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.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Alumina-doped alginate gel as a cell carrier for ethanol production in a packed-bed bioreactor

Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.     

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.     

The effect of size of polystyrene particles on their retention within the rat peritoneal compartment, and on their interaction with rat peritoneal macrophages in vitro

Polystyrene particles (size range 300 nm-3 microns diameter) were radioiodinated and their capture by rat peritoneal macrophages measured in vitro. For unmodified particles, most efficient accumulation was observed using a diameter of 600 nm (Endocytic Index (E.I.) = 16.4 +/- 2.9 microliters/10(6) cells/h). Particles (3 microns diameter) which had been modified to become more hydrophilic by hydroxymethylation showed an increased rate of capture (E.I. = 136.6 +/- 91.2 microliters/10(6) cells/h). Following intraperitoneal administration to rats, unmodified 3 micron particles showed selective accumulation in the omentum (18.4% injected dose/g), and this was increased for the hydroxymethylated bead (35.3% dose/g). The smaller (800 nm) particles were better able to leave the peritoneal compartment. Radiolabelled particles isolated from a peritoneal wash after 5 h were mostly cell-associated (72-86%, depending on the type of particle).     

Development of macrocapsules containing bioflavors generated in situ by immobilized cells

An alternative approach to microbial production of bioflavors, eliminating the need for lengthy product purification, is presented. It is based on co-immobilization of precursors for bioflavor generation by microbial cells, traditionally employed for food and beverage processing, within beads made of food-grade gel matrix. Following incubation under controlled conditions the bioflavor — or bioflavor mixture — is generated and accumulated within the beads. The flavor-retaining bead may then be employed as a food additive. A feasibility study demonstrated this approach with ethanol production by baker's yeast co-immobilized with glucose medium. Means and conditions for bead preparation and control of ethanol levels and production rate are presented. Complex bioflavor generation was also demonstrated by baker's yeast co-immobilized with apple juice, generating cider flavors. Beads providing beer taste were also readily made via co-immobilization of commercial brewing yeast with malt. Furthermore, the potential inherent in bioflavor generation by co-immobilization of filamentous fungi with an emulsion of oily precursor was demonstrated by γ-decalactone production from castor oil.     

Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position.

We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.     

The effect of size of polystyrene particles on their retention within the rat peritoneal compartment,and on their interaction with rat peritoneal macrophages in vitro

Polystyrene particles (size range 300nm-3μm diameter) were radioiodinated and their capture by rat peritoneal macrophages measured in vitro. For unmodified particles, most efficient accumulation was observed using a diameter of 600nm (Endocytic Index (E.I.) = 16.4 ± 2.9μl/10⁶cells/h). Particles (3μm diameter) which had been modified to become more hydrophilic by hydroxymethylation showed an increased rate of capture (E.I. = 136.6 ± 91.2μl/10⁶cells/h). Following intraperitoneal administration to rats, unmodified 3μm particles showed selective accumulation in the omentum (18.4% injected dose/g), and this was increased for the hydroxymethylated bead (35.3% dose/g). The smaller (800 nm) particles were better able to leave the peritoneal compartment. Radiolabelled particles isolated from a peritoneal wash after 5h were mostly cell-associated (72–86%, depending on the type of particle).     

Stability of alginate-immobilized algal cells

Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead characteristics were found to be affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation was found to involve a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable calcium alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5muM. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.     

Removal of diacetyl from beer by adsorbants and diacetyl reductase

Removal of diacetyl from beer with adsorbants like cellulose, silica gel, activated charcoal, calcium phosphate gel, anion- and cation-exchange resins, and silicylic acid black soil bed (SABSB) was attempted in comparison with the enzyme diacetyl reductase (EC 1.1.1.5). Diacetyl could be removed from beer by the adsorbants but they had undesirable effect on the beer quality such as color, pH, and alcohol levels. These adverse effects were not observed with the use of diacetyl reductase. The results favor the enzymatic removal of diacetyl from beer as a superior approach.     

A new dimension in retrograde flow: centripetal movement of engulfed particles

Centripetal motion of surface-adherent particles is a classic experimental system for studying surface dynamics on a eukaryotic cell. To investigate bead migration over the entire cell surface, we have developed an experimental assay using multinuclear giant fibroblasts, which provide expanded length scales and an unambiguous frame of reference. Beads coated by adhesion ligands concanavalin A or fibronectin are placed in specific locations on the cell using optical tweezers, and their subsequent motion is tracked over time. The adhesion, as well as velocity and directionality of their movement, expose distinct regions of the cytoplasm and membrane. Beads placed on the peripheral lamella initiate centripetal motion, whereas beads placed on the central part of the cell attach to a stationary cortex and do not move. Careful examination by complementary three-dimensional methods shows that the motion of a bead placed on the cell periphery takes place after engulfment into the cytoplasm, whereas stationary beads, placed near the cell center, are not engulfed. These results demonstrate that centripetal motion of adhering particles may occur inside as well as outside the cell. Inhibition of actomyosin activity is used to explore requirements for engulfment and aspects of the bead movement. Centripetal movement of adherent particles seems to depend on mechanisms distinct from those driving overall cell contractility.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.     

啤酒生产酵母全循环新工艺的研究

以改善浅色啤酒质量,降低生产成本,提高经济效益的目的,提出一种新颖的浅色啤酒的酿造方法。采用煮－浸法糖化工工艺及５０％麦芽和５０％大米作为啤酒酿造的原料及辅料。在糖化过程中添加啤酒酵母提取物作为补充氮源,不仅使所酿造的成品啤酒色泽浅,口味淡爽,纯正,泡沫洁白细腻,持久挂杯,而且具有较显著的经济效益和社会效益。     

Reaction and diffusion in a gel membrane reactor containing immobilized cells

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel-immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well-mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel-containing immobilized cells. A mathematical model with time- and position-dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel-entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel-immobilized cells. (c) 1992 John Wiley & Sons, Inc.     

Feeding experiments on Penilia avirostris dana (cladocera: crustacea)

Feeding in the marine cladoceran, Penilia avirostris Dana, was investigated using plastic micronic beads as a controlled particle source. The range of bead size ingested by P. avirostris was largely limited to diameters of 20 μm and below, perhaps a competitive advantage in nature, was found that: (1) as the concentration of beads increased, the number of beads ingested increased: (2) as the number of P. avirostris increased, the number of beads ingested increased: and (3) the number of beads ingested also increased with body size. The size of P. avirostris, however, did not appear to limit the size of bead ingested, while larger sized animals may be better competitors for particles.     

Aldehydic derivatives of bead cellulose--relationships between matrix structure and function in immobilization of enzymes catalyzing hydrolysis of high molecular substrates

Benzaldehyde derivatives of bead cellulose (BAC) were prepared and used, in parallel with bead cellulose oxidized by periodate (OC), for trypsin immobilization by reductive alkylation. Influence of pH (range 5-9) on formation and reduction of Schiff base as well as the importance of the reducing agent type-cyanoborohydride or borohydride-were tested. Borohydride reduction was more advantageous at pH 7 and pH 9 for BAC and OC, respectively. Residual activity of trypsin immobilized in this way was evaluated with substrates in the range 435 相似文献   

An Intensified Vibratory Milling Process for Enhancing the Breakage Kinetics during the Preparation of Drug Nanosuspensions

As a drug-sparing approach in early development, vibratory milling has been used for the preparation of nanosuspensions of poorly water-soluble drugs. The aim of this study was to intensify this process through a systematic increase in vibration intensity and bead loading with the optimal bead size for faster production. Griseofulvin, a poorly water-soluble drug, was wet-milled using yttrium-stabilized zirconia beads with sizes ranging from 50 to 1500 μm at low power density (0.87 W/g). Then, this process was intensified with the optimal bead size by sequentially increasing vibration intensity and bead loading. Additional experiments with several bead sizes were performed at high power density (16 W/g), and the results were compared to those from wet stirred media milling. Laser diffraction, scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, and dissolution tests were used for characterization. Results for the low power density indicated 800 μm as the optimal bead size which led to a median size of 545 nm with more than 10% of the drug particles greater than 1.8 μm albeit the fastest breakage. An increase in either vibration intensity or bead loading resulted in faster breakage. The most intensified process led to 90% of the particles being smaller than 300 nm. At the high power intensity, 400 μm beads were optimal, which enhanced griseofulvin dissolution significantly and signified the importance of bead size in view of the power density. Only the optimally intensified vibratory milling led to a comparable nanosuspension to that prepared by the stirred media milling.     

Synthesis of hydroxypropylcellulose-poly(acrylic acid) particles with semi-interpenetrating polymer network structure

To develop a novel type of semi-IPN particles using biocompatible materials, hydroxypropylcellulose-poly(acrylic acid) (HPC-PAA) particles with semi-interpenetrating polymer network structure and a porosity-structural surface were prepared by direct polymerization of acrylic acid monomer in the reaction system comprised of HPC and AA monomer and N,N'-methylenebisacrylamide (MBAAm). The properties of HPC-PAA gel particles were characterized by dynamic light scattering, FT-IR, transmission electron microscopy, and atomic force microscope. It is found that the formation of HPC-PAA gel particles is driven by the hydrogen bonding interaction between proton-donating PAA and proton-accepting HPC. These HPC-PAA gel particles exhibit thermo and pH dual-responsive behaviors. Depending on the chemical composition and the degree of cross-linking, the thermo-responsive property of HPC-PAA gel particles can be shifted from the UCST to the LCST property, and particle sizes can be changed from 100 to 1 microm in a controllable way. Successful loading of the gel particles with oxaliplatin, a hydrophilic antitumor drug, was achieved by take advantage of the complex interaction between the platinum atom of oxaliplatin and the carboxylic group of PAA in the gel particles. In vitro cytotoxicity assay indicates that the oxalipatin-loaded HPC-PAA gel particles have high anticancer activity. Considering the good biosafety, simple and mild preparation strategy and tunable size as well as the stimuli-responsive properties, the HPC-PAA gel particles should be a promising candidate for the drug delivery system.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Immobilized pepsin: Properties and use to prevent haze formation in beer and wine

Summary The characteristics of a new immobilized pepsin preparation are shown as a function of pH-value and temperature. Optimal working conditions for beer and wine treatment are derived from the stability and activity data. It was found that good protein stability of the beverage results when at least 50 g immobilized enzyme per hl beer per h (alternatively 20 g per hl wine per hour) are applied in a special type of packed bed reactor. Since the foam stability of beer is decreased by the enzyme treatment, better perspectives for industrial application are seen in wineries than in breweries.