Gabriela Kuncová

ENCAPSULATION OF MICROBIAL CELLS INTO SILICA GEL

This work deals with changes in microbial phenol degradation and cell proliferation caused by immobilization into silica gel. Mixed microbial culture and the yeast Candida tropicalis were immobilized in silica layers and pieces prepared by mixing of prepolymerized tetraethoxysilane with cell suspension. The phenol degradation rate of cells entrapped in silica gel was compared with those immobilized into an organic polymer-polyurethane. The phenol degradation efficiency decreased in the following order: free cell suspension > cells entrapped into polyurethane foam > cells entrapped into prepolymerized TEOS. Inside the silica there was no growth observed by optical microscope. The immobilization of bacterium Pseudomonas species 2 into silica gel, cells which co-metabolize PCBs with biphenyl, did not result in substantial change of intermediate concentration.

Fiber-Optic Chemical Sensors and Fiber-Optic Bio-Sensors

This review summarizes principles and current stage of development of fiber-optic chemical sensors (FOCS) and biosensors (FOBS). Fiber optic sensor (FOS) systems use the ability of optical fibers (OF) to guide the light in the spectral range from ultraviolet (UV) (180 nm) up to middle infrared (IR) (10 µm) and modulation of guided light by the parameters of the surrounding environment of the OF core. The introduction of OF in the sensor systems has brought advantages such as measurement in flammable and explosive environments, immunity to electrical noises, miniaturization, geometrical flexibility, measurement of small sample volumes, remote sensing in inaccessible sites or harsh environments and multi-sensing. The review comprises briefly the theory of OF elaborated for sensors, techniques of fabrications and analytical results reached with fiber-optic chemical and biological sensors.

The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol

Abstract A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (qsmax) decreased in the order: PBRC (598 mg l−1 h−1) > PBR (PU, 471 mg l−1 h−1) > PBR (SG, 394 mg l−1 h−1) > FBR (PU, 161 mg l−1 h−1) > FBR (SG, 91 mg l−1 h−1). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100–200 μm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO2 enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing.

Growth Model and Metabolic Activity of Brewing Yeast Biofilm on the Surface of Spent Grains: A Biocatalyst for Continuous Beer Fermentation

In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate ( qim) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble‐column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (Xactim) of the total immobilized biomass ( Xim) was subsequently estimated at approximately Xactim = 0.12 gIB gC−1 (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell‐to‐carrier adhesion and cell‐to‐cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (Xactim) and the maximum attainable biomass load (Xmaxim) was included into the model. Since the average biofilm thickness was estimated at ca. 10 μm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble‐column reactor. Good agreement between model simulations and experimental data was achieved.

Lipase immobilized in organic-inorganic matrics

Enzyme lipase was immobilized with ferrite powder and deposited in layers on glass slides from lipase to a solution of silicone alkoxides. The highest hydrolytical activity was observed with the magnetic lipase prepared by mixing the paste of ferrite powder and lipase with tetramethoxysilane, 3-aminopropyltriethoxysilane and propyltrimethoxysilane. In a mixed reactor, the particles of the magnetic lipase were desintegrated by mechanical stirring which caused loosing the lipase linked to magnetic material and resulted in a significant drop of activity after magnetic separation. Transparent layers were prepared by dip- or spin-coating from partially hydrolyzed tetraethoxysilane and solutions containing methyltriethoxysilane with 3-aminopropyltriethoxysilane or tetraethoxysilane with 3-mercaptopropyltriethoxysilane. The lipases immobilized in films with magnetic particles were active in tests with 4-nitrophenyl butyrate and were not inhibited by 0,0-dimethyl-0-(2,2-dichlor-vinyl)-phosphate.

Optical fibre biosensors using enzymatic transducers to monitor glucose

The construction and performance of a novel enzyme based optical sensor for in situ continuous monitoring of glucose in biotechnological production processes is presented. Sensitive optical coatings are formed from inorganic?organic hybrid polymers (ORMOCER?s) combined with a flurophore (ruthenium complex) and an enzyme, and applied to lenses, declad polymer optical fibre (POF) and polymer clad silica fibre (PCS). The enzyme, glucose oxidase, catalyzes oxidization of glucose to gluconic acid by depleting oxygen. Oxygen consumption is determined by measuring the fluorescence lifetime of metal organic ruthenium complexes which are quenched by oxygen. The coatings developed were designed to adhere to glass and polymer surfaces, to be compatible with enzymes and ruthenium complexes, and were demonstrated both as double- and single-layer structures. The sensor response to gaseous oxygen, dissolved oxygen and dissolved glucose was measured via fluorescence lifetime changes. A best detection limit of 0.5% (vol) has been determined for gaseous O2 with selected ORMOCER? sensing layers. Glucose concentrations were measured to a detection limit of 0.1 mmol L?1 over a range up to 30 mmol L?1. The sensor was usable for 30 days in a bioreactor. The opto-electronic instrumentation and performance in laboratory bioreactors and in an industrial reactor are evaluated.

Response of the bioluminescent bioreporter Pseudomonas fluorescens HK44 to analogs of naphthalene and salicylic acid.

Pseudomonas fluorescens HK44 is alux-based bioluminescent bioreporter capable of selective luminescence in the presence of naphthalene and/or salicylic acid intermediate of its metabolism. We attempted to induce bioluminescence (BL) in this strain with 72 compounds,viz. substituted naphthalenes, naphthalene-like compounds (e.g., quinoline), substituted salicylic acids, salicylic acid-like compounds (e.g., 2-anthranilic acid), oligocyclic aromates, and intermediates of naphthalene metabolism to better discriminate response specificity. From them, 42 induced BL significantly lower as compared to naphthalene, three (viz. isoquinoline,o-cresol, and salicylamide) induced BL significantly greater than naphthalene, and 27 yielded no bioluminescent response whatsoever. Strain HK44 is therefore not prone to extensive false-positive signaling and can serve as a fairly specific indicator organism for naphthalene bioavailability. At elevated concentrations, 41 compounds inhibited BL. Thus, the inclusion of constitutive bioreporter controls as indicators of sample toxicity is vital to successful biosensing application.

Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

A study of the preparation and properties of copper-containing optical planar glass waveguides

Abstract We have studied the fabrication and properties of the Cu + -containing waveguides. These waveguides were fabricated in a special soda-lime silicate glass as well as in commercial optical glass substrates, by using an ion exchange in the melts containing either Cu + or Cu 2+ , at temperatures from 350°C to 500°C, and for periods from 5 min to 21 h. The optical properties of the fabricated waveguides were characterized using both mode and photoluminescence spectroscopy. The composition of waveguiding layers was studied using Rutherford Backscattering Spectrometry (RBS) and ESCA. The presence of divalent copper was determined using Electron Paramagnetic Resonance (EPR). After ion exchange, the refractive index increased, depending on the fabrication conditions, up to Δ n =+0.0693, and the waveguides supported up to 16 TE and TM modes. The depths of the fabricated waveguides varied between 6.0 and 27.5 μm. The most intensive blue-green luminescence was achieved with the samples that were ion-exchanged in the Cu 2 Cl 2 ·ZnCl 2 melt, where the presence of Zn 2+ strongly impeded the oxidation of Cu + to Cu 2+ . Both oxidation states of copper (Cu + as well as Cu 2+ ) were found in the waveguides fabricated in the pure Cu 2 Cl 2 . The main advantage of the copper-containing waveguides is the possibility of integrating the passive and active functions of the waveguides on the same substrate.

The influence of immobilization of Pseudomonas sp. 2 on optical detection of polychlorinated biphenyls

Abstract The cells Pseudomonas sp. 2 were immobilized on porous glass surface (S), encapsulated into alginate beads (A), encapsulated into alginate beads coated with a silica layer (AC) and entrapped into silica gel (C). The silica layer thickness of 2.5 μm was prepared from prepolymerized tetramethoxysilane by dipping of the alginate beads. The production of stable yellow intermediates of biodegradation of PCBs, congeners with three chlorine atoms, was used for the detection of commercial mixture of PCBs, Delor 103. In the initial stage of biodegradation of Delor 103, the highest and the most rapid increase of concentration of yellow color was observed in the case of biodegradation with AC but the selectivity of the detection method was decreased by the simultaneous production of orange compounds. The necessary conditions for production of orange intermediates were concentration of biphenyl or PCBs with biphenyl 5 g/l together with 2–6 vol.% of methanol. Trihydroxyphenyl pyridine, phenyl pyridine, dimethylhydrazide of benzoic acid, pyridine carboxylic acid and trimethylindane were identified in the lyophilized solution of the orange compounds. The metabolic pathways of the creation of orange compounds are unknown and for that reason, utilization of these metabolites for optical detection of PCBs needs further research.