to drift to the high‐cell‐density steady state. Inspection of the steady‐state rate expressions for the ammonium ion transport/assimilation network (in polynomial form) suggests that at low ammonium ion concentrations, two steady states are possible. One corresponds to heavy use of the glutamine synthetase‐glutamate synthase (GLNS–GS) branch and the second to heavy use of the glutamate dehydrogenase (GDH) branch. Realization of the predicted intracellular steady states is also found to be dependent on the parameters of the transport process. Moreover, the two steady states differ in where their energy intensity lies. To explain the drift, GLNS, which is inducible under low ammonium ion concentrations, is suggested to be a “memory element.” A chemostat‐based model is developed to illustrate that perturbations in dilution rate can lead to drift between the two steady states provided that the disturbance in dilution rate is sufficiently large and/or long in duration.
Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium‐alginate beads: I. Batch and fed‐batch fermentations
Authors: El‐Sayed Abdel‐Halim M. M. , Mahmoud Wafaa M. , Coughlin Robert W. 
Published: December 2004

AbstractIn batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized‐cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell‐free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.
A new biochemical engineering approach to the fractional precipitation of proteins
Authors: Richardson P. , Hoare M. , Dunnill P. 
Published: December 2004

AbstractA biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double‐cut fractional precipitation is obtained as the gradient of an appropriate operating tie‐line. A computer algorithm has been devised to maximize the tie‐line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.
Growing Saccharomyces cerevisiae in calcium‐alginate beads induces cell alterations which accelerate glucose conversion to ethanol
Authors: Galazzo Jorge L , Bailey James E. 
Published: December 2004

AbstractNongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi‐steady‐state pathway stoichiometry shows that alginate‐grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two‐, two‐, and ninefold larger, respectively, than in suspension‐grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension‐ and alginate‐grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate‐grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension‐grown cells. When placed in a calcium alginate matrix, alginate‐grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension‐grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended‐grown cells in suspension.