Effective diffusion coefficient of sucrose in calcium alginate gel.

The effective diffusion coefficient of sucrose in 5% calcium alginate gel containing 41.6 g.d.c. l-1. Saccharomyces cerevisiae was investigated. Both free and immobilized S. cerevisiae in 0.175 cm and 0.3 cm diameter particles were used and the reactions were achieved in a medium containing 100 g l-1 sucrose and 0.05 M CaCl2. With the assumption that the microorganisms did not grow or die in this medium, the results were analyzed according to Michaelis-Menten kinetics and the values of the parameters were determined as: Vm = 0.256 g ml-1 gel h-1, Km0 = 0.097 g ml-1, Km1 = 0.125 g ml-1, and Km2 = 0.165 g ml-1. Using these values, effectiveness factors were calculated as eta 1 = 0.89 and eta 2 = 0.76, and effective diffusion coefficients for sucrose in calcium alginate gel were determined as De1 = 4.1 X 10(-6) cm2 s-1 and De2 = 4.0 X 10(-6) cm2 s-1, for the particle size involved.     

Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain

A Pseudomonas luteola strain possessing azoreductase activity was utilized to decolorize a reactive azo dye (C. I. Reactive Red 22) with fed-batch processes consisting of an aerobic cell growth stage and an anaerobic fed-batch decolorization stage. The fed-batch decolorization was conducted with different agitation and aeration rates, initial culture volumes, dye loading strategies, and yeast extract to dye (Y/D) ratios, and the effect of those operation parameters on azo dye decolorization was evaluated. Dissolved oxygen strongly inhibited the azo reduction activity; thus aeration should be avoided during decolorization but slight agitation (around 50 rpm) was needed. With the periodical feeding strategy, the specific decolorization rate (v(dye)) and overall decolorization efficiency (eta(dye)) tended to increase with increasing feeding concentrations of dye, whereas substrate inhibition seems to arise when the feeding concentration exceeded 600 mg dye/L. In the continuous feeding mode, higher initial culture volume resulted in better eta(dye) due to higher biomass loading, but lower v(dye) due to lower dye concentration in the bioreactor. With a volumetric flow rate (F) of 25 mL/h, both v(dye) and eta(dye) increased almost linearly with the increase in the loading rate of dye (F(dye)) over the range of 50-200 mg/h, while further increase in F(dye) (400 mg/h) gave rise to a decline in v(dye) and eta(dye). As the F was doubled (50 mL/h), the v(dye) and eta(dye) increased with F(dye) only for F(dye) < 80 mg/h. The best v(dye) (113.7 mg dye g cell(-)(1) h(-)(1)) and eta(dye) (86.3 mg dye L(-)(1) h(-)(1)) were achieved at F(dye) = 200 mg/h and F = 25 mL/h. The yield coefficient representing the relation between dye decolorized and yeast extract consumed was estimated as 0.8 g/g. With F(dye) = 75 mg/h, the Y/D ratio should be higher than 0.5 to ensure sufficient supply of yeast extract for stable fed-batch operations. However, performance of the fed-batch decolorization process was not appreciably improved by raising the Y/D ratio from 0.5 to 1.875 but was more sensitive to the changes in the dye loading rate.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Adsorptive immobilization of a Pseudomonas strain on solid carriers for augmented decolourization in a chemostat bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml-1 carrier) in comparison with PVA particles (4.8 mg VS ml-1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factorin the thicker biofilms (effectiveness factor eta = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml-1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l-1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80-81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to approximately 80% by the augmented system.     

Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.     

Biosorption behavior of azo dye by inactive CMC immobilized Aspergillus fumigatus beads

The biosorption equilibria and kinetics of an azo dye (reactive brilliant red K-2BP) were examined in this study using inactive carboxylmethylcellulose (CMC) immobilized Aspergillus fumigatus beads as the biosorbent. It was found that the biosorption capacity was at maximum when dye solution pH was about 2.0, that the sorption was spontaneous and endothermic with insignificant entropy changes, and that the Freundlich isotherm model fitted well to the biosorption equilibrium data. The biosorption rates were found to be consistent with a pseudo-second-order model. An intraparticle diffusion-based Weber-Morris model was applied to evaluate rate-limiting steps of the biosorption processes. The results suggested that the diffusion controlled the overall biosorption process, but the boundary layer diffusion of dye molecules could not be neglected. External mass transfer coefficients (beta(I)S) obtained by both Mathews and Weber model and Frusawa and Smith model were consistent.     

Pore diffusion model for a two-substrate enzymatic reaction: Application to galactose oxidase immobilized on porous glass particles

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.     

Removal of Astrazon Yellow 7GL from aqueous solutions by adsorption onto wheat bran

Adsorption kinetic and equilibrium of a basic dye (Astrazon Yellow 7GL) from aqueous solutions at various initial dye concentration (50-300 mg/l), pH (4-10), adsorbent dosage (2-8 g/l), particle size (354-846 microm) and temperature (30-50 degrees C) on wheat bran were studied in a batch mode operation. The result showed that the amount adsorbed of the dye increased with increasing initial dye concentration and contact time, whereas particle size and pH had no significant affect on the amount of dye adsorbed by the adsorbent. A comparison of kinetic models on the overall adsorption rate showed that dye/adsorbent system was best described by the pseudo second-order rate model. The removal rate was also dependent on both external mass transfer and intra-particle diffusion. The low value of the intraparticle diffusivity, 10(-11) cm2/s, indicated the significant influence of intraparticle diffusion on the kinetic control. The adsorption capacity (Q0) calculated from the Langmuir isotherm was 69.06 mg/g for at pH 5.6, 303 K for the particle size of 354 microm. The experimental data yielded excellent fits with Langmuir and Tempkin isotherm equations. Different thermodynamic parameters showed that the reaction was spontaneous and endothermic in nature.     

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.     

固定化产氨短杆菌MA-2、黄色短杆菌MA-3反应动力学的研究

多年来虽然有不少学者对固定化细胞生产Ｌ 苹果酸的方法进行过探讨[1～ 6] ,但对过程动力学的研究报道并不多见[2 ,7] ,在富马酸铵转化体系中的表观动力学及本征动力学模型还未见报道 ,本文对富马酸铵转化体系中固定化产氨短杆菌ＭＡ 2、黄色短杆菌ＭＡ 3细胞的动力学进行了探讨 ,测定了两种固定化细胞的表观动力学常数 ,并进一步求解了相应的本征动力学常数 ,这一结果便于从理论上指导富马酸铵转化过程的工业化生产。1　材料和方法1 1　试剂富马酸 ,工业级 ,苏州合成化工厂 ,碳酸钙 ,工业级 ,泗联化工厂。1 2　菌株本文所用的菌株是由我院…     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Diffusion of (de)acylated antibiotic A40926 in alginate and carrageenan beads with or without cells and/or soybean meal

Effective diffusion coefficients (D(e)) of antibiotic A40926 and its deacylated derivative were determined in Ca-alginate (2% wt/wt) and kappa-carrageenan (2.6% wt/wt) gel beads with or without immobilized Actinoplanes teichomyceticus cells and/or soybean meal (SBM). The method used was based on transient concentration changes in a well-stirred antibiotic solution in which gel beads, initially free of solute, were suspended. Unsteady-state diffusion in a sphere was applied and D(e) determined from the best fit of experimental data. A40926 showed markedly different diffusion characteristics than its deacylated derivative. Diffusivity of deacyl-A40926 in alginate or carrageenan gel beads was six to seven times that of A40926. Large differences in partition coefficients (Kp) were also found. In case of beads without additions, A40926, in contrast to deacyl-A40926, strongly partitioned to the liquid phase. Introduction of SBM and/or mycelium in the gel beads decreased the effective diffusivity of deacyl-A40926, but increased its partitioning to the solid phase. Our findings indicate that a relatively moderate structural change of a lipoglycopeptide molecule could lead to a major change in its diffusion/partition characteristics.     

Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels

A fluorescence-based method for simultaneously determining the diffusion coefficients of two proteins is described, and the diffusion coefficient of insulin-like growth factor (IGF-I) and ribonuclease (RNase) in a 0.27% fibrin hydrogel is reported. The method is based on two-color imaging of the relaxation of the protein concentration field with time and comparing the results with a transport model. The gel is confined in a thin (200 μm) capillary and the protein is labeled with a fluorescent dye. The experimentally determined diffusion coefficient of RNase (D = 1.21 × 10⁻⁶ cm²/s) agrees with literature values for dilute gels and bulk aqueous solutions, thus indicating the gel and the dye had a negligible effect on diffusion. The experimental diffusion coefficient of IGF-I (D = 1.59 × 10⁻⁶ cm²/s), in the absence of binding to the fibrin matrix, is consistent with the dimensions of the molecule known from x-ray crystallography and a correlation between D and molecular weight based on 14 other proteins. The experimental method developed here holds promise for determining molecular transport properties of biomolecules under a variety of conditions, for example, when the molecule adsorbs to the gel or is convected through the gel by fluid transport.     

Sorptive removal of Methylene blue from aqueous solution using palm kernel fibre: Effect of fibre dose

The use of palm kernel fibre, a readily available agricultural waste product for the sorption of Methylene blue from aqueous solution and the possible mechanism of sorption has been investigated at various fibre doses. The extent of dye removal and the rate of sorption were analyzed using two kinetic rate models (pseudo-first and pseudo-second-order kinetic models) and two diffusion models (intraparticle and external mass transfer models).

Analysis of the kinetic data at different sorbent dose revealed that the pseudo-first order kinetics fitted to the kinetic data only in the first 5 min of sorption and then deviated from the experimental data. The pseudo-second-order kinetic model was found to better fit the experimental data with high correlation coefficients at the various fibre dose used. The dye sorption was confirmed to follow the pseudo-second-order model by investigating the relationship between the amount of dye sorbed and the change in hydrogen ion concentration of the dye solution and also the dependence of dye uptake with solution temperature. It was found that the change in hydrogen ion concentration and increase in sorption temperature were directly related to the amount of dye sorbed, and activation energy was calculated to be −39.57 kJ/mol, indicating that the dye uptake is chemisorption, involving valence forces through sharing or exchange of electrons between sorbent and sorbate as covalent forces.

The intraparticle diffusion plots showed three sections indicating that intraparticle diffusion is not solely rate controlling. The intraparticle diffusion and mass transfer rate constants where observed to be well correlated with sorbent dose in the first 5 min of sorption, indicating sorption process is complex. It was found that at low sorbent dose the mass transfer is the main rate controlling parameter. However at high sorbent dose, intraparticle diffusion becomes rate controlling.     

Influencing effect of intra-granule mass transfer in expanded granular sludge-bed reactors treating an inhibitory substrate

Two expanded granular sludge-bed (EGSB) reactors (superficial velocity u s=6.0 and 9.0m/h) were used to treat an inhibitory substrate phenol. The granule diameter (dp) increased with increasing organic loading rate (OLR) and u(s). At the OLRs of 1.67-4.44 kg phenol/m3 d, the accumulation of volatile fatty acids (VFAs) was insignificant; whereas at the OLR of 5.11 kg phenol/m3 d, both the accumulation of VFAs and the washout of large hollow granules (average dp=2.90-3.12 mm) occurred. The comparative experimental and simulated results showed that the proposed kinetic model is suitable for design and predicting purposes. The calculation results of mass transfer parameters (Thiele modulus, Biot number, diffusion layer thickness, and overall effectiveness factor) and parametric sensitivity analysis results (half-saturation constant Ks and dp) showed that the intra-granule mass transfer would lead to a more influencing effect than the external mass transfer on the overall substrate removal rate in EGSB reactors.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Studies on the respiration rate of free and immobilized cells of Cephalosporium acremonium in cephalosporin C production

Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes. However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen. In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina. The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally. By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium. The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero‐order effectiveness factor, considering the dead core model. Furthermore, experimental results obtained with immobilized cells in a 1.7‐L tower bioreactor were compared with those obtained in 5‐L conventional fermentor with free cells. It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca. 2.5 mm) and sucrose consumption‐based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 593–600, 1999.