Entrapment of microbial cells within a gelatin matrix: A comparison of three procedures

Summary Three of the methods in which a gelatin matrix is used to entrap microbial cells are compared. The advantages and disadvantages of each method when used for immobilizing invertase-active cells of yeast (Saccharomyces cerevisae) are discussed.     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Immobilization of microbial cells in polyurethane matrices

Summary Microbial cells were immobilized in polyurethane foam as well as polyurethane gel using liquid hydrophilic polyisocyanates which react with only the addition of water. Cell concentration is about 10% (w/v) and relative activity is about 50% depending on the type of biocatalytic reaction.     

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.     

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads

Summary Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 g·g^–1 FWB) within beads and excreted it into the culture medium (up to 8 g·g^–1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 g·g^–1 FWB), no scopolin has been found into the culture medium.Abbreviations ANA -Naphthaleneacetic acid - HPLC high performance liquid chromatography - FWB fresh weight biomass - LS medium Linsmaier and Skoog medium - MS mass spectroscopy - NMR nuclear magnetic resonance - r² coefficient of determination - s standard deviation     

Degradation of carbazole by microbial cells immobilized in magnetic gellan gum gel beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and kappa-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe(3)O(4) nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g(-1) saturation magnetization. When the mixture of gellan gel and the Fe(3)O(4) nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe(3)O(4) nanoparticles was 9 mg ml(-1) and the saturation magnetization of magnetically immobilized cells was 11.08 emu g(-1). Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

Bioanode of polyurethane/graphite/polypyrrole composite in microbial fuel cells

Polyurethane (PU) foams were coated with graphite, and pyrrole monomer was subsequently polymerized onto its surface by chemical oxidization to obtain nanostructured polyurethane/graphite/polypyrrole (PU/Graph/PPy) composites, which were used for anaerobic microorganisms grown and tested as anodes in microbial fuel cells (MFC) using municipal wastewater as fuel. The effects of oxidizing agent type (ammonium persulfate and FeCl3) used in pyrrole polymerization on the performance of electrodes in MFC were studied. Composites were characterized by Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM), and by the four-point probes to determine conductivity. It was observed from SEM analysis that globular nanostructures of PPy were formed onto PU surface with average diameters between 120 and 450 nm, which are typical of aqueous polymerization of pyrrole monomer. The highest output power density observed in MFCs was 305.5 mW/m³ for the composite synthesized using FeCl₃ as the oxidant, and 128.6 mW/m³ using the composite obtained with ammonium persulfate as oxidizing; the corresponding chemical oxygen demand (COD) removal were 48.2 and 45.5%, respectively. The calculated coulombic efficiency for PU/Graph/PPy composite obtained with FeCl₃ as oxidant was of 9.4%. Internal resistance of MFC using the composite obtained with FeCl₃ as oxidant was determined by linear sweep voltammetry (LSV) and the variable resistance (VR) methods, giving 4.8 and 2.9 kO, respectively, with average maximum power density of 237.5 mW/m³.     

Comparative study on the diffusion of an IgG from various hydrogel beads

Summary The diffusion of an IgG was measured from a number of carrageenan and alginate gels. Variations in diffusion rates were high, ranging from no diffusion in a food grade carrageenan to a rate approaching the maximum theoretical value in a low viscosity alginate. Differences in the mechanical stability of beads were also observed.     

Design and development of hydrogel beads for targeted drug delivery to the colon

The purpose of this research was to develop and evaluate a multiparticulate system of chitosan hydrogel beads exploiting pH-sensitive property and specific biodegradability for colon-targeted delivery of satranidazole. Chitosan hydrogel beads were prepared by the cross-linking method followed by enteric coating with Eudragit S100. All formulations were evaluated for particle size, encapsulation efficiency, swellability, and in vitro drug release. The size of the beads was found to range from 1.04±0.82 mm to 1.95±0.05 mm. The amount of the drug released after 24 hours from the formulation was found to be 97.67%±1.25% in the presence of extracellular enzymes as compared with 64.71%±1.91% and 96.52%±1.81% release of drug after 3 and 6 days of enzyme induction, respectively, in the presence of 4% cecal content. Degradation of the chitosan hydrogel beads in the presence of extracellular enzymes as compared with rat cecal and colonic enzymes indicates the potential of this multiparticulate system to serve as a carrier to deliver macromolecules specifically to the colon and can be offered as a substitute in vitro system for performing degradation studies. Studies demonstrated that orally administered chitosan hydrogel beads can be used effectively for the delivery of drug to the colon. Published: July 13, 2007     

Fluorescence lifetime spectroscopy of a pH-sensitive dye encapsulated in hydrogel beads

A pH-sensing dye, carboxy seminaphthofluorescein-1 (C-SNAFL-1), was immobilized in poly(ethylene glycol) (PEG) microparticles via ester-amine reaction. Following photopolymerization, the hydrogel particles were then immersed in buffered pH solutions of varying pH with added polystyrene. Measurements of phase shift and amplitude attenuation of the generated and multiply scattered fluorescent light were attained as a function of modulation frequency of the incident excitation light. Upon regressing the measured data to the coupled optical diffusion equations, the average lifetimes of protonated and deprotonated forms of C-SNAFL-1 were obtained and compared to the values acquired from conventional fluorescence lifetime spectroscopy in nonscattering media. The results demonstrate the ability to perform analyte sensing with fluorescence lifetime without the confounding effect of fluorophore loading or the use of a "reference" measurement within multiply scattering systems. When extended to the immobilized fluorophore-enzymatic systems, fluorescence lifetime spectroscopy with multiply scattered light may provide a new ultrasensitive approach for analyte or toxin screening.     

Interface resistances of anion exchange membranes in microbial fuel cells with low ionic strength

The interface resistances between an anion exchange membrane (AEM) and the solution electrolyte were measured for low buffer (or ionic strength) of electrolytes typical of microbial fuel cells (MFCs). Three AEMs (AFN, AM-1, and ACS) having different properties were tested in a flat-plate MFC to which 5-mM acetate was fed to the anode and an air-saturated phosphate buffer (PB) solution was fed to the cathode. Current density achieved in the MFCs was correlated inversely with independently measured membrane-only resistances. However, the total interfacial resistances measured by current-voltage plots were approximately two orders higher than those of the membrane-only resistances, although membranes had the same order as with the membrane-only resistance. EIS spectra showed that the resistances from electric-double layer and diffusion boundary layer were the main resistances not the membrane's resistance. The electric-double layer and diffusion boundary layer resistances of the AEMs were much larger in the 10 mM PB electrolyte, compared to 100 mM PB. EIS study also showed that the resistance of diffusion boundary layer decreased due to mechanical stirring. Therefore, the interface resistance that originates from the interaction between the membrane and the catholyte solution should be considered when designing and operating MFC processes with an AEM. The AEMs allowed transport of uncharged O(2) and acetate, but the current losses for both were low during normal MFC operation.     

Unexpected distribution of immobilized microorganisms within alginate beads

Immobilization refers to the prevention of free cell movement by natural or artificial means. It has always been assumed that immediately after an immobilization procedure is performed, cells are distributed homogeneously in the beads that entrap them. However, in this study, Escherichia coli and Trichoderma asperellum distribution in alginate-gel beads was found to be nonhomogeneous. In fact, there was a greater presence of cells on the surface of the alginate beads than in their cores.     

Preparation of pure monoclonal antibody to interleukin-2 by cultivation of hybridoma cells entrapped in novel composite hydrogel beads

A simple and reliable technique was developed to prepare pure monoclonal antibody (MAb) to interleukin-2 using cells entrapped in novel composite poly(N-vinyl caprolactam)-calcium alginate beads. Flow cytometry was applied to study cell size and cell cytoplasm granularity distribution. Maximum MAb production by the gel-entrapped cells in serum free medium was 2-3-fold higher compared to free suspension culture in serum containing medium. The only contaminating protein in culture supernatant was transferrin at 5% w/v.     

Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.     

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.     

Long-term viability of photosynthetic cells stacked in a hydrogel film within a polydimethylsiloxane microfluidic device

Immobilizing cells while maintaining their long-term viability is important to utilize cells in biosensors and energy devices. In this study, we fabricated a hydrogel film of 10 μm thickness immobilizing photosynthetic cells, using a polydimethylsiloxane microfluidic device, and we monitored the viability of the cells for 30 days. Cell viability was measured by chronoamperometry using two electrodes located in the microfluidic device and was compared between hydrogel-immobilized and non-immobilized cells. The non-immobilized cells showed variation in viability. In contrast, the hydrogel-immobilized cells remained viable for 30 days. A simulation of the oxygen distribution changes by photosynthesis of the cells and mass transfer of cell culture nutrients (NaNO₃) suggested that a proper environment for cell survival was effectively established inside the hydrogel. We successfully fabricated a photosynthetic cell-laden hydrogel with potential use in next-generation photosynthesis-based solar cells and sensors.     

Mechanism of microbial utilization of biphenyl sorbed to polyacrylic beads

 A microbial consortium mineralized biphenyl sorbed to polyacrylic beads faster than the slow rate at which much of the compound was desorbed. Pure cultures of bacteria isolated from the consortium mineralized biphenyl in solution but not the sorbed compound. However, combinations of two strains did degrade biphenyl. The consortium did not reduce the surface tension in media containing sorbed biphenyl or biphenyl in solution, and addition of synthetic and microbially produced surfactants to pure cultures did not result in utilization of sorbed biphenyl by isolates able to use the soluble molecule. Cells from the consortium that were attached to continuously washed beads degraded the substrate. We suggest that bacteria may act on sorbed compounds without the necessity of an initial desorption and that the mechanism may involve cells attached to the particles rather than the excretion of a surfactant. Received: 23 May 1995/Received revision: 17 October 1995/Accepted: 30 October 1995     

New imidazole-coordinated chemotherapeutics with low epithelial toxicity

The new imidazole-coordinated chemotherapeutics with low epithelial toxicity (NICE) presented in this article feature innovative drugs that combine epithelial toxicity comparable with that of carboplatin with novel carrier ligands optimized for DNA interaction. Recent identification of the pivotal role of basolateral organic cation transporters (OCTs) in cisplatin nephrotoxicity by a new model system (electrical resistance breakdown assay) facilitated the search for substances with a favorable organotoxic profile. The assay uses the high transepithelial electrical resistance (TEER) of the C7-clone of Madin-Darby canine kidney (MDCK) cells and the exclusive basolateral expression of OCT2 in these cells. TEER and caspase-3 activity of MDCK-C7-cells grown on microfilter membranes were monitored in response to exposure of either the apical or basolateral plasma membrane to platinum complexes. The impact of complexes on cancer cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bomide tests. Effects of substituents on pharmacological properties of NICE were systematically investigated by introducing sterically demanding groups as well as electron-donating and electron-withdrawing groups. Derivatives of NICE showed different renal epithelial toxic profiles and effects on cancer cells. NICE were significantly less toxic than cis-or oxaliplatin. The chlorine substituted NICE had no effect on epithelial integrity but markedly cytotoxic activity against amelanotic melanoma cells. Together, side effect targeted screening for new anticancer drugs with the electrical resistance breakdown assay offers an interesting approach for identifying and investigating new compounds. NICE feature the first group of platinum-based cytostatics discovered by using this system for systematic screening of new chemotherapeutics with low renal epithelial toxicity.