Diffusion of lactose in kappa-carrageenan/locust bean gum gel beads with or without entrapped growing lactic acid bacteria

Effective diffusion coefficients (De) of lactose in kappa-carrageenan (2.75% wt/wt)/locust bean gum (0.25% wt/wt) (LBG) gel beads (1.5-2.0-mm diameter)with or without entrapped lactic acid bacteria (LAB) were determined at 40 degrees C. The effects of lactose concentration, bacteria strain (Streptococcus salivarius subsp. thermophilus and Lactobacillus casei subsp. casei) and cell content at various steps of the fermentation process (after immobilization, pre-incubation of the beads and successive fermentations) were measured on De as a first step for process modelling. Results were obtained from transiend concentration changes n well-stirred lactose solutions in which the beads were suspended. A mathematical model of unsteady-state diffusion in a sphere was used, and De was obtained from the best fit of the experimental data. Diffusivity of lactose in cell-tree beads was significantly lower than in pure water mainly because of the obstruction effect of the polymer chains and the hydration region. Furthermore, effective diffusivity and equilibrium partition factor were independent of lactose concentration in the range from 12.5 to 50 g/L. No significant difference was found for De (effective diffusivity) and Kp (partition) coefficients between beads entrapping S. thermophilus (approximately 5 x 10(9) CFU/mL) and cell-free beads. On the other hand higher cell counts obtained with L. casei (close to 1.8 x 10(11) CFU/mL) increased mass transfer resistance resulting in lower effective diffusivities and Kp. Finally, the effects of the type of bacteria and their distribution in the beads on the diffusivity were also discussed.     

Counter-diffusion of lactose and lactic acid in kappa-carrageenan/locust bean gum gel beads with or without entrapped lactic acid bacteria.

The effective diffusion coefficient (De) and equilibrium partition factor (Kp) for lactose and lactic acid in k-carrageenan (2.75% w/w)/locust bean gum (0.25% w/w) (LBG) gel beads (1.5-2.0 mm diameter), with or without entrapped Lactobacillus casei subsp. casei (L. casei), were determined at 40 degrees C. Results were obtained from transient concentration changes in well-stirred solutions of finite volume in which the beads were suspended. Mathematical models of unsteady-state diffusion into and/or from a sphere and appropriate boundary conditions were used to calculate effective diffusion coefficients of lactose and lactic acid from the best fit of the experimental solute concentration changes. The effective diffusivities of lactose and lactic acid were 5.73 x 10(-10) and 9.96 x 10(-10) m2 s-1, respectively. Furthermore, lactic acid was found to modify gel structure since lactose diffusion characteristics (De and Kp) differed significantly from an earlier study and in the literature. In gel beads heavily colonized with L. casei, the effective diffusion coefficients of lactose and lactic acid were respectively 17% and 24% lower than for cell-free beads. Partition coefficients also confirmed the obstruction effect due to the cells, and decreased from 0.89 to 0.79, and from 0.98 to 0.87, for lactose and lactic acid, respectively. External mass transfer was estimated by an unsteady-state model in infinite volume using the Biot number. The effect of external mass transfer resistance on De results and the data reported in the literature are discussed.     

Diffusion and partitioning of proteins in charged agarose gels.

The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.     

Evaluation of effective diffusion coefficient and intrinsic kinetic parameters on azo dye biodegradation using PVA-immobilized cell beads

An immobilized mixed culture (Aeromonas hydrophila, Comamonas testosteroni, and Acinetobacter baumannii) was prepared by entrapment into phosphorylated polyvinyl alcohol (PVA) gel beads. The unsteady-state diffusion mechanism in a gel bead was applied to estimate the effective diffusion coefficients (D(e)) and the partition coefficients (K(p)) of azo dye. In addition, a simple method was developed to determine the intrinsic kinetic parameters of immobilized cells from observed reaction rates and the intrinsic kinetic parameters were then verified by fitting the experimental data into the reaction-diffusion model in a batch reactor running at a well-stirred state. The calculated effectiveness factor (eta(cal)) approached unity at Thiele modulus (Phi) < 0.3 (i.e., d(p) < 0.475 mm). The experimental effectiveness factor (eta(exp)) was in the range of 0.71-0.45 for a corresponding sphere diameter (d(p)) of 1.91 +/- 0.16 to 4.43 +/- 0.07 mm at an initial dye concentration of 200 mg/L. The results show that intraparticle diffusion resistance has a significant effect on the azo dye biodegradation rate.     

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Diffusion of acetophenone and phenethyl alcohol in the calcium-alginate-bakers' yeast-hexane system

The intrabead diffusion coefficients of acetophenone and phenethyl alcohol were measured at 30 degrees C in the triphasic immobilized yeast-water-hexane system. Saccharomyces cerevisiae cells were deactivated with hydrochloric acid and entrapped in calcium-alginate beads. Measurements of dry cell loss during deactivation, shrinkage of the beads during deactivation and the final porosity of the beads were made for various cell loadings. Final concentrations of wet cells in the beads ranged from approximately 0.25 to 0.30 g/mL. Mass transfer in the hexane phase, external to the beads, was eliminated experimentally. The estimated error of 5% to 10% in the diffusion coefficients is within the experimental error associated with the bead method. The effect of significant sampling volumes on the diffusivities was estimated theoretically and accounted for experimentally. The intrabead concentration of acetophenone and phenethyl alcohol was 150 to 800 ppm. The deactivated cells were shown to be impervious to acetophenone so that the measured diffusivities are extracellular parameters. The cell volume fraction in the beads ranged from 0.70 to 0.90, significantly higher than previously reported data. The effective diffusion coefficients conform to the random pore model. No diffusional interaction between acetophenone and phenethyl alcohol was observed. The addition of 2 vol% ethanol or methanol slightly increased the diffusivities. The thermodynamic partition coefficients were measured in the bead-free water-organic system and found to be an order of magnitude lower than the values calculated from the analysis of the diffusion data for the organic-bead system, suggesting that bead-free equilibrium data cannot be used in triphasic systems. (c) 1994 John Wiley & Sons, Inc.     

Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations.

The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.     

Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.     

Continuous biotransformation of glycopeptide antibiotic A40926 in a cascade of three airlift bioreactors using immobilized Actinoplanes teichomyceticus cells

Immobilized cells of Actinoplanes teichomyceticus ATCC 31121 were used to selectively cleave the acyl group of A40926 yielding the deacylated form of the molecule. The feasibility of this particular biotransformation in a series of three perfectly mixed airlift bioreactors with immobilized cells was examined. A continuously operated airlift cascade was designed using a model for a series of reactors with immobilized biocatalyst beads obeying Michaelis–Menten kinetics. In independent experimental runs the cascade bioreactor system was operated continuously for 56 days with an overall conversion of 99%. Model estimates for reactor volumes and relative conversions were found to be in a good agreement with the experimental results.     

Determination of glucose and ethanol effective diffusion coefficients in Ca-alginate gel.

Glucose and ethanol diffusion coefficients in 2% Ca-alginate gel were measured using the experimental technique based on solute diffusion into or out of gel beads in a well-stirred solution. The aim of the study was to make the measurements under typical conditions found in alcoholic fermentations, such as the concentrations of glucose (100 g l-1) and ethanol (50 g l-1), the simultaneous counter-diffusion of glucose and ethanol, and the presence of cells in the gel beads at a level of 10(9) cells g-1 of beads. Previously, an evaluation of the error associated with the methodology used indicated how the experimental procedure would minimize the error. The individual measurement of glucose and ethanol coefficients in 2% Ca-alginate with no cells gave values of 5.1 and 9.6 x 10(-6) cm2 s-1, respectively, which are lower than those in water. When the effect of counter-diffusion was investigated, both coefficients decreased: glucose by 14% and ethanol by 28%. When cells were incorporated into the beads, only the ethanol coefficient decreased significantly, while the glucose coefficient apparently increased its value to 6.9 10(-6) cm2 s-1.     

The nonelectrolyte permeability of planar lipid bilayer membranes

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.     

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.     

High-yield production of lipoglycopeptide antibiotic A40926 using a mutant strain Nonomuraea sp. DP-13 in optimized medium

The lipoglycopeptide antibiotic A40926 produced by Nonomuraea sp. is a complex of structurally related components differing in the fatty acid moiety. Besides showing an intrinsic antibacterial activity, A40926 is the precursor of the semisynthetic antibiotic Dalvance. In this work, A40926 production by a mutant strain Nonomuraea sp. DP-13 was investigated. It was found that A40926 production was markedly promoted by using poorly assimilated carbon source maltodextrin and nitrogen source soybean meal. Addition of Cu²⁺ resulted in a stimulation of A40926 production, but Co²⁺ had an inhibitory effect. L-Leucine addition greatly improved total A40926 production and modified the complex composition toward factor B0. An optimized production medium IM-3 was developed and a maximum A40926 production of 1096 mg/L was obtained in the 10-L fermenter. This was the highest A40926 productivity so far reported.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Diffusion characteristics of substrates in Ca-alginate gel beads

The diffusion characteristics of several substrates of varying molecular sizes into and from Ca-alginate gel beads in well-stirred solutions were investigated. The values of the diffusion coefficient (D) of substrates such as glucose, L-tryptophan, and alpha-lactoalbumin [with molecular weight (MW) less than 2 x 10(4)] into and from the gel beads agreed with those in the water system. Their substrates could diffuse freely into and from the gel beads without disturbance by the pores in the gel beads. The diffusion of their substrates into and from the gel beads was also not disturbed by increasing the Ca-alginate concentration in the beads and the CaCl(2) concentration used in the gel preparation. In the case of higher molecular weight substances such as albumin (MW = 6.9 x 10(4)), gamma-globulin (MW = 1.54 x 10(5)) and fibrinogen (MW = 3.41 x 10(5)), the diffusion behaviors of the substrates into and from the gel beads were very different. No diffusion of their substrates into the gel beads from solutions was observed, and only albumin was partly absorbed on the surface of the gel beads. The values of D of their substrates from the gel beads into their solutions were smaller than their values in the water system, but all their substrates could diffuse from the gel beads. The diffusion of high molecular weight substrates was limited more strongly by the increase of Ca-alginate concentration in the gel beads than by the increase of the CaCl(2) concentration used in the gel preparation. Using these results, the capacity of Ca-alginate gel as a matrix of immobilization was discussed.     

Biotransformation of the lipoglycopeptide antibiotic A40926 byActinoplanes teichomyceticus cells

Compound A40926, produced byActinomadura ATCC 39727, is a lipoglycopeptide antibiotic complex which inhibits Gram-positive bacteria andNeisseria species. Individual components of the complex have an identical glycopeptide core but differ in the acid chains attached to the amino group of the glucuronic moiety. Suspension cultures and resting cells ofActinoplanes teichomyceticus ATCC 31121 were able to deacylate compound A40926 factors to yield the glycopeptide nucleus, which can be then synthetically reacylated to form new analogs. In an optimized fedbatch deacylation process, 0.5 g L^–1 of compound A40926 was almost completely converted into the deacyl derivative. Under the same conditions, deacylation was also accomplished withtert-butoxycarbonyl (tert-BOC) A40926, in which the amino group at C15 was blocked to prevent formation of diacyl analogs during reacylation. The deacylase is an endoenzyme whose preliminary characterization is presented.