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.     

Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90-day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium- or barium-crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium- and barium-crosslinked alginates were similar at 60 days-350 +/- 20 g and 300 +/- 60 g, respectively-indicating equivalence in their stability. The stability of calcium-crosslinked sodium alginate gels over a 60-day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium-crosslinked alginate gels was estimated, using a semi-empirical model, to increase from approximately 176 to 289 A over a period of 60 days. (c) 1996 John Wiley & Sons, Inc.     

Optical anisotropy of calcium-induced alginate gels

Alginate gels formed by diffusion of calcium ions into solutions of sodium alginate were found to exhibit optical anisotropy depending on preparation conditions. When observed under crossed nicols, the anisotropic alginate gels showed a birefringence pattern which is characteristic of radial orientation of polymer chains. Calcium alginate gels were prepared from different concentrations of sodium alginate and calcium ion, and the conditions for formation of the anisotropic gels were determined. The gel-formation process was measured by monitoring the development of the birefringent layer and was compared with the model in which the diffusion of calcium ions dominates gel formation.     

Determination of hematocrit based on diffusion of an inert molecular probe from agarose gels into whole blood

Whole blood hematocrit was determined by an approach which depends on the diffusion of an inert probe, to which red blood cells are impermeable, from a small agarose gel into a stirred, much larger blood sample. Blood cells influence the diffusion rate of the probe by, on the average, physically blocking a fraction of the gel surface. The blocking effect increases with the hematocrit. Cyanocobalamin (B-12) was found to be a suitable probe because it did not penetrate, bind to, or lyse blood cells and was not bound by plasma solutes. The loss of B-12 from gels in contact with blood was monitored by determination of the absorbance change at 540 nm of gels which had been quickly rinsed. The visible spectrum of B-12 in agarose gels was identical to the spectrum in water. Beer's Law was obeyed in 1-mm thick agarose gels over a concentration range of 0.1-0.8 mM. Based on the results from 48 blood samples covering the hematocrit range 25-69, a least-squares line was generated with a slope, -3.46 X 10(-3) delta A/hematocrit unit, a Y intercept of 0.295, and a correlation coefficient of 0.971. The precision of the technique was +/- 9.7%. The assay was insensitive to mean corpuscular volume and sample volume as long as the latter was 50-fold larger than the gel volume. The diffusion coefficient for B-12 in 1% agarose gels was found to be 1.4 +/- 0.2 X 10(-6) cm2 sec-1.     

Conjugative plasmid transfer between Pseudomonas strains within alginate bead microcosms: effect of the internal gel structure.

Because microorganisms frequently live in an immobilized state in natural habitats, a cell-confined system was used to study bacterial conjugation. Two Pseudomonas putida strains were introduced together within calcium alginate gels. Different alginate beads were designed by varying the polysaccharide and the gelation solution concentrations. Microscopic examinations showed that 2% gels were quite homogeneous, but that 1.5% and 1% gels were rather heterogeneous. In these two last cases, shaft-shaped macrostructures were present. They were colonized during the culture by great densities of highly motile bacteria. Gene transfers due to conjugation were investigated in such alginate gel bead microcosms, in batch and continuous cultures. High-initial transfer frequencies were detected whatever the gel, but no conjugation events seemed to occur with further growth in the beads. Transfer frequency values were roughly similar in the different tested systems. Alginate gels used as artificial microcosms may be valuable to study the effect of cell microenvironment on genetic transfers in complex systems.     

Thermostable amylase production by immobilized thermophilic Bacillus sp.

Agar, agarose and alginate immobilized cells of thermophilic Bacillus sp. WN11 produced 7.0, 6.2, and 10.5 U/ml of thermostable amylase in shake flasks, respectively. Alginate entrapped cells released high level of amylase (10.5 U/ml) than freely suspended cells (8.0 U/ml). Amylase production was stable in five successive batches with productivity of 9-12 U/ml for alginate, 5.5-8.8 U/ml for agar, and 4.8-8.0 for agarose immobilized cells.     

Barnacle Settlement on Hydrogels

Settlement of cultured Balanus amphitrite cyprid larvae was tested on different non-solid hydrogel surfaces. Gels consisting of alginate (highly anionic), chitosan (highly cationic), polyvinyl alcohol substituted with light-sensitive stilbazolium groups (PVA-SbQ; very low cationic) and agarose (neutral) were applied in cell culture multi-well plates. Polystyrene served as a solid surface reference. Preliminary experiments were performed to determine whether any substances leaching out of the gels could inhibit barnacle settlement. Whilst leachate from the gels revealed no toxicity towards Artemia salina nauplius larvae, PVA-SbQ in solution at and above a concentration of 0.4 ppm inhibited B. amphitrite cyprid settlement. Gels were therefore washed to avoid such effects during further testing, and toxicity and settlement tests with B. amphitrite nauplii and cyprids, respectively, applied to verify that washing was effective. Settlement was tested directly on the different test materials, followed by a quality test of non-settled larvae. All gels inhibited barnacle settlement compared to the polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed promising antifouling properties. Although some settlement occurred on 2.5% PVA-SbQ gels, metamorphosis was clearly inhibited. Only 10% of the larvae had settled on 0.5% agarose gels after 8 d. Less than 40% settlement occurred on alginate gels, as well as on 2% chitosan gels. Quality testing showed that the majority of remaining non-settled larvae in all gel experiments were able to settle when offered a suitable solid substratum.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Degradation of limonin by entrappedRhodococcus fascians cells

Summary Limonin degradingRhodococcus fascians was immobilized by entrapment in alginate, k-carrageenan, agarose and polyacrylamide gels. Except this latter, gels were used both with and without polyethyleneimine treatment followed by glutaraldehyde crosslinking. Coated derivatives showed lower activity and stability and higher diffusional limitations that uncoated ones. Immobilized cells in k-carrageenan gave, globally, the best results.     

Degradation of partially oxidized alginate and its potential application for tissue engineering

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon-carbon bond of the cis-diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (approximately 5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross-linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate-derived hydrogels greatly improved cartilage-like tissue formation in vivo, as compared to alginate hydrogels.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions

A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.     

Alginate as immobilization material: III. Diffusional properties

The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of approximately 23 kJ x mol(-1).     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

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.     

Effect of Dynamic Loading on the Transport of Solutes into Agarose Hydrogels

In functional tissue engineering, the application of dynamic loading has been shown to improve the mechanical properties of chondrocyte-seeded agarose hydrogels relative to unloaded free swelling controls. The goal of this study is to determine the effect of dynamic loading on the transport of nutrients in tissue-engineered constructs. To eliminate confounding effects, such as nutrient consumption in cell-laden disks, this study examines the response of solute transport due to loading using a model system of acellular agarose disks and dextran in phosphate-buffered saline (3 and 70 kDa). An examination of the passive diffusion response of dextran in agarose confirms the applicability of Fick's law of diffusion in describing the behavior of dextran. Under static loading, the application of compressive strain decreased the total interstitial volume available for the 70 kDa dextran, compared to free swelling. Dynamic loading significantly enhanced the rate of solute uptake into agarose disks, relative to static loading. Moreover, the steady-state concentration under dynamic loading was found to be significantly greater than under static loading, for larger-molecular-mass dextran (70 kDa). This experimental finding confirms recent theoretical predictions that mechanical pumping of a porous tissue may actively transport solutes into the disk against their concentration gradient. The results of this study support the hypothesis that the application of dynamic loading in the presence of growth factors of large molecular weight may result in both a mechanically and chemically stimulating environment for tissue growth.     

The Effect of Sterilization, pH, Filler and Spore Inoculum Concentration on the Preparation of Alginate Pellets

Alginate encapsulation of an atoxigenic strain of Aspergillus flavus was studied in order to optimize encapsulation of fungal inocula with alginic acid. Sterilization by autoclaving is known to depolymerize sodium alginate. Buffered solutions (pH = 7-8) reduced this effect. Autoclaving the alginate solution with a filler/nutrient further inhibited the depolymerization reaction. Autoclaving under optimal conditions allowed a less expensive alginate (medium viscosity) to be used at a lower concentration (1%) to produce a stable product. The lowest cost pellets resulted from use of 1% medium viscosity sodium alginate with 10% cotton-seed meal. Further savings may be achieved by performing fermentations directly in alginate-nutrient mixtures and thus eliminating the mixing and blending steps. In such formulations, the nutrient composition and length of fermentation must be adjusted to prevent alginate hydrolysis. The ultimate composition of alginate pellets is influenced by the diffusion of nutrients during gelation. Up to 65% of water-soluble nutrients were lost from alginate pellets during gelation. Once pellets are introduced into the environment, organisms other than the formulated agent compete for pelleted nutrients. A minimum concentration of the biocontrol agent must be present to ensure the agent excludes competitors and successfully converts the nutrients to biomass. For A. flavus, 5000 spores g^-1 were required.     

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization.

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.