Macroporous Carriers Research Articles

Upgrading Epoxy Supports for Enzyme Immobilization by Affinity Function Doping—A Case Study with Phenylalanine Ammonia-Lyase from Petroselinum crispum

This article provides a method to upgrade epoxy-functionalized carriers for covalent enzyme immobilization to selective carriers suitable for covalent immobilization of metal affinity-tagged enzymes without the need of preliminary enzyme purification. Affinity function doping of the epoxy-functionalized surface introduces an advanced possibility to avoid the costly and time-consuming downstream processes required for efficient immobilization on non-selective epoxy carriers. Our approach is based on the partial functionalization of surface epoxides via a proper diamine-derived linker and an ethylenediaminetetraacetic dianhydride-based chelator charged with cobalt ions. The solid macroporous carriers, doped with metal affinity functions, have both coordinative binding ability (rapid anchoring the metal affinity-tagged enzymes to the surface) and subsequent covalent bond-forming ability (preferred binding of the tagged enzyme to the surface after proper washing by the residual epoxide functions), enabling a single operation for the enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. The immobilized PcPAL was applied in the ammonia elimination of racemic phenylalanine, 4-chlorophenylalanine, and 4-bromophenylalanine to produce the corresponding d-phenylalanines, in addition to the formation of (E)-cinnamates, as well as in ammonia addition reactions to (E)-cinnamates, yielding the corresponding enantiopure l-phenylalanines.

Biological properties of macroporous cryostructurate based on extracellular matrix components

Objective: to study the biological properties of macroporous cryostructurate from multicomponent concentrated collagen-containing solution (MCCS) as a promising matrix for the formation of cell- and tissue-engineered constructs.Materials and methods. A macroporous spongy carrier was obtained by cryostructuring of collagencontaining extract, prepared by acetic acid hydrolysis of chicken connective tissue (BIOMIR Service, Russian Federation). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (Sigma-Aldrich, USA) was used to make the cryostructurate water insoluble. The micromorphology of the sponge surface was studied using scanning electron microscopy. The cytotoxicity of the carrier was evaluated by reaction of the mouse NIH 3T3 fibroblast cell culture using automated microscope IncuCyte ZOOM (EssenBioscience, USA). Biocompatibility of the macroporous carrier was studied on cultures of human adipose tissue-derived mesenchymal stromal cells (AD-MSC), human hepatocellular carcinoma cell line HepG2 and human umbilical vein endothelial cell line EA.hy926. The metabolic activity of cells was determined using PrestoBlue™ reagents (Invitrogen™, USA). Cell population development during long-term cultivation of the cell-engineered construct (CEC) was assessed by fluorescencelifetime imaging microscopy over the entire surface of the sample using a Leica Dmi8 inverted microscope with Leica Thunder software (Leica Microsystems, Germany).Results. Optical microscopy and scanning electron microscopy (SEM) showed the presence of pores of different sizes in the resulting biopolymer material: large pores with 237 ± 32 μm diameter, medium-sized pores with 169 ± 23 μm diameter, and small-sized pores with 70 ± 20 μm diameter; large and medium-sized pores were predominant. The studied media did not exhibit cytotoxicity. Cell adhesion and proliferation on the surface of the material and their penetration into the underlying layers during long-term cultivation were observed. The highest metabolic activity of the cells was observed for human AD-MSC on day 14, which corresponds to the normal dynamics of development of a population of cells of this type. The functional activity of HepG2 cells – albumin and urea production – was shown in the liver CEC model.Conclusion. The good adhesion and active proliferation that were shown for the three cell types indicate that the resulting biopolymer carrier is biocompatible, and that the spread of the cells into the inner volume of the sponge and active population of the sponge under prolonged culturing indicates that this material can be used to create cell- and tissue-engineered constructs.

Fabrication of a Zircon Microfiltration Membrane for Culture Medium Sterilization

Multilayer ceramic membranes to be used for bacteria removal by filtration were prepared from ceramic materials. They consist of a macro-porous carrier, an intermediate layer and a thin separation layer at the top. Tubular and flat disc supports were prepared from silica sand and calcite (natural raw materials), using extrusion and uniaxial pressing methods, respectively. Making use of the slip casting technique, the silica sand intermediate layer and the zircon top-layer were deposited on the supports, in this order. The particle size and the sintering temperature for each layer were optimized to achieve a suitable pore size for the deposition of the next layer. Morphology, microstructures, pore characteristics, strength and permeability were also studied. Filtration tests were conducted to optimize the permeation performance of the membrane. Experimental results show that the total porosity and average pore size of the porous ceramic supports sintered at different temperatures within the range (1150-1300 °C), and lie in the ranges of 44-52% and 5-30 μm, respectively. For the ZrSiO4 top-layer, after firing at 1190 °C, a typical average pore size of about 0.3 μm and a thickness of about 70 μm were measured, while water permeability is estimated to a value of 440 lh-1m-2bar-1. Finally, the optimized membranes were tested in the sterilization of a culture medium. Filtration results show the efficiency of the zircon-deposited membranes for bacteria removal; indeed, the growth medium was found to be free of all microorganisms.

Cryostructuring of Polymeric Systems: 62 Preparation and Characterization of Alginate/Chondroitin Sulfate Cryostructurates Loaded with Antimicrobial Substances.

Targeted drug release is a significant research focus in the development of drug delivery systems and involves a biocompatible polymeric carrier and certain medicines. Cryostructuring is a suitable approach for the preparation of efficient macroporous carriers for such drug delivery systems. In the current study, the cryogenically structured carriers based on alginate/chondroitin sulfate mixtures were prepared and their physicochemical properties and their ability to absorb/release the bactericides were evaluated. The swelling parameters of the polysaccharide matrix, the amount of the tightly bound water in the polymer and the sulfur content were measured. In addition, FTIR and UV spectroscopy, optical and scanning microscopy, as well as a standard disk diffusion method for determining antibacterial activity were used. It was shown that alginate/chondroitin sulfate concentration and their ratios were significant factors influencing the swelling properties and the porosity of the resultant cryostructurates. It was demonstrated that the presence of chondroitin sulfate in the composition of a polymeric matrix slowed down the release of the aminoglycoside antibiotic gentamicin. In the case of the NH2-free bactericide, dioxidine, the release was almost independent of the presence of chondroitin sulfate. This trend was also registered for the antibacterial activity tests against the Escherichia coli bacteria, when examining the drug-loaded biopolymeric carriers.

Carrier type induces anammox biofilm structure and the nitrogen removal pathway: Demonstration in a full-scale partial nitritation/anammox process

In this study, two typical carrier types, microporous and macroporous carriers, were collected from a full-scale partial nitritation/anammox reactor for analysis and comparison of the biofilm structure characteristics, performance and removal nitrogen pathway. For microporous carriers, a thicker biofilm (>5 mm) was obtained with higher biomass and abundance of anammox bacteria as well as a higher nitrogen removal efficiency due to the integration of denitrifying and anammox bacteria. In addition, higher microbial community stability can be expected under varying environmental conditions. In comparison, macroporous carrier biofilm exhibited a lower thickness (0.4–2.3 mm) and lower microbial richness, with a strong network correlation among genera. Analysis showed that the mainly positive correlation between anammox bacteria and ammonium oxidizing bacteria, enhancing coupling partial nitritation and anammox. These findings help further our understanding of the mechanisms of anammox biofilm nitrogen removal and provide a baseline for optimization of the design of carrier structures.

Functionalization of Porous Cellulose with Glyoxyl Groups as a Carrier for Enzyme Immobilization and Stabilization.

The functionalization of the internal surface of macroporous carriers with glyoxyl groups has proven to highly stabilize a large variety of enzymes through multipoint covalent immobilization. In this work, we have translated the surface chemistry developed for the fabrication of glyoxyl-agarose carriers to macroporous cellulose (CEL). To that aim, CEL-based microbeads were functionalized with glyoxyl groups through a stepwise alkoxylation (or alkylation)/oxidation synthetic scheme. This functionalization sequence was analyzed by solid-state NMR, while the scanning electron miscroscopy of CEL microbeads reveals that the mild oxidation conditions negligibly affect the morphological properties of the material. Through the optimal functionalization protocol using rac-glycidol, we introduce up to 200 μmols of aldehyde groups per gram of wet CEL, a similar density to the one obtained for the benchmarked agarose-glyoxyl carrier. This novel CEL-based carrier succeeds to immobilize and stabilize industrially relevant enzymes such as d-amino acid oxidase from Trigonopsis variabilis and xylanases from Trichoderma reseei. Remarkably, the xylanases immobilized on the optimal CEL-based materials present a half-life time of 51 h at 60 °C and convert up to 90% of the xylan after four operation cycles for the synthesis of xylooligosaccharides.

PEGylation and macroporous carrier adsorption enabled long-term enzymatic transesterification

A novel enzyme catalyst with excellent stability and catalytic efficiency expected to be used in the ERD industry was prepared.

Absorbable hemostatic hydrogels comprising composites of sacrificial templates and honeycomb-like nanofibrous mats of chitosan

The development of hemostatic technologies that suit a diverse range of emergency scenarios is a critical initiative, and there is an increasing interest in the development of absorbable dressings that can be left in the injury site and degrade to reduce the duration of interventional procedures. In the current study, β-cyclodextrin polyester (CDPE) hydrogels serve as sacrificial macroporous carriers, capable of degradation under physiological conditions. The CDPE template enables the assembly of imprinted chitosan honeycomb-like monolithic mats, containing highly entangled nanofibers with diameters of 9.2 ± 3.7 nm, thereby achieving an increase in the surface area of chitosan to improve hemostatic efficiency. In vivo, chitosan-loaded cyclodextrin (CDPE-Cs) hydrogels yield significantly lower amounts of blood loss and shorter times to hemostasis compared with commercially available absorbable hemostatic dressings, and are highly biocompatible. The designed hydrogels demonstrate promising hemostatic efficiency, as a physiologically-benign approach to mitigating blood loss in tissue-injury scenarios.

KINETICS OF POLYMER CARRIER IMPREGNATION BY TRIALKYLAMINE

The immediacy of the problem under study is due to the lack of data on the kinetics of polymer carrier impregnation with extraction agents. The article is aimed at obtaining the kinetic characteristics of the impregnation of a polymeric macroporous carrier—a weakly acidic cation resin with carboxyl groups—with trialkylamine and its solutions in acetone. The integral kinetic curves of the extraction agent adsorption on the carrier were obtained. This allowed estimating the time of equilibrium by the limited solution volume method. This time was 25-30 hours, decreasing with the application of a dilution agent and with the increase in temperature. The kinetic impregnation characteristics were calculated by means of the pseudo-second-order, Elovich, and internal diffusion models. The pseudo-second-order model describes the experimental kinetic data with a high correlation degree (R2 0.995-0.997) and points out that interaction with two sorption centres possibly takes place with the adsorption of a single trialkylamine molecule. The impregnation rate constants via an extraction agent were calculated using all kinetic models. Based on the apparent activation energy magnitude (37±14 kJ/mol), it can be assumed that diffusion is the rate-limiting step of the impregnation process. This article can be useful for creating impregnates containing extraction agents of various classes.

Roles of an easily biodegradable co-substrate in enhancing tetracycline treatment in an intimately coupled photocatalytic-biological reactor

Intimately coupled photocatalysis and biodegradation (ICPB) was realized in a macroporous carrier in which a photocatalyst was present on the outer surface, while a biofilm accumulated inside the carrier. In ICPB, photocatalysis products are rapidly biodegraded by a protected biofilm, leading to mineralization of the refractory organics, such as antibiotics. However, mineralization in ICPB could be compromised if the photocatalysis products remain refractory or are inhibitory. To address this, we attempted to increase metabolic activity by providing a readily biodegradable co-substrate (acetate) that could act as a source of energy and electrons to improve biotransformation and mineralization of the refractory antibiotic tetracycline (TCH). When we added acetate during ICPB of TCH, TCH removal increased by ∼5%, mineralization increased by ∼20%, and almost all photocatalysis products disappeared. Acetate addition also led to an increase in active biomass, an increase in the biomass's respiratory activity, and evolution of the microbial community to having more members able to biodegrade photocatalysis and biotransformation intermediates. Thus, providing an easily biodegradable co-substrate was an effective means for enhancing TCH removal and mineralization with the ICPB technology.

Preparation of pumice-loaded CeO2/Bi2WO6 photocatalysts and treatment of tetracycline wastewater with a continuous flow photocatalytic reactor

Pumice-loaded CeO2/Bi2WO6 photocatalysts were prepared with a typical solvothermal method by using pumice as the macroporous carrier. The photocatalysts were characterized by X-ray diffraction, scanning electron microscopy, energy dispersive spectrometry and element mapping. The preparation conditions were optimized and the photoactivity was evaluated via photocatalytic tetracycline degradation under visible light radiation. Moreover, the photocatalysts were applied to a continuous flow reactor and the optimal conditions were studied. It was found the CeO2/Bi2WO6 catalysts were successfully loaded on the surfaces and pores of pumice. The optimal catalyst/pumice loading ratio was 1:4 mmol/g, and the average load rate of the photocatalysts was 37.24%. Under visible-light irradiation, the pumice-loaded CeO2/Bi2WO6 catalysts could well photocatalytically degrade tetracycline, with a degradation rate within 90 min up to 91.72%, and were stable photocatalytically. Experiments with the continuous flow reactor showed the optimal catalyst quantity, light source intensity, pollutant concentration and hydraulic retention time were 10 g/L, 140,000 lx, 50 mg/L and 6 h, respectively, when the tetracycline degradation rate was stably improved to 90.00%.

Evaluation of Fixed-Bed Cultures with Immobilized Lactococcus Lactis ssp. Lactis on Different Scales

Fixed-bed processes, where cells are immobilized within macroporous carriers, are a promising alternative to processes with suspended cells. A scale-up concept is presented in order to evaluate the performance as part of process design of fixed-bed processes. Therefore,Lactococcus lactiscultivation in chemostat and batch mode was compared to fixed bed cultures on three different scales, the smallest being the downscaledMultifermwith 10 mL fixed bed units, the second a 100 mL fixed-bed reactor and the third a pilot scale reactor with 1 L fixed bed volume. As expected, the volume specific lactate productivity of all cultivations was dependent on dilution rate. In suspension chemostat culture a maximum of 2.3 g·L-1·h-1was reached. Due to cell retention in the fixed-beds, productivity increased up to 8.29 g·L-1·h-1at a dilution rate of D = 1.16 h-1(corresponding to 2.4·µmax) on pilot scale. For all fixed bed cultures a common spline was obtained indicating a good scale-up performance.

Effects of macroporous hydroxyapatite carriers on the growth and function of human hepatoblasts derived from fetal hepatocytes

Improvement of three-dimensional (3D) culture conditions, including substrates for cell growth, is needed for various cell-based applications. In this study, we developed hydroxyapatite (HAp) macroporous carriers having several pore size distributions and tried to obtain the findings about the effective pore sizes for the growth and function of hepatoblasts derived from human fetal hepatocytes. Cellular CYP3A4 activity was significantly enhanced when 20% HAp macroporous carrier was used, reaching 1.49±0.28pmol/10(6)cells/min of benzyloxyresorufin-O-dealkylation activity, which is comparable to that of primary human hepatocytes from livers of adult donors. Analysis of the pore size (the radius of curvature) distribution of each HAp carrier using a 3D-electron beam surface roughness analyzer revealed two peaks of pore size distribution at 30-40μm and 70-80μm, respectively. Thirty-five percent of the pores in the 20% carrier had a size distribution within 50-80μm. Especially, pores of 70-80μm were more abundant in the 20% HAp carrier than in the 10% and 30% HAp carriers. These results suggested that a HAp carrier with the pore size distribution of 50-80μm might be effective for cell growth and function in human hepatoblasts derived from fetal hepatocytes.

Структурное состояние печени после имплантации макропористых матриц с криоконсервированными клетками фетальной печени крысам с экспериментальной печеночной недостаточностью

Структурное состояние печени после имплантации макропористых матриц с криоконсервированными клетками фетальной печени крысам с экспериментальной печеночной недостаточностью

Meso- and macroporous materials modified with amines for CO2 storage

Adsorbents of a new type for CO2 absorption on amine-modified meso- and macroporous carriers are more efficient than amine water solutions. Their adsorption capacity with respect to CO2 reaches 13–15 wt %, they are recyclable, and the rates of CO2 absorption and evolution are higher than those for the liquid systems of CO2 storage.

Perspectives of treatment of anemias with cells of fetal liver, immobilized in macroporous alginate-gelatin carriers.

Aim of the work was to study possibility of erythropoiesis stimulation by transplantation of fetal liver cells, seeded into macro­porous carriers to the rats with post-hemorrhargic anemia, induced by 70% hepatectomy. Fetal liver cells (FLC) were isolated from fetuses of rats with 15 days’ gestation and were cryopreserved. Decryopreserved FLC were seeded into macroporous spongy alginate-gelatin scaffolds, which were covered by alginate capsule and implanted into omentum of rats with modeled liver insufficiency. It was shown that fetal liver cells, immobilized in macroporous scaffolds after implantation have positive effect on red blood count and hemoglobin content, indicating that this approach is promising for the development of new methods of anemia treatment.

Al-MCM-41 inside a glass–ceramic scaffold: A meso–macroporous system for acid catalysis

Abstract Al-MCM-41 was incorporated, through a sol–gel based dipping approach, inside a glass–ceramic scaffold belonging to the SiO 2 –CaO–Na 2 O–Al 2 O 3 (SCNA) system, to obtain an acidic system with bimodal porosity in the meso- and macro-range. First, SCNA was investigated by means of wide-angle X-ray diffraction (XRD), differential thermal analysis (DTA) and hot stage microscopy (HSM) in order to select the optimal sintering condition to fabricate the macroporous foam-like carrier for Al-MCM-41. Then, Al-MCM-41 was incorporated into the SCNA scaffold; the pH value of Al-MCM-41 synthesis solution, used for the scaffold impregnation process, was shown to be a crucial parameter, on identical scaffold dipping time, to determine the distribution and the stability of the incorporated phase. The systems were characterized by means of XRD, N 2 adsorption/desorption, TGA and SEM coupled with EDS. The Al-MCM-41-containing scaffold is active in the acid-catalyzed sucrose hydrolysis, chosen as model reaction, at variance with the scaffold as such.

Developing an efficient TiO2-coated biofilm carrier for intimate coupling of photocatalysis and biodegradation

Intimate coupling of photocatalysis and biodegradation (ICPB) shows promise to remove recalcitrant organic compounds from water, because photocatalysis breaks the compounds into biodegradable intermediates that are immediately mineralized by microorganisms inside a macroporous carrier, where they are protected from UV light and free radicals. Key to successful ICPB is a carrier capable of accumulating biofilm in its interior and strongly adhering photocatalyst on its exterior. We employed a low-temperature sintering method, the O method, to adhere TiO2 to sponge-type macroporous carriers. The O method achieved a 7-fold increase of TiO2 density, compared to the previously used sol–gel method, and it conserved the macropores for biofilm accumulation. Although the O-method carrier met the basic requirements of ICPB when degrading 2,4,5 trichlorophenol, it had low photocatalytic activity for breaking down more complex aromatics, like reactive dyes. Therefore, we improved the sintering method in two steps called the D and DN methods: reducing the TiO2 concentration and then removing trimesic acid in the coating solution. The photocatalytic efficiency towards reactive black 5 increased 5 fold for the D method and 10 fold for DN method, and the DN carrier had superior TiO2 adherence during long-term operation.

Surface-layer composite sorbents for the rapid preconcentration of volatile organic substances from aqueous solutions and gas atmospheres

The regularities of the dynamic sorption of volatile organic substances from aqueous solutions and atmospheric air on surface-layer composite sorbents, in which a finely dispersed sorption-active material is retained due to adhesion on the surface of a relatively coarsely dispersed macroporous carrier, were determined. The analytical capabilities of such sorbents were studied, and they were found more effective than traditional porous bulk sorbents used for the concentration of volatile organic compounds from aqueous solutions and gas atmospheres.

Degradation of reactive dyes in a photocatalytic circulating‐bed biofilm reactor

Decolorization and mineralization of reactive dyes by intimately coupled TiO₂-photocatalysis and biodegradation (ICPB) on a novel TiO₂-coated biofilm carrier were investigated in a photocatalytic circulating-bed biofilm reactor (PCBBR). Two typical reactive dyes--Reactive Black 5 (RB5) and Reactive Yellow 86 (RY86)--showed similar first-order kinetics when being photocatalytically decolorized at low pH (~4-5) in batch experiments. Photocatalytic decolorization was inhibited at neutral pH in the presence of phosphate or carbonate buffer, presumably due to electrostatic repulsion from negatively charged surface sites on TiO₂, radical scavenging by phosphate or carbonate, or both. Therefore, continuous PCBBR experiments were carried out at a low pH (~4.5) to maintain high photocatalytic efficiency. In the PCBBR, photocatalysis alone with TiO₂-coated carriers could remove target compound RB5 and COD by 97% and 47%, respectively. Addition of biofilm inside macroporous carriers maintained a similar RB5 removal efficiency, but COD removal increased to 65%, which is evidence of ICPB despite the low pH. ICPB was further proven by finding microorganisms inside carriers at the end of the PCBBR experiments. A proposed ICPB pathway for RB5 suggests that a major intermediate, a naphthol derivative, was responsible for most of the residual COD, while most of the nitrogen in the azo-bonds (-N=N-) was oxidized to N₂.