Chromate reduction by Microbacterium liquefaciens immobilised in polyvinyl alcohol

A polyvinyl alcohol-based immobilisation technique has been utilised for entrapping the newly-isolated chromate-reducing bacterium, Microbacterium liquefaciens MP30. Three immobilisation methods were evaluated: PVA-nitrate, PVA-borate and PVA-alginate. Chromate reduction was studied in batch and continuous-flow bioreactors, where the beads maintained integrity during continuous operation. PVA-borate and PVA-alginate cell beads showed a higher rate and extent of chromate reduction than PVA-nitrate cell beads in batch experiments. With the former 100 M Cr(VI) was removed within 4 days, while only 40 M Cr(VI) was removed using the latter, and with no increase in Cr(VI) removal subsequently. Cell activity was maintained during immobilisation but the rate of Cr(VI) removal by immobilised cells was only half that of an equivalent mass of free cells. Using PVA-alginate cell beads in a continuous-flow system, chromate removal was maintained at 90–95% from a 50 M solution over 20 days without signs of bead breakdown.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Enhanced biodegradation of phenol by magnetically immobilized <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis>

Aromatic compounds are abundant in aqueous environments due to natural resources or different manufacturer’s wastewaters. In this study, phenol degradation by the yeast, Trichosporon cutaneum ADH8 was compared in three forms namely: free cells, nonmagnetic immobilized cells (non-MICs), and magnetically immobilized cells (MICs). In addition, three different common immobilization supports (alginate, agar, and polyurethane foams) were used for cell stabilization in both non-MICs and MICs and the efficiency of phenol degradation using free yeast cells, non-MICs, and MICs for ten consecutive cycles were studied. In this study, MICs on alginate beads by 12 g/l Fe₂O₃ magnetic nanoparticles had the best efficiency in phenol degradation (82.49%) and this amount in the seventh cycle of degradation increased to 95.65% which was the highest degradation level. Then, the effect of magnetic and nonmagnetic immobilization on increasing the stability of the cells to alkaline, acidic, and saline conditions was investigated. Based on the results, MICs and non-MICs retained their capability of phenol degradation in high salinity (15 g/l) and acidity (pH 5) conditions which indicating the high stability of immobilized cells to those conditions. These results support the effectiveness of magnetic immobilized biocatalysts and propose a promising method for improving the performance of biocatalysts and its reuse ability in the degradation of phenol and other toxic compounds. Moreover, increasing the resistance of biocatalysts to extreme conditions significantly reduces costs of the bioremediation process.     

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC₅₀ (10.04 ± 0.75 $\text{μg}$/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 $\text{μg}$/ml). The EC₅₀value reached 12.6 ± 1.51 $\text{μg}$/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.     

Kinetics of invertase immobilised on poly(phe-lys) coated polystyrene beads

Summary Surface of polystyrene beads was modified by poly(phe-lys) for invertase immobilisation. The optimum immobilisation conditions of invertase were; 0.01% (w/v) poly(phe-lys), 2% (v/v) glutaraldehyde at 25 °C and pH 4.5. The kinetics of sucrose hydrolysis by free and immobilised invertase in a batch reactor at pH 4.5 and 55 °C gave K_m and V_max values for sucrose with free and immobilised invertase of 81, 114 mM and 10.1, 9.2 mol glucose/min.mg enzyme, respectively. The deactivation rate constants of free and immobilised invertase were 0.0347 and 0.0098 min^–1, respectively.     

Immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase by a combination of heat treatment in the presence of minerals and entrapment in calcium alginate gels

A method of immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase was devised, based on techniques of heat fixation in the presence of minerals and, entrapment in calcium alginate gels. The optimum activity of the enzyme was obtained when the cells were heat-fixed at 60°C for 10 min in the presence of 50 mmol/L MgSO₄·7H₂O and 5 mmol/L CoCl₂·6H₂O and then cast into calcium alginate beads using 2% sodium alginate.     

Biosorption of Cr (VI) with Trichoderma viride immobilized fungal biomass and cell free Ca-alginate beads

Ability of Cr (VI) biosorption with immobilized Trichoderma viride biomass and cell free Ca-alginate beads was studied in the present study. Biosorption efficiency in the powdered fungal biomass entrapped in polymeric matric of calcium alginate compared with cell free calcium alginate beads. Effect of pH, initial metal ion concentration, time and biomass dose on the Cr (VI) removal by immobilized and cell free Ca-alginate beads were also determined. Biosorption of Cr (VI) was pH dependent and the maximum adsorption was observed at pH 2.0. The adsorption equilibrium was reached in 90 min. The maximum adsorption capacity of 16.075 mgg(-1) was observed at dose 0.2 mg in 100 ml of Cr (VI) solution. The high value of kinetics rate constant Kad (3.73 x 10(-2)) with immobilized fungal biomass and (3.75 x 10(-2)) with cell free Ca- alginate beads showed that the sorption of Cr (VI) ions on immobilized biomass and cell free Ca-alginate beads followed pseudo first order kinetics. The experimental results were fitted satisfactory to the Langmuir and Freundlich isotherm models. The hydroxyl (-OH) and amino (-NH) functional groups were responsible in biosorption of Cr (VI) with fungal biomass spp. Trichoderma viride analysed using Fourier Transform Infrared (FTIR) Spectrometer.     

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K _m and V _max of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min^?1 mg^?1, respectively; whereas, the K _m and V _max of free enzyme were 18.26 mM and 434.78 μmol min^?1 mg^?1, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.     

Encapsulation of astaxanthin-rich Xanthophyllomyces dendrorhous for antioxidant delivery

Calcium alginate gel (CAG) beads were used to entrap the antioxidant astaxanthin-rich Xanthophyllomyces dendrorhous (ASX) by ionic gelation. ASX-CAG bead entrapment efficiency and release behavior, as influenced by alginate and CaCl₂ concentration and hardening time, were investigated. The optimized bead preparation conditions that gave rise to an efficient ASX release pattern were 1.5% alginate, 50 mM CaCl₂, and a 5 min hardening time. The antioxidant activity of non-encapsulated ASX was maintained for 4 days and then sharply decreased, whereas encapsulated ASX was maintained for 6 days. These results revealed that physical entrapment of ASX within CAG beads could be an effective technique for protecting the antioxidant activity of ASX from lipid peroxidation.     

Carbon nanotubes promote Cr(VI) reduction by alginate-immobilized Shewanella oneidensis MR-1

Bioreduction of hexavalent chromium (Cr(VI)) into trivalent one (Cr(III)) based on microbial immobilization techniques has been recognized as a promising way to remove Cr contaminants from wastewater. However, such a bioreduction process is inefficient due to limited electron transfer through the immobilization matrix. In this study, a modified immobilization process was proposed by impregnating carbon nanotubes (CNTs) into Ca-alginate beads, which were then used to immobilize Shewanella oneidensis MR-1 for enhanced Cr(VI) reduction. Compared with the free cells and the beads without CNTs, the AL/CNT/cell beads showed up to 4 times higher reduction rates, mainly attributed to an enhanced electron transfer by the CNTs. In addition, the dose of CNTs greatly improved the stability of beads, suggesting a high feasibility of the AL/CNT/cell beads for repeated use. The optimized CNT concentration, temperature and pH for Cr(VI) reduction by the AL/CNT/cell beads were 0.5%, 30 °C and 6.0–7.0, respectively.     

Removal of toxic chromate using free and immobilized Cr(VI)-reducing bacterial cells of Intrasporangium sp. Q5-1

Chromate-reducing microorganisms with the ability of reducing toxic chromate [Cr(VI)] into insoluble trivalent chromium [Cr(III)] are very useful in treatment of Cr(VI)-contaminated water. In this study, a novel chromate-reducing bacterium was isolated from Mn/Cr-contaminated soil. Based on morphological, physiological/biochemical characteristics and 16S rRNA gene sequence analyses, this strain was identified as Intrasporangium sp. strain Q5-1. This bacterium has high Cr(VI) resistance with a MIC of 17 mmol l⁻¹ and is able to reduce Cr(VI) aerobically. The best condition of Cr(VI) reduction for Q5-1 is pH 8.0 at 37°C. Strain Q5-1 is also able to reduce Cr(VI) in resting (non-growth) conditions using a variety of carbon sources as well as in the absence of a carbon source. Acetate (1 mmol l⁻¹) is the most efficient carbon source for stimulating Cr(VI) reduction. In order to apply strain Q5-1 to remove Cr(VI) from wastewater, the bacterial cells were immobilized with different matrices. Q5-1 cells embedded with compounding beads containing 4% PVA, 3% sodium alginate, 1.5% active carbon and 3% diatomite showed a similar Cr(VI) reduction rates to that of free cells. In addition, the immobilized Q5-1 cells have the advantages over free cells in being more stable, easier to re-use and minimal clogging in continuous systems. This study provides potential applications of a novel immobilized chromate-reducing bacterium for Cr(VI) bioremediation.     

Dried alginate-entrapped enzymes (DALGEEs) and their application to the production of fructooligosaccharides

A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (a_w) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low a_w media.     

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca²⁺, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.     

Comparative Study of Cr(VI) Removal by Exiguobacterium sp. in Free and Immobilized Forms

Cr(VI) is a toxic environmental pollutant. To determine the potential role of microbes towards chromate bioremediation, two bacterial strains, E1 and E4, that could tolerate Cr(VI) at levels up to 2250 μg ml^?1 were isolated from the soil of a tannery. They were identified as Exiguobacterium sp. To estimate the removal of Cr(VI) using immobilized bacterial cells, 2% sodium alginate and 2.5% agar were used as immobilizing matrices. In the case of sodium alginate, 89% and 93% of Cr(VI) removal by E1 and E4, respectively, were observed. When agar beads were used as an immobilizing matrix, removal was recorded as 39% and 48% for E1 and E4, respectively. Removal of Cr(VI) was also estimated in sterile and nonsterile tannery effluent. More Cr(VI) removal was noted in the nonsterile effluent than in the sterile effluent. The maximum uptake of Cr(VI) of bound cells of E1 and E4 was found to be 17.54 and 20.04 μg ml^?1, respectively. Fourier transform infrared (FTIR) spectra of cells of E4 with Cr(VI), without Cr(VI), and immobilized cells depicted several absorption peaks, mainly for P?OH group, C?H bending, C?O bond, and amide II groups, reflecting the complex nature of the bacterial cells and the contribution of these functional groups to the Cr(VI) binding process.     

Investigation of alginate gel inhomogeneity in simulated gastro-intestinal conditions using magnetic resonance imaging and transmission electron microscopy

The inhomogeneity of alginate gel beads prepared by an external diffusion method has been characterised using spatially resolved nuclear magnetic resonance or “magnetic resonance imaging” (MRI) and transmission electron microscopy (TEM). The beads exhibited various degrees of inhomogeneity although reducing the length of exposure to the gelling bath and the presence of non-gelling ions decreased gel inhomogeneity. In order to gain information regarding the gastro-intestinal functionality of these beads for in vivo applications, they were exposed to simulated gastro-intestinal conditions. The increased polymer concentration at the edge of the beads was shown to persist throughout our gastro-intestinal model despite the centre of the bead becoming progressively more porous in nature. The porosity of the alginate gels has been quantified by image analysis of transmission electron micrographs and shown to depend on both location within the bead and gastro-intestinal conditions. We suggest that such changes in porosity of these alginate beads during simulated gastro-intestinal conditions may make these an attractive option for controlled delivery applications in vivo.     

Chromium (VI) uptake by grape stalks wastes encapsulated in calcium alginate beads: equilibrium and kinetics studies

Abstract

The removal of hexavalent chromium from aqueous solution using grape stalks wastes encapsulated in calcium alginate (GS–CA) beads was investigated. Cr(VI) sorption kinetics were evaluated as a function of chromium initial concentration and grape stalks (GS) content in the calcium alginate (CA) beads. The process follows pseudo second-order kinetics. Transport properties of hexavalent chromium on GS–CA beads was characterised by calculating chromium diffusion coefficient using the Linear Absorption Model (LAM). Langmuir isotherms, at pH 3.0 were used to describe sorption equilibrium data as a function of GS percentage in the CAbeads. Maximum uptake obtained was 86.42 mmol of Cr(VI) per L of wet sorbent volume. Results indicated that both kinetic and equilibrium models describe adequately the adsorption process.     

Cr(VI) reduction by Pseudomonas aeruginosa immobilized in a polyvinyl alcohol/sodium alginate matrix containing multi-walled carbon nanotubes

Pseudomonas aeruginosa (P. aeruginosa) was immobilized with polyvinyl alcohol (PVA), sodium alginate and multiwalled carbon nanotubes (MCNTs). After immobilization, the beads were subjected to freeze-thawing to enhance mechanical strength. When exposed to 80 mg/L Cr(VI), the immobilized bacteria were able to reduce 50% of them in 84 h, however the free cells were deactivated at this concentration. The beads were used to reduce 50 mg/L Cr(VI) for nine times, with the reduction efficiency above 90% in the first five times and 65% in the end.     

Papain entrapment in alginate beads for stability improvement and site-specific delivery: Physicochemical characterization and factorial optimization using neural network modeling

This work examines the influence of various process parameters (like sodium alginate concentration, calcium chloride concentration, and hardening time) on papain entrapped in ionotropically cross-linked alginate beads for stability improvement and site-specific delivery to the small intestine using neural network modeling. A 3³ full-factorial design and feed-forward neural network with multilayer perceptron was used to investigate the effect of process variables on percentage of entrapment, time required for 50% and 90% of the enzyme release, particle size, and angle of repose. Topographical characterization was conducted by scanning electron microscopy, and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Times required for 50% (T₅₀) and 90% (T₉₀) of enzyme release were increased in all 3 of the process variables. Percentage entrapment and particle size were found to be directly proportional to sodium alginate concentration and inversely proportional to calcium chloride concentration and hardening time, whereas angle of repose and degree of cross-linking showed exactly opposite proportionality. Beads with >90% entrapment and T₅₀ of <10 minutes could be obtained at the low levels of all 3 of the process variables. The inability of beads to dissolve in acidic environment, with complete dissolution in buffer of pH≥6.8, showed the suitability of beads to release papain into the small intestine. The shelf-life of the capsules prepared using the papain-loaded alginate beads was found to be 3.60 years compared with 1.01 years of the marketed formulation. It can be inferred from the above results that the proposed methodology can be used to prepare papain-loaded alginate beads for stability improvement and site-specific delivery. Published: September 30, 2005     

Immobilisation of Solanum chrysotrichum plant cells withinCa-alginate gel beads to produce an antimycotic spirostanol saponin

Plant cell cultures of the Mexican species Solanum chrysotrichum can be established  from friable calli. These cells produce an antifungal spirostanol saponin designated SC1. Plant cell immobilisation within Ca-alginate gel beads can lead to an enhancement in secondary metabolite production. Different matrices, obtained by varying calcium (0.1 to 0.8 mol·L^–1) and alginate (1 to 1.5 % w/v) concentrations were used to study the influence of immobilisation parameters on the behaviour of S. chrysotrichum cells. Throughout the growth cycle and in the various immobilisation conditions, fresh and dry weight, SC1 yield and viability were measured. Important metabolic, morphological and physiological modulation seemed to be generated by the gel bead networks and by the cell-cell interactions inherent to the microenvironmental constraints. An accumulation of starch grains was observed through amyloplasts. Immobilised S. chrysotrichum cells accumulated 5 mg·g^–1 dry weight of SC1. Although this was four times less than the free cells, the desired metabolite was excreted into the surrounding medium (around 40 % of the total production).     

Immobilisation of Pseudomonas gessardii acidic lipase derived from beef tallow onto mesoporous activated carbon and its application on hydrolysis of olive oil

The study was carried out to immobilise the acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon (MAC₄₀₀) for the application of hydrolysis of olive oil. MAC₄₀₀ was prepared from rice husk by the two stages process. P. gessardii was isolated from the beef tallow acclimatised soil. The acidic lipase (ALIP) was produced from a slaughterhouse waste, namely beef tallow as a substrate and immobilised onto MAC₄₀₀. The maximum immobilisation capacity of the MAC₄₀₀ was 3570 U/g at optimum immobilisation conditions; time (180 min), pH (5.0) and temperature (30 °C). The immobilised lipase had better thermal stability and reusability than the free lipase. The immobilisation of ALIP onto MAC₄₀₀ (MAC₄₀₀–ALIP) followed pseudo-first-order rate kinetics with rate constant 0.012/min. The Michaelis–Menten constant of MAC₄₀₀–ALIP was lower than that of the ALIP, which confirmed the higher affinity between enzyme and substrate. The immobilization of acidic lipase onto MAC₄₀₀ was confirmed by Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) pattern analysis. Reusability study of MAC₄₀₀–ALIP on olive oil hydrolysis showed 82% of hydrolysis up to 13 runs and 50% of hydrolysis up to 35 cycles of reuse. This work concludes that the acidic lipase immobilised mesoporous activated carbon matrix can be considered as a potential biocatalyst for the hydrolysis of olive oil. Thus, the enzyme immobilised matrix has potential industrial applications.”