Porous Glass Beads Research Articles

Spectrophotometric Microdetermination of Urea in a Rice Wine by Using an Immobilized Acid Urease Column FIA System

Determination of urea in rice wines was performed by using a spectrophotometric flow-injection analysis (FIA) system introducing an acid urease column as a recognition element. An acid urease, having specific properties of showing catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans, was covalently immobilized onto porous glass beads and then, packed into a small polymer column. This flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, a gas-diffusion unit, and a flow-through quartz cell attached to a UV/VIS detector. Standard urea solutions were measured through monitoring variations in absorbance resulting from pH shift due to ammonia molecules enzymatically generated. A wide, linear relationship was obtained between the concentration of urea (7.8 µM - 1.0 mM) and the change in absorbance. Followed by several investigations for application of this FIA system for measurement of urea in commercially available rice wines, the real samples were injected into the FIA system and urea in the samples were determined. These results were compared with those obtained with use of an F-kit method which was widely used for determination of urea. Comparative studies exhibited that this FIA system might be a powerful tool for urea determination in alcoholic beverages.

A composite polyaniline-containing silica sorbent for DNA isolation

A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles. It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick. It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins. The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma. The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent.

Immobilization of chiral enzyme inhibitors on solid supports by amide-forming coupling and olefin metathesis

The question whether phage display can be used as a selection method in the directed evolution of enantioselective enzymes has not been answered satisfactorily to date. In order to be able to test this in a specific case, namely in the hydrolytic kinetic resolution of the acetate derived from α,β-isopropylideneglycerol (IPG) catalyzed by the lipase from Bacillus subtilis, suicide enzyme inhibitors anchored on porous glass or polymer beads were designed and synthesized. These are immobilized phosphonates, which bear a leaving group and also contain the chiral substrate ( d) and ( l)-IPG. Modified SIRAN ® (porous glass) and Tentagel ® (polymer) were chosen as carriers, attachment occurring via amide-forming coupling or Ru-catalyzed olefin metathesis. Initial lipase inhibition studies are also reported.

Productivity of Immobilized Yeast Reactors with Very-High-Gravity Worts

In the competitive world of beer production, efficient utilization of raw materials and equipment together with smooth operation are advantageous. In this study, data from laboratory-scale main fermentations with original gravities between 15 and 24°P and immobilized yeast are presented. The worts were prepared from malt extract, and lower original gravities were obtained by diluting the 24°P wort with brewery water. The yeast strain used was a production strain from a brewery. Porous glass beads were used as the carrier. The optimal gravity found in regard to ethanol productivity was between 18 and 21°P. The highest ethanol concentration in the beers was over 10% (v/v). The aroma compound profiles of the beers produced were quite similar, except for acetaldehyde. The laboratory-scale fermentations using immobilized yeast may be an effective tool to screen for ethanol-tolerant strains or to find process parameters that facilitate very-high-gravity brewing.

Covalent binding of genetically engineered microorganisms to porous glass beads

Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×10 8 cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density. Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.

Transformation of porous glass beads into MFI-type containing beads

Macroporous glass beads (Na 2O–B 2O 3–SiO 2) with a particle diameter of ∼300 μm and an average pore diameter of 92 nm, obtained by the VYCOR-process, were transformed into MFI-type containing beads, that retained the original shape. The conversion was achieved through a hydrothermal treatment with an aqueous mixture of an Al-source (sodium aluminate or aluminum sulfate) and a template (tetrapropylammonium bromide (TPABr) or dipropylamine (DPA)). The resulting products were characterized by X-ray diffraction, 11B MAS NMR, N 2-adsoption and SEM. The filtrates were analyzed by ICP-OES. Using two different templates and two kinds of Al-source MFI-type beads with varying properties were obtained. The boron atoms, which are constituents of the glass material, were used as probe atoms for the characterization of the synthesis procedure. With DPA the crystallization took place inside and on the outer surface of the spheres, while TPABr led to a shell like crystallization starting from the outer surface of the glass particles. The type of Al-source influences the pH value of the reaction mixture and varies the Si/B ratio in the obtained products.

UV-irradiated DNA matrixes selectively bind endocrine disruptors with a planar structure.

DNA has unique chemical properties and is readily purified from salmon milts and shellfish gonads. DNA has few commercial uses and is generally discarded as an industrial waste. Recently, we prepared water-insoluble and nuclease-resistant DNA-films by UV irradiation. The DNA-films removed DNA-intercalating compounds. Here, we immobilized double-stranded DNA onto porous glass beads by UV irradiation and prepared DNA-immobilized glass bead columns. The DNA-immobilized columns effectively accumulated more DNA-intercalating materials than the DNA-films. The DNA-immobilized columns bound endocrine disruptors with a planar structure, such as dioxin-derivatives, polychlorobiphenyl (PCB)-derivatives, and benzo[a]pyrene. Bisphenol A and diethylstilbestrol, which lack a planar structure, did not bind to the DNA-immobilized columns. These results suggest that DNA-immobilized glass bead columns could selectively remove chemical compounds with a planar structure by intercalation into the double-stranded DNA. DNA-immobilized glass beads and DNA-films prepared by UV irradiation have the potential to serve as useful biomaterials for medical, engineering, and environmental applications.

Synthesis of Amphiphilic Polymer Particles by Seed Polymerization and Their Application for Lipase Immobilization

To develop excellent immobilized enzymes, monodisperse amphiphilic polymer particles that have both hydrophilic guanidino groups and hydrophobic acyl groups were synthesized and characterized. The monodisperse amphiphilic particles, which had an average diameter of 7.92 μm, had macropores with diameters ranging from 50 nm to 500 nm. The amount of Rhizopus delemar lipase immobilized on the monodisperse amphiphilic polymer particles was 150-8 700 times those of the immobilized lipases prepared by previous investigators using Dowex MWA-1, porous glass beads, and Sepharose 4B. The specific transesterification activity of the immobilized lipase prepared with the monodisperse amphiphilic polymer particles was 93.4 times that of the lyophilized lipase.

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20α-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.

Remediation of acidic waste waters using immobilised, acidophilic sulfate‐reducing bacteria

AbstractAcidic waste waters from industrial and mining activities constitute a wordwide environmental hazard. ‘Acid mine drainage’ (AMD) waters are often highly acidic (pH < 4), contain elevated concentrations of sulfate and dissolved metals, and are toxic to most life forms. Whilst bioremediation of acidic, metal‐contaminated waters using sulfate‐reducing bacteria (SRB) has been successfully demonstrated in pilot‐scale and full‐scale operations during the past decade, the sensitivity of characterised SRB to acidity limits their use in AMD remediation. In the current study, we have evaluated the potential use of novel acidophilic SRB for remediating acidic waste waters, in comparision with, and in conjuction with, neutrophilic SRB. Three SRB‐populations (a mixed population of acidophilic isolates, a neutrophilic culture and a mixed acidophilic/neutrophilic consortium) were immobilised on porous glass beads, packed into perspex columns and the three bioreactors percolated with synthetic medium for several months. Energy and carbon source utilisation, and tolerance to acid stress of the different consortia were evaluated. Acidophilic SRB were more efficient than the neutrophilic culture in coupling ethanol oxidation to sulfate reduction and all of the substrates tested were oxidised to acetic acid. The bioreactors containing acidophilic SRB reduced sulfate and generated alkalinity when challenged with influent at pH 3 and above, indicating that such bacteria have potential for bioremediating highly acidic waste waters. Average conversion rates of 0.25–0.30 g SO42− reduced dm−3 day−1 were achieved with bioreactors containing acidophilic SRB, percolated with a pH 4 liquid medium.© 2001 Society of Chemical Industry

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum.

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.

A chemiluminometric FIA procedure for the enzymatic determination of l-aspartate

An enzymatic FIA procedure was developed for the chemiluminometric determination of l-aspartate in a medium for mammalian cell cultivation. Packed bed flow reactors containing aspartate:aminotransferase and l-glutamate oxidase, respectively, immobilized on sieved porous glass beads were combined with a peroxidase modified fibre optic sensor to detect the produced hydrogen peroxide. Peroxidase from Arthromyces ramosus was immobilized on a preactivated microporous nylone membrane and catalyzes the light generating oxidation of luminol by hydrogen peroxide. To improve the selectivity of the l-aspartate determination a l-glutamate eliminating reactor was prepared by coimmobilization of l-glutamate oxidase and catalase in a packed bed enzyme reactor. Under FIA conditions at least 0.5 mM l-glutamate can be quantitatively eliminated from the preconditioned sample solution. l-Aspartate can be determined in the range between 5 and 1000 μM under FIA conditions. The determination of l-aspartate was optimized with respect to pH, cosubstrate concentration and residence time in the packed bed enzyme reactors. The proposed procedures show high recoveries between 96 and 100% for the l-aspartate determination in a medium for mammalian cell cultivations after the elimination of l-glutamate.

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry.

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.

Metabolic differences between attached and free-living marine bacteria: inadequacy of liquid cultures for describing in situ bacterial activity

Marinobacter sp. strain CAB was cultivated with or without porous glass beads as solid support. Two substrates were used: the hydrophilic sodium lactate and a hydrophobic C 18-isoprenoid ketone (6,10,14-trimethylpentadecan-2-one (TMP)). The substrate adsorption onto the beads was measured. Bacterial adhesion was determined by a direct count technique and amounted to 70% of total cells. In the immobilised cell cultures (ICC), generation times were 1.5 and 1.8 times shorter than in the planktonic cultures (FCC) with sodium lactate and with TMP, respectively. In ICC, the growth yields were lower (15.3 FCC×10 9 and 0.8 ICC×10 9 bacteria mg −1 of sodium lactate; 50 FCC×10 9 and 35 ICC×10 9 bacteria mg −1 of TMP). The mineralisation of substrates was estimated after mass spectrometric determination of the CO 2 production rates of both free and immobilised cell cultures. The results indicated a higher specific CO 2 production rate in the ICC with sodium lactate (3.1 FCC±0.2 and 3.5 ICC±0.3 nmol CO 2 mg −1 protein min −1) but not in the ICC with TMP (1.9 FCC±0.7 and 0.5 ICC±0.3 nmol CO 2 mg −1 protein min −1). The affinities for the two substrates were lower in the presence of the solid support ( K m,ICC=18.2±0.2 μM and 37.1±2.0 μM, for sodium lactate and TMP, respectively) than without support ( K m,FCC=8.5±1.5 μM and 8.4±1.2 μM, for sodium lactate and TMP, respectively). Moreover, the presence of a solid support showed a lower inhibition by the TMP ( K i,FCC=3.8±1.0 μM and K i,ICC=12.2±2.5 μM) which may explain why the immobilised cell cultures degraded hydrophobic TMP more efficiently than the planktonic cultures.

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.

Accumulation and removal of heavy metal ions by insolubilized-DNA and its interaction.

DNA was immobilized onto a porous glass bead by a treatment with UV irradiation. The immobilized DNA was insoluble in water and used for accumulation of heavy metal ion. When DNA-immobilized glass bead was added into aqueous solution containing heavy metal ions, such as Hg2+, Cd2+, Pb2+, Zn2+, Cu2+ and Fe3+, the concentration of these metal ions in the solution was decreased. However, the concentration of Mg2+ in the solution was not affected by the addition of the DNA-immobilized glass bead. These results suggested that UV-irradiated DNA selectively accumulated heavy metal ions.

Microreactor and electrochemical detectors fabricated using Si and EPON SU-8

An electrochemiluminescence (ECL) detector and a microenzymatic reactor (MER) combining Si and SU-8 technologies are described. Both devices were fabricated using standard processing techniques to produce on-wafer sensor elements, which were composed of a platinum or, alternatively, carbon interdigitated electrode array. The platinum array was resting on top of an Si pn photodiode. After these elements were completed, the entire wafer was modified with SU-8, which was structured to form a series of shaped spacers surrounding each device. For the ECL detector, a simple flow-channel was defined whereas for the microreactor a more complex layout defining two chambers separated by a series of SU-8 columns was employed. The upstream chamber of the microreactor was packed with porous glass beads modified with immobilised enzyme glucose oxidase whereas the downstream chamber contained the electrochemical detector. The performance of the ECL detector was assessed by the detection of codeine using ruthenium (II) tris(2,2′-bipyridyl). A detection limit of 100 μM was obtained and pharmaceutical preparations were successfully assayed. The MER was first evaluated by electrochemical determination of glucose and as a next step, a miniature ECL detector was placed on line downstream the MER to perform glucose measurements by ECL. Glucose was determined with detection limits of 2 and 50 μM by electrochemistry and ECL, respectively. This system was found to have a lifetime of at least 1 month when stored at 4°C.

A new method of characterising liquid uptake within particles over short time periods

A method of measuring both the capacity and rate of absorption of liquid by powders of small particles over short time periods (of the order of a few seconds or less) is presented. The method is based on the measurement of the isothermal transient ionic current in a sample cell containing the absorbant material and the liquid. The method has been tested on solid glass beads, porous glass beads and cellulose agglomerates. Properties such as the instantaneous absorption capacity and rate can be characterised within a few seconds. No other technique is currently able to measure these fast outcomes.

Primary Fermentation with Immobilized Yeast: Some Effects of Carrier Materials on the Flavour of the Beer

Three carrier materials (porous glass beads, DEAE-cellulose, diatomaceous earth) for immobilized primary fermentation of beer were tested on a 1.6 litre scale. The influence of the carrier materials on flavour compounds in the green beer was analysed. The nature of the carrier affected the concentrations of some flavour compounds in a yeast strain specific manner. The findings suggest that the most appropriate material for immobilized primary fermentation of beer depends on the yeast strain used and the beer characteristics desired.

Microbial sulfate reduction in a liquid-solid fluidized bed reactor

A liquid-solid fluidized bed reactor was used to carry out sulfate reduction with a mixed culture of sulfate reducing bacteria. The bacteria were immobilized on porous glass beads. Stable fluidized bed operation with these biofilm-coated beads was possible. The low specific gravity of the hydrated beads allowed operation at low liquid recirculation rates. H(2)S level in the reactor was controlled by N(2) sparging, which also served as the location for liquid feed and removal. Ethanol was used as the electron donor/carbon source for the bacteria. Sulfate reduction rates up to 6.33 g sulfate L(-1) day(-1) were attained in the reactor at a hydraulic retention time of 5.1 h. The effect of hydraulic retention time and biomass loading on the beads, on reactor performance, and efficiency were examined. The efficiency of sulfate reduction increases considerably as the hydraulic retention increases, until the bacteria became very strongly substrate-limited at 55h HRT. The effect of bead biomass loading on bed expansion at various liquid superficial velocities was studied. A model for the reactor was developed. Simulations of the continuous flow experiments indicate that the model can describe the system well, and thus could be used in the design/scale-up of such reactors. The model suggests that a significant increase in the sulfate reduction capacity of the system is possible by increasing the volume of the bed relative to the total liquid volume of the system.