bluuebunny/crossref_metadata_2025 huggingface dataset:
Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues
Authors: Chum H. L. , Johnson D. K. , Black S. , Baker J. , Grohmann K. , Sarkanen K. V. , Wallace K. , Schroeder H. A.
Published: December 2004

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H2SO4 and H3PO4) such that the cooking liquor pH ⩽ 3 are easily digested by enzymes. The total yields of hydrolysis residues (pulps) are in the 40–60% range; the acid‐catalyzed delignification followed by enzyme hydrolysis can generate 70–88% of the original six‐carbon sugars contained in the wood. Glucomannan and arablnogalactan are dissolved into the pulping liquor in the pH range of 2–4.5. Lower pH (⩽3) leads to additional solubilization of six‐carbon sugars. These sugars may be fermented directly. From the insoluble hydrolysis residues, 36–41% conversions of wood into fermentable sugars were obtained after enzyme hydrolysis; the starting feedstocks contain 50.8 and 46.6% hexosans, respectively, for aspen and black cotton‐wood. The kinetics of enzymatic hydrolysis of cellulose can be formally treated as two simultaneous pseudo‐first‐order reactions in which fast and slow hydrolyses of cellulose occur. Correlations between the glucan digestibility and the effect of the pretreatment have been made. The higher residual xylan content reduces the amount of the rapidly hydrolyzable glucan fraction and lowers the glucan digestibility. The proposed simple kinetic treatment is very helpful in assessing the effect of the pretreatment on pulp enzyme hydrolyzability.
pH and trace‐elements content in raisin extract industrial‐scale alcoholic fermentation
Authors: Akrida‐Demertzi K. , Demertzis P. G. , Koutinas A. A.
Published: December 2004

The ability of all kinds of Greek raisins for the industrial‐scale fermentation related to pH and trace‐element contents is discussed. In the case of raisin extracts of pH 3.2 larger ethanol productivities, yields, and a continuous use of the same yeast culture at a level of 300 batches was obtained. Metals such as Co, Fe, Zn, Cu, Cd, and Pb in all kinds of raisins have been determined using atomic absorption spectroscopy. The raisin variety Trechumena contains the smallest amount of Cu, and the variety Psilla contains 7 times higher concentration than Trechumena. This is in agreement with the fact that Psilla is the worst kind of raisin and Trechumena the best for spirit production. This observation is related with experimental results obtained by alcoholic fermentation in a series of synthetic media containing sacharose and different concentrations of copper.
Two‐Liquid phase reactor studies of 11α‐hydroxylation of progesterone by Aspergillus ochraceus
Authors: Ceen E. G. , Dunnili P. , Herrmann J. P. R.
Published: December 2004

The 11α‐hydroxylation of progesterone by free cells of Aspergillus ochraceus has been examined in a reactor with an aqueous and a natural oil phase. As the proportion of oil is increased, the point of inversion from a continuous aqueous phase is affected by the cell concentration, as is the optimum ratio of the phases for conversion. High oil ratios are not productive because of poor mass transfer to the concentrated cells in the aqueous phase. The cells are stable in the presence of oleic acid for at least 24 h.
The effective diffusive permeability of a nonreacting solute in microbial cell aggregates
Authors: Libicki Shari B. , Salmon Peter M. , Robertson Channing R.
Published: December 2004

Aggregates of Escherichia coli confined within hollowfiber reactors were either formed in place by culturing cells within the reactors, or were prepared by injecting a cell suspension that had been concentrated by centrifugation. The effective diffusive permeability of an uncharged nonreacting tracer, nitrous oxide, within the cell aggregates was calculated from measurements of the tracer flux through the aggregates. Estimates of the hydraulic permeability were also obtained for the aggregates that were grown in place. The effective diffusive permeability was found to decrease with increasing cell volume fraction to a value, for aggregates comprising 95% cells, of ca. 30% that obtained for cell‐free buffer solution. The dependence on the cell volume fraction was described adequately by the well‐known HashinShtrikman bounds for a two‐phase medium. The transport properties of aggregates cultivated in place were not significantly different from those of aggregates prepared by centrifugation. Furthermore, the effective diffusive permeabilities of the tracer in aggregates prepared from cells treated with detergent or disrupted by dehydration and grinding differed only slightly from the values obtained for aggregates formed from untreated cells. The results suggest that the method of formation of the cell aggregate and the details of the structure of the cells have little influence on the effective diffusive permeability. These findings should be applicable to the transport of other small uncharged solutes, such as oxygen, that can diffuse through cells. The hydraulic permeability estimates for the aggregates cultured in place were several orders of magnitude larger than the values predicted by a theory formulated with the assumption that the cells are impervious to flow and homogeneously distributed within the aggregates. Two possible reasons for this discrepancy are, first, that there is some flow through the cells themselves, and second, that the cells may form discrete clusters separated by relatively open regions.
Bacterial leaching of a sulfide ore by Thiobacillus ferrooxidans and Thiobacillus thiooxidans: I. Shake flask studies
Authors: Lizama Hector M. , Suzuki Isamu
Published: December 2004

Bacterial leaching of a sulfide ore containing pyrite, chalcopyrite, and sphalerite was studied in shake flask experiments using Thiobacillus ferrooxidans and Thiobacillus thiooxidans strains isolated from mine sites. The Fe2+grown T. ferrooxidans isolates solubilized sphalerite preferentially over chalcopyrite leaching 7–10% Cu, 68–76% Zn, and 10–22% Fe from the ore in 18 days. The sulfur grown T. thiooxidans isolates leached Zn much more slowly and very little Fe, with a Cu–Zn extraction ratio twice the value obtained with T. ferrooxidans. The ore adapted T. ferrooxidans started solubilizing Cu and Zn without a lag period. The ore‐adapted T. thiooxidans extracted Cu as well as T. ferrooxidans, but the extraction of Zn or Fe was still much slower in the low‐phosphate medium, while in the high‐phosphate medium it approached the value obtained with T. ferrooxidans. A high Cu–Zn extraction ratio of 0.34 was obtained with T. thiooxidans in the low phosphate medium. In the mixed‐culture experiments with T. ferrooxidans and T. thiooxidans, the culture behaved as T. thiooxidans in the low‐phosphate medium with a higher Cu–Zn extraction ratio and as T. ferrooxidans in the high‐phosphate medium with a lower Cu–Zn extraction ratio. It is concluded that T. ferrooxidans and T. thiooxidans solubilize sulfide minerals by different mechanisms.
Immobilization of glucose oxidase on polymer membranes treated by low‐temperature plasma
Authors: Kawakami Mitsuyasu , Koya Hidekazu , Gondo Shinichiro
Published: December 2004

Low‐temperature plasma was employed for activation of polymer membranes as a carrier for enzyme immobilization. Glucose oxidase was immobilized on polypropylene (PP), polyvinylidene fluoride (PVDF), or polytetrafluoroethylene (PTFE) membrane surfaces treated by nitrogen or ammonia gas plasma using glutaraldehyde as a linking agent. Enzyme activity was evaluated by the response of glucose sensor composed of the immobilized enzyme membrane and a dissolved oxygen electrode. The sensor response was found to depend on the kind of carrier membrane and to become maximum at suitable conditions of plasma treatment.
Effect of ethanol on glucose transport, key glycolytic enzymes, and proton extrusion in Saccharomyces cerevisiae
Authors: Pascual Carlos , Alonso Alberto , Garcia Isabel , Romay Cheyla , Kotyk Arnošt
Published: December 2004

The effect of ethanol on the activities of the key enzymes of the glycolytic pathway and on two membrane functions related with fermentation, the glucose uptake system, and proton extrusion rate are examined. The results indicate that ethanol, up to 2M, does not cause any change of the glucose uptake velocity nor any substantial change in the key glycolytic enzyme activities while the fermentation rate is reduced by about 50%. In a cell extract 3M ethanol as well as incubation of yeast cells with 4M ethanol caused a considerable decrease of pyruvate kinase and hexokinase activities. Phosphofructokinase remained unchanged even at higher ethanol concentrations. Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.
Effect of hydrogen and carbon dioxide partial pressures on growth and sulfide production of the extremely thermophilic archaebacterium Pyrodictium brockii
Authors: Parameswaran A. K. , Schicho R. N. , Soisson J. P. , Kelly R. M.
Published: December 2004

The effect of hydrogen and carbon dioxide partial pressure on the growth of the extremely thermophilic archaebacterium Pyrodictium brockii at 98°C was investigated. Previous work with this bacterium has been done using an 80:20 hydrogen‐carbon dioxide gas phase with a total pressure of 4 atm; no attempt has been made to determine if this mixture is optimal. It was found in this study that reduced hydrogen partial pressures affected cell yield, growth rate, and sulfide production. The effect of hydrogen partial pressure on cell yield and growth rate was less dramatic when compared to the effect on sulfide production, which was not found to be growth‐associated. Carbon dioxide was also found to affect growth but only at very low partial pressures. The relationship between growth rate and substrate concentration could be correlated with a Monod‐type expression for either carbon dioxide or hydrogen as the limiting substrate. The results from this study indicate that a balance must be struck between cell yields and sulfide production in choosing an optimal hydrogen partial pressure for the growth of P. brockii.
Continuous production of maltotetraose using immobilized Pseudomonas stutzeri amylase
Authors: Kimura Takashi , Ogata Masafumi , Yoshida Masahiro , Nakakuki Teruo
Published: December 2004

A continuous production process of maltotetraose was investigated by using immobilized maltotetraose (G4)‐ forming amylase (1,4‐α‐D‐glucan maltotetraohydrolase, EC3.2.1.60) from Pseudomonas stutzeri adsorbed on a macroporous hydrophobic resin. The maximum reaction rate was obtained at 55°C and the activation energy of hydrolysis by immobilized G4‐forming amylase was calculated to be 8.45 kcal/mol. The maltotetraose yield was greatly influenced by the flow rate of substrate solution, its concentration, and the immobilized enzyme activity. The newly defined factor “specific space velocity” was successfully introduced to normalize the operating parameters. Using this factor, the immobilized enzyme reactor then can be simulated and the operating dynamics can be determined.
Immobilization of proteins on organic polymer beads
Authors: Ampon Kamaruzaman , Means Gary E.
Published: December 2004

A new method is described for the immobilization of biologically active proteins onto several types of organic polymer beads. First, the soluble protein is modified by reaction with an excess of a hydrophobic imidoester, for example methyl 4‐phenyl‐butyrimidate, at ca. pH 9 and 0°. Excess imidoester and side products resulting from imidoester hydrolysis are separated from the hydrophobic protein derivative by exclusion chromatography or dialysis. A suspension of polymer beads (e.g. Amberlite XAD‐7) is then added to a solution of the modified protein at room temperature or below and stirred gently for 1‐2 h. The polymer beads are allowed to settle, separated from the solution by decantation or filtration, washed, and resuspended in an appropriate buffer. Quantitative adsorption of protein to the polymer beads is observed under such conditions. The synthesis of seven hydrophobic imidoesters and their use for the immobilization of trypsin onto several types of porous polymer beads is described. The immobilizations of trypsin, yeast alcohol dehydrogenase, and E. coli asparaginase by this procedure with high recoveries of catalytic activity, suggests that it will be applicable to a large number of biologically active proteins.
Microalgae production: Technical and economic evaluations
Authors: Tapie P. , Bernard A.
Published: December 2004

A review of the various literature data for large‐scale algae production costs is described. Costs were updated and recomputed in order to compare the different schemes. Total production costs of a nonprocessed biomass range from US$0.15 to US$4.0 kg−1, according to various authors. Process performance hypotheses and proposed technologies are analyzed to explain these variations. A cost analysis for a tubular bioreactor system is then presented that shows that, assuming a productivity of 60 tons/ha yr, production costs would range from FF24 to FF29 kg−1 for such a system. Operating costs as well as fixed charges account for approximately 50% of the cost. Parametric sensitivity of these costs is then analyzed: If productivity would be 30, 45, or 90 tons/ha yr, total cost would be around FF48, FF33 and FF19 kg−1. Advantages and disadvantages of the proposed tubular technology are finally discussed.
Optimization of enzymatic lysis of yeast
Authors: Liu Lee‐Cheng , Prokopakis George J. , Asenjo Juan A.
Published: December 2004

In enzymatic lysis of yeast for the recovery of intracellular proteins, the rupture of whole cells is caused by the action of a lytic system consisting primarily of protease and glucanase. A first‐principles mechanism for the lytic reaction based on a two‐layer model of the wall structure and a burst model for the disruption of cells is pre sented. The fed‐batch model results in a dynamic optimization problem, with the enzymes, activities being the control variables. Orthogonal collocation is applied to discretize the state equations, and the resulting non linear program is solved using successive quadratic pro gramming to determine the enzyme and protease inhibitor add‐in rates and pH control profiles that maximize the recovery of intracellular protein. Applying the proposed approach, optimal profiles were determined such that a significant increase of the production of proteins in a fed‐batch reactor is realized. Also, the optimal control policies in a series of continuous‐flow stirred tank reactors (CFSTRs) are determined.
Production of catalytic cells for formaldehyde production and alcohol oxidase by a catabolite repression‐insensitive mutant of a methanol yeast Candida boidinii A5
Authors: Tani Yoshiki , Sawai Tohru , Sakai Yasuyoshi
Published: December 2004

A catabolite repression‐insensitive mutant of Candida boidinii A5, strain ADU‐15, was investigated as to alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production. The mutant strain ADU‐15 showed higher cell productivity and higher alcohol oxidase activity when grown on mixed substrates (glucose–methanol), especially with a high concentration of glucose in the medium. Thus, even under substrate (glucose–methanol)‐limited chemostat conditions, where the glucose concentration was low, partial derepression of alcohol oxidase by glucose in mutant strain ADU‐15 was detected. The chemostat culture conditions with the glucose–methanol medium were optimized for alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production, respectively. By means of chemostat culturing on mixed substrates, we improved the alcohol oxidase productivity 5.0‐fold and the productivity of cells exhibiting the maximum catalytic activity for formaldehyde production 3.8‐fold, in comparison with the parent strain chemostat cultured with methanol as the single substrate.
Simplified models for packed‐bed biofilm reactors
Authors: Skowlund Christopher T. , Kirmse Dale W.
Published: December 2004

For a packed‐bed biofilm reactor two reactor models are proposed. One model is for the limiting case of a biofilm with a constant biofilm thickness in which diffusion within the biofilm is shown to be negligible. The second model assumes that the thickness of the biofilm is limited by the concentration of substrate within the biofilm. The analytical solutions for these reactor models are shown to agree very well with the numerical solutions to the exact differential equations.
Cell growth and α‐amylase production characteristics of Bacillus amyloliquefaciens
Authors: Roychoudhury Siddhartha , Parulekar Satish J. , Weigand William A.
Published: December 2004

Growth and α‐amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.
Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli
Authors: Betenbaugh Michael J. , Beaty Christine , Dhurjati Prasad
Published: December 2004

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature‐sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.
Characterization of a cellodextrin glucohydrolase with soluble oligomeric substrates: Experimental results and modeling of concentration‐time‐course data
Authors: Schmid G. , Wandrey C.
Published: December 2004

A previously isolated cellodextrin glucohydrolase (β‐glucosidase) from Trichoderma reesei QM 9414 is characterized using β‐1,4‐glucose oligomers with defined degrees of polymerization as soluble substrates. The enzyme splits off glucose units from the nonreducing chain ends of cellooligomers. Besides this hydrolytic activity there is also evidence for transfer activity depending on the concentration and degree of polymerization of substrates. Concentration‐time‐course data have been gathered for the degradation of cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose covering a wide range of enzyme and substrate concentrations. A Michaelis‐Menten type kinetic model has been developed, which is able to satisfactorily describe the complex system of parallel and series reactions during the conversion of oligomers to glucose. The only kind of inhibition considered is competitive inhibition by the final product glucose. The model takes into account the formation of multiple enzyme‐substrate complexes and is limited to those conditions, in which no transglucosylation products are observed. Cellodextrins with higher degrees of polymerization are found to be better substrates for this enzyme than is the dimer cellobiose.
Periodic operation of immobilized cell systems: Analysis
Authors: Sayles Gregory D. , Ollis David F.
Published: December 2004

The authors' mathematical model of transient immobilized cell growth and product formation is applied here to examine the performance of an immobilized cell system subject to periodic cycling of the rate‐limiting substrate supply. The model system consists of a single hydrogel‐like (porous) particle entrapping viable microorganisms. Proper nutrient cycling is shown to yield a relaxed periodic system and to virtually eliminate the leakage of biomass from the support that is commonly observed experimentally in steady (continuous nutrient supply) operation of these systems. The use of cyclic operation is evaluated by calculating the average product yield (the ratio of product formed to substrate consumed) and the average product flux from the particle (a measure of the total productivity of the system), for various cycling rates. Cycling increased the average product yield by at least a factor of three in nongrowth‐related fermentations, relative to steady operation, without any significant sacrifice in average total productivity. Growth‐related fermentations lost significant total productivity under most cycling conditions, while the average product yield was approximately unchanged at all cycling rates. Thus, immobilization in conjunction with periodic operation should be considered as an alternative process design for the production of nongrowth‐related products such as penicillin and monoclonal antibodies.
Cybernetic modeling of bacteriol cultures at low growth rates: Single‐substrate systems
Authors: Turner Brian G. , Ramkrishna D. , Jansen Norman B.
Published: December 2004

The cybernetic framework developed by Ramkrishna and co‐workers has been expanded to include the effects of cellular maintenance energy requirements on biomass levels in slow‐growing, carbon‐substrate‐limited cultures. A simple structured model, based on the existence of distinct key enzymes for growth and maintenance functions, is presented. Comparisons of the model with experimental data for the growth of Klebsiella oxytoca in constant fed‐batch culture on glucose, fructose, arabinose, and xylose show good agreement. In addition, perturbed fed‐batch culture experiments indicate that slow‐growing cultures respond less rapidly to a removal of the growth limitation than do faster‐growing ones. The possibility of a growth‐rate dependent “critical resource” is discussed.
Expression of β‐lactamase by recombinant Escherichia coli strains containing plasmids of different sizes—effects of pH, phosphate, and dissolved oxygen
Authors: Ryan Wen , Parulekar Satish J. , Stark Benjamin C.
Published: December 2004

The characteristics of growth and synthesis of plasmid‐encoded protein were studied for strains of recombinant E. coli JM103 which carried the β‐lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying‐sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0–8.0, the bulk β‐lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7–8.7 kb), at which β‐lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for β‐lactamase synthesis. The bulk β‐lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and β‐lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid‐encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.
Autoradiographic determination of mass‐transfer limitations in immobilized cell reactors
Authors: Karel Steven F. , Robertson Channing R.
Published: December 2004

Pseudomonas putida cells were grown in confined volumes in dual‐membrane immobilized cell reactors constructed from microporous polyethylene hollow fibers and silicone rubber tubules as a model system for the study of mass transport in microbial aggregates. Local cell concentrations in the reactors reached 300 g dry mass/L. Pulse‐chase radioisotope labeling with 35SO42− was used to estimate the rates of cell mass synthesis and degradation. Sulfur incorporation consistently exceeded sulfur release, implying that the cell mass concentration continually increases. The location and size of the cell growth region was determined using liquid emulsion autoradiography of thin sections prepared from labeled reactors. Cell growth occurs in a region less than 25 μm in depth adjacent to the oxygen supply, and the expansion of the cells caused by cell growth promotes convection of the cell mass into regions of the reactor where starving cells accumulate. The combination of mass‐balance and spatial distribution measurements that can be made using radioisotope tracers provides a versatile method for determining metabolic rates and limitations caused by mass transfer in immobilized cell reactors.
Semicontinuous cultivation of Digitalis lanata cells: Production of β‐methyldigoxin in a 300‐L airlift bioreactor
Authors: Reinhard E. , Kreis W. , Barthlen U. , Helmbold U.
Published: December 2004

Selected Digitalis lanata cell lines cultivated in 1‐L shake flasks or 20‐L airlift bioreactors converted β‐methyldigitoxin into β‐methyldigoxin with almost no side reactions. This biotransformation process was optimized with regard to substrate supply and culture medium composition, and was then scaled up to a volume of 210 L using a 300‐L airlift bioreactor. A semicontinuous process was developed in which 513.3 g β‐methyldigoxin were produced after 89 days of cultivation.
Xylose fermentation by yeasts. 5. Use of ATP balances for modeling oxygen‐limited growth and fermentation of yeast Pichia stipitis with xylose as carbon source
Authors: Rizzi Manfred , Klein Christian , Schulze Constanze , Bui‐Thanh Ngoc‐Anh , Dellweg Hanswerner
Published: December 2004

Kinetic studies are presented for the growth and fermentation of the yeast Pichia stipitis with xylose as the carbon source. Ethanol is produced from xylose under anaerobic as well as under oxygen‐limiting conditions but only at dissolved oxygen concentrations up to 3 μmol/L Maximum yields and production rates were obtained under oxygen‐limiting conditions, where the xylose metabolism may be considered to be consisted of three different components (assimilation, respiration, fermentation). The contribution of each pathway is determined by the availability of oxygen and the energy yield of each pathway. In order to describe the course of oxygen‐limited fermentations, a mathematical model has been developed with the assumption that growth is coupled to the energy production. The resulting model requires only four independent parameters (Y, Y, mATP, and P/O). These parameters were estimated on the basis of eight separate batch fermentations.
Reduced product formation following perturbation of ethanol‐ and propionate‐fed methanogenic CSTRs
Authors: Smith Daniel P. , McCarty Perry L.
Published: December 2004

Energetic analysis was applied to reduced product formation following perturbation of ethanol‐ and propionate‐fed methanogenic continuous stirred tank reactors (CSTRs). Formation and dissipation of longer‐chained n‐carboxylic acids corresponded with the variation in Gibbs free energy change associated with beta‐oxidation reactions. Formation appeared to occur from acetate and propionate by reductive back‐reactions, made energetically favorable by elevated hydrogen partial pressure (P), and possibly mediated by biosynthetic enzymes. The formed longer‐chained acids dissipated when the P fell and equilibrium shifted to favor beta‐oxidations. n‐Propanol was found to be produced from propionate in a coupled ethanol oxidation/propionate reduction reaction, mediated by ethanol‐oxidizing organisms during high rates of ethanol utilization and elevated P. When P declined, n‐propanol was oxidized back to its precursor propionate. Both reaction energetics and intracellular diffusion of the electron carrier may effect transient mediation of this coupled reaction.
Urea hydrolysis by immobilized urease in a fixed‐bed reactor: Analysis and kinetic parameter estimation
Authors: Moynihan H. J. , Lee C. K. , Clark W. , Wang N.‐H. L.
Published: December 2004

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed‐bed reactor has been studied. A modified Michaelis–Menten rate expression is used to describe the pH‐dependent, substrate‐ and product‐inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug‐flow reactor model has been developed. An effectiveness factor is used to describe the reaction–diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best‐fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the Vmax (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.
A comparison of three models for the diffusion of oxygen in electrolyte solutions
Authors: Holtzapple Mark T. , Eubank Philip T. , Matthews Michael A.
Published: December 2004

Diffusion of oxygen through aqueous solutions is of great importance in biological systems. In this work, three models for the diffusion of oxygen through aqueous salt solutions are compared. One model uses mole fraction as the driving force (Fick's Law) and another uses chemical potential. The third model uses the gradient in oxygen activity as the driving force. This new model was chosen because of the availability of oxygen electrodes which directly measure oxygen activity in aqueous solution. These models have been used to reevaluate the technique of measuring O2 diffusivities. We show that Pick's Law diffusion coefficients do not vary strongly with salt concentration as was erroneously reported in the literature. In addition, we compare the predicted O2 fluxes of the three models over a wide range in O2 concentrations. For oxygen concentrations of biological interest, the three models give identical predictions of the flux.
Uptake of cadmium and zinc by the alga Chlorella vulgaris: Part 1. Individual ion species
Authors: Ting Y. P. , Lawson F. , Prince I. G.
Published: December 2004

The ability of algae and bacteria to accumulate heavy metals from the surrounding environment is a widely recognized phenomenon that has a number of important implications. This work reports on the development of a quantitative model that addresses the basic mechanisms inherent in many uptake processes. The model postulates two mechanisms: an initial rapid metal ion uptake due to attachment onto the cell wall followed by a relatively slow uptake due to membrane transport of the metal into the cell. The mathematical model has been tested using the alga Chlorella vulgaris in the presence of cadmium and zinc in solution under various experimental conditions.
Properties of a reversible soluble–insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose
Authors: Taniguchi Masayuki , Kobayashi Mikio , Fujii Michihiro
Published: December 2004

Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product—the soluble sugar solution—by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble–insoluble carrier for immobilizing enzyme is promising, not only for cellulose–cellulase systems, but also for other heterogeneous reaction systems.
Dynamics of plasmid maintenance in a CSTR upon square‐wave perturbations in the dilution rate
Authors: Weber Allison E. , San Ka‐Yiu
Published: December 2004

The effects of forced oscillations in the dilution rate on a population of Escherichia coli K12 harboring the plasmid pBR322 in a chemostat with a nonselective medium were studied. In the constant dilution rate control experiments, the percentage of plasmid‐containing cells decreased after a long lag time. Eventually, the culture approached a population consisting of 100% plasmid‐free ells. However, under forced perturbations of the dilution rate, the culture maintained a mixed population of plasmid‐free and plasmid‐carrying cells for a longer period of ime. An unstructured model was developed to describe the above observations. Our results indicate that transient conditions created by dilution‐rate perturbations provide a favorable environment for the plasmid‐carrying population. In addition, experiments with different cycling frequencies suggest that adaptation by the culture to these transient conditions will reduce or totally eliminate such an advantage.
Acetone–butanol–ethanol (ABE) production in a novel hollow fiber fermentor–extractor
Authors: Shukla R. , Kang W. , Sirkar K. K.
Published: December 2004

Acetone–butanol–ethanol (ABE) production by immobilized C. acetobutylicum cells is studied in a novel microporous hollow fiber based tubular fermentor–extractor. The solvent 2‐ethyl‐l‐hexanol is used for in situ dispersion‐free extraction of products. A mathematical model for simultaneous fermentation and extraction of the products has been investigated. The predicted as well as experimental data follow the same trend. The experimentally observed value of total solvent productivity increased by more than 40% as a result of in situ solvent extraction.
Enzymatic oxidation of ethanol in the gaseous phase
Authors: Barzana Eduardo , Karel Marcus , Klibanov Alexander M.
Published: December 2004

The enzymatic conversion of gaseous substrates represents a novel concept in bioprocessing. A critical parameter in such systems is the water activity, Aw The present article reports the effect of Aw on the catalytic performance of alcohol oxidase acting on ethanol vapors. Enzyme activity in the gas‐phase reaction increases several orders of magnitude, whereas the thermostability decreases drastically when Aw is increased from 0.11 to 0.97. The enzyme is active on gaseous substrates even at hydration levels below the monolayer coverage. Enhanced thermostability at lower hydrations results in an increase in the optimum temperature of the gas‐phase reaction catalyzed by alcohol oxidase. The apparent activation energy decreases as Aw increases, approaching the value obtained for the enzyme in aqueous solution. The formation of a pread‐sorbed ethanol phase on the surface of the support is not a prerequisite for the reaction, suggesting that the reaction occurs by direct interaction of the gaseous substrate with the enzyme. The gas‐phase reaction follows Michaelis–Menten kinetics, with a Km value almost 100 times lower than that in aqueous solution. Based on vapor–liquid equilibrium data and observed Km values, it is postulated that during the gas‐phase reaction the ethanol on the enzyme establishes an equilibrium with the ethanol vapor similar to that between ethanol in water and ethanol in the gas phase.
Structure of activated sludge floes
Authors: Li Da‐Hong , Ganczarczyk Jerzy J.
Published: December 2004

Relatively large activated sludge floes (larger than about 100 μm) were stabilized, using a histological tissue specimen preparation procedure, and then were sliced into sections of 3 to 6 μm thick. The study of these sections, after staining, revealed the internal structure of the activated sludge floes. No uniformity of this structure was found. The distribution of microorganisms and of extracellular polymers (EPs) in the floes varied randomly on the plane of the sections and along the dimension perpendicular to the plane, leaving large water channels and reservoirs in some of the floes. The lack of a characteristic size for the water gaps in the floes and a general self‐similar appearance of the sections suggested that the activated sludge floes might be characterized by the fractal concept within a certain size limit. Direct observation of the interior of the floes indicated an abundant presence of extracellular polymers (EPs) in amorphous forms, surrounding microorganisms in most of the floes.
Lipolytic activities of a lipase immobilized on six selected supporting materials
Authors: Shaw Jei‐Fu , Chang Rey‐Chang , Wang Fung Fang , Wang Yng Jiin
Published: December 2004

Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase‐coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45‐55°C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half‐life of the PVC immobilized lipase operating at 30°C in a packed‐bed reactor was estimated to be about 400 h.
Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross‐flow membrane filtration
Authors: Uribelarrea Jean‐Louis , Winter Jacques , Goma Gérard , Pareilleux Alain
Published: December 2004

The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single‐stage process with recycle of the cells via cross‐flow micro‐filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.

Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.
Protein refolding in reversed micelles
Authors: Hagen Anna J. , Hatton T. Alan , Wang Daniel I. C.
Published: December 2004

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant‐free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.
Protein refolding in reversed micelles: Interactions of the protein with micelle components
Authors: Hagen Anna J. , Hatton T. Alan , Wang Daniel I. C.
Published: December 2004

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, γ‐interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since γ‐interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.
Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation
Authors: Hecht V. , Bischoff L. , Gerth K.
Published: December 2004

To study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10‐L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.
Enzymatic catalysis in microemulsions: Enzyme reuse and product recovery
Authors: Larsson Karin M. , Adlercreutz Patrick , Mattiasson Bo , Olsson Ulf
Published: December 2004

A technique for enzyme reuse and product recovery from enzymatic catalysis in microemulsions is demonstrated. The enzymatic reaction is performed in a homogeneous isotropic microemulsion; AOT (sodium bis‐(2‐ethyl‐ hexyl)sulfosuccinate)/isooctane/buffer or C12E5(penta ethylene glycol dodecyl ether)/heptane/buffer. By small temperature changes the systems are shifted to two phase regions, where an oil‐rich phase, containing the product, coexists with a water‐rich phase containing surfactant and enzyme. The oil‐rich phase may be replaced by an oil solution containing new substrate. Thus, the reaction may be continued and the enzyme reused. This procedure was repeated nine times in the present study. Data on phase behavior in presence and in absence of protein, partitioning of the components and a radioactive‐labelled protein between the phases, and the repeated use of horse liver alcohol dehydrogenase (HLADH) in the microemulsions are presented.