Ca-alginate Gel Research Articles

Stabilities of Immobilizedβ-galactosidase ofAspergillussp. AF for the Optimal Production of Galactooligosaccharides from Lactose

Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storage stability of the enzyme activity were investigated and compared with those of the free enzyme. The study on the improvement of mechanical strength of the alginate beads was carried out through various methods, which demonstrates that the hardening process, where the alginate beads were treated with 0.225 M CaCl(2) solution after three batches to compensate the lost of calcium in the beads, provided a high mechanical stability for repeated use in large-scale production. The experiment results show that GOS yield increased with the increase of lactose concentration, and also increased with excessive addition of lactose (exceeding its solubility) at the beginning of the reaction. The immobilization of beta-galactosidase of Aspergillus sp. AF crude homogenate is cheap in processing cost and easy to carry out, and the immobilized enzyme possesses high performance for industrial application.

Effective degradation of tannic acid by immobilized rumen microbes of a sika deer ( Cervus nippon yesoensis) in winter

In winter seasons, wild sika deer ( Cervus nippon yesoensis) inhabiting the Shiretoko Peninsula of Hokkaido Island, Japan, mainly graze woody materials (bark and twigs, etc.) as their feed source. Most of the tree species that they feed upon contain a high level of hydrolysable tannins within the inner bark. Tannins generally lead to low protein digestion and nutrient loss to these herbivorous mammals due to tannization of proteins. In winter months, it is speculated that wild sika deer develop a mechanism to degrade the tannins which are contained in their feed sources, but rumen fluid obtained from sika deer in winter months did not exhibit any ability to degrade tannins in liquid culture medium. However, constant degradation of hydrolysable tannin was observed when Ca-alginate gel beads were used for microbial immobilization and culturing. The gel beads that had been impregnated with 0.6×10 4 fold-diluted rumen fluid of sika deer in winter and pre-incubated for 24 h under anaerobic conditions supplemented with a 1.5 g/L sugar were reacted with 5 g/L tannic acid solution. Under these conditions, the immobilized rumen bacteria grown in the macrogel beads effectively hydrolyzed tannic acid to release gallic acid monomers. Major bacterial colonies emerging in the Ca-alginate gel beads were identified as Streptococcus macedonicus and this bacterium (EC-D140) was regarded as the most likely candidate as the tannin-degrading bacterium.

Tensile properties of yeast cell-loaded Ca-alginate gel layers

The tensile properties of Ca-alginate gel strips (2.0% (w/v) alginate) containing varying amounts of yeast cells (0.1–5.0%, w/v) were investigated. The stress, strain and energy at failure of yeast-filled alginate samples showed very similar evolutions as a function of the initial cell content, i.e., they peaked at a cell content of 0.5–0.8% (w/v). By contrast, no clear effect of cell loading on Young's modulus could be highlighted at moderate yeast concentration (≤1.0%, w/v) but the elastic modulus significantly decreased at higher cell loading. These results are discussed in light of literature data on particulate–polymer composites.

Biological sulfide oxidation using autotrophicThiobacillussp.: evaluation of different immobilization methods and bioreactors

Evaluation of various immobilization methods and bioreactors for sulfide oxidation using Thiobacillus sp. was studied. Ca-alginate, K-carrageenan and agar gel matrices (entrapment) and polyurethane foam and granular activated carbon (adsorption) efficacy was tested for the sulfide oxidation and biomass leakage using immobilized Thiobacillus sp. Maximum sulfide oxidation of 96% was achieved with alginate matrix followed by K-carrageenan (88%). Different parameters viz. alginate concentration (1%, 2%, 3%, 4% and 5%), CaCl(2) concentration (1%, 2%, 3%, 4% and 5%), bead diameter (1, 2, 3, 4 and 5 mm), and curing time (1, 3, 6, 12 and 18 h) were studied for optimal immobilization conditions. Repeated batch experiments were carried out to test reusability of Ca-alginate immobilized beads for sulfide oxidation in stirred tank reactor and fluidized bed reactor (FBR) at different sulfide concentrations. The results proved to be promising for sulfide oxidation using Ca-alginate gel matrix immobilized Thiobacillus sp. for better sulfide oxidation with less biomass leakage. Biological sulfide oxidation is gaining more importance because of its simple operation. Present investigations will help in successful design and operation of pilot and industrial level FBR for sulfide oxidation.

Biodegradation of coumaphos, chlorferon, and diethylthiophosphate using bacteria immobilized in Ca-alginate gel beads

Calcium-alginate immobilized cell systems were developed for the detoxification and biodegradation of coumaphos, an organophosphate insecticide, and its hydrolysis products, chlorferon and diethlythiophosphate (DETP). Optimum bead loadings for bioreactor operation were found to be 200 g-beads/L for chlorferon degradation and 300 g-beads/L for DETP degradation. Using waste cattle dip (UCD) solution as substrate, the degradation rate for an immobilized consortium of chlorferon-degrading bacteria was five times greater than that for freely suspended cells, and hydrolysis of coumaphos by immobilized OPH + Escherichia coli was 2.5 times greater. The enhanced degradation of immobilized cells was due primarily to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of the cells caused by Ca-alginate immobilization may have contributed to increased reaction rates. Degradation rates for repeated operations increased for successive batches indicating that cells became better adapted to the reaction conditions over time.

Impact of gel layer formation on colloid retention in membrane filtration processes

Colloidal particles in the feed streams of membrane filtration processes control membrane fouling rate in many instances. In this study, the non-gelling colloidal Na-alginate and the gelling colloidal Ca-alginate are employed to investigate the significance of gel layer formation in membrane filtration processes in terms of contribution to membrane fouling and supplementary impurity removal. The results show that contribution of colloidal particles to membrane fouling depends on the gelling propensity of the colloids and the operational mode (constant pressure or constant flux) implemented. A small dose of either Na-alginate or Ca-alginate was found to greatly increase membrane fouling rate during constant pressure filtration. Both the resistance to removal by application of shear and the lower susceptibility of the concentration polarization layer to shear resulted in more severe fouling during constant flux filtration in the presence of Ca-alginate assemblages than in the presence of Na-alginate. Apparent channel sizes of the Ca-alginate gel layer were calculated from the material properties of the fouling layer. Incomplete catalase retention highlighted the likely heterogeneity in size of liquid transport pathways. Adsorption also contributed to the trapping of colloidal particles according to the retention behaviour of BSA by the Ca-alginate gel layer. Gel layer formation propensity should be seriously considered for the operation of membrane filtration processes. Two simple methods based on (i) a permeability recovery experiment and (ii) comparison of dead-end filtration behaviour with and without shear application are proposed for evaluation of the gelling propensity of colloidal dispersions.

Mechanical Properties of C-5 Epimerized Alginates

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

Biosorption of copper (II) onto immobilized cells of Pycnoporus sanguineus from aqueous solution: Equilibrium and kinetic studies

The ability of white-rot fungus, Pycnoporus sanguineus to adsorb copper (II) ions from aqueous solution is investigated in a batch system. The live fungus cells were immobilized into Ca-alginate gel to study the influence of pH, initial metal ions concentration, biomass loading and temperature on the biosorption capacity. The optimum uptake of Cu (II) ions was observed at pH 5 with a value of 2.76 mg/g. Biosorption equilibrium data were best described by Langmuir isotherm model followed by Redlich–Peterson and Freundlich models, respectively. The biosorption kinetics followed the pseudo-second order and intraparticle diffusion equations. The thermodynamic parameters enthalpy change (10.16 kJ/mol) and entropy change (33.78 J/mol K) were determined from the biosorption equilibrium data. The FTIR analysis showed that OH, NH, C H, C O, COOH and C N groups were involved in the biosorption of Cu (II) ions onto immobilized cells of P. sanguineus. The immobilized cells of P. sanguineus were capable of removing Cu (II) ions from aqueous solution.

Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca‐Alginate gel beads

Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca-alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 x 10(-11) m(2)/s and 4.28 x 10(-11) m(2)/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium-alginate gel beads with radii of 1.65-1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca-alginate gel beads were reaction-limited.

Preparation of Calcium Alginate Microgel Beads in an Electrodispersion Reactor Using an Internal Source of Calcium Carbonate Nanoparticles

An electrodispersion reactor has been used to prepare calcium alginate (Ca-alginate) microgel beads in this study. In the electrodispersion reactor, pulsed electric fields are utilized to atomize aqueous mixtures of sodium alginate and CaCO3 nanoparticles (dispersed phase) from a nozzle into an immiscible, insulating second liquid (continuous phase) containing a soluble organic acid. This technique combines the features of the electrohydrodynamic force driven emulsion processes and externally triggered gelations in microreactors (the droplets) ultimately to yield soft gel beads. The average particle size of the Ca-alginate gels generated by this method changed from 412 +/- 90 to 10 +/- 3 microm as the applied peak voltage was increased. A diagram depicting structural information for the Ca-alginate was constructed as a function of the concentrations of sodium alginate and CaCO3 nanoparticles. From this diagram, a critical concentration of sodium alginate required for sol-gel transformation was observed. The characteristic highly porous structure of Ca-alginate particles made by this technique appears suitable for microencapsulation applications. Finally, time scale analysis was performed for the electrodispersion processes that include reactions in the microreactor droplets to provide guidelines for the future employment of this technique. This electrodispersion reactor can be used potentially in the formation of many reaction-based microencapsulation systems.

Influence of Oligoguluronates on Alginate Gelation, Kinetics, and Polymer Organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.

Palatinose production by free and Ca-alginate gel immobilized cells of Erwinia sp.

Palatinose is a non-cariogenic disaccharide obtained from the enzymatic conversion of sucrose, used in food industries as a sugar substitute. Free and Ca-alginate immobilized cells of Erwinia sp. D12 were used to produce palatinose from sucrose. Palatinose production was studied in a repeated-batch process using different immobilized biocatalysts: whole cells, disrupted cells and glucosyltransferase. Successive batches were treated with the immobilized biocatalyst, but a decrease in palatinose production was observed. A continuous process using a packed-bed reactor was investigated, and found to produce 55–66% of palatinose during 17 days using immobilized cells treated with glutaraldehyde and a substrate flow speed of 0.56 ml min −1. However, immobilized cells in a packed-bed reactor failed to maintain the palatinose production for a prolonged period. The free cells showed a high conversion rate using batch fermentation, obtaining a palatinose yield of 77%. The cells remained viable for 16 cycles with high palatinose yields (65–77%). Free Erwinia sp. D12 cells supported high production levels in repeated-batch operations, and the results showed the potential for repeated reuse.

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V (max)) and Michaelis-Menten constant (K (m)) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca-alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 degrees C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.

Biosynthesis of γ-aminobutyric acid (GABA) using immobilized whole cells of Lactobacillus brevis

On an industrial scale, the production of γ-aminobutyric acid (GABA) from the cheaper sodium L-glutamate (L-MSG) is a valuable process. By entrapping Lactobacillus brevis cells with higher glutamate decarboxylase (GAD) activity into Ca-alginate gel beads, the biotransformation conditions of L-MSG to GABA were optimized with the immobilized cells. The cells obtained from a 60-h culture broth showed the highest biotransformation efficiency from L-MSG to GABA. The optimal cell density in gel beads, reaction pH and temperature were 11.2 g dry cell weight (DCW) l−1, 4.4 and 40°C respectively. The thermal stability of immobilized cells was significantly higher than free cells. Under the optimized reaction conditions, the yield of GABA reached above 90% during the initial five batches and the yield still remained 56% in the tenth batch. Continuous production of GABA was realized with a higher yield by incorporating cell re-cultivation using the packed bed reactor.

Intact and permeabilized cells of the yeast Hansenula polymorpha as bioselective elements for amperometric assay of formaldehyde

Intact and permeabilized yeast cells were tested as the biorecognition elements for amperometric assay of formaldehyde (FA). For this aim, the mutant C-105 ( gcr1 catX) of the methylotrophic yeast Hansenula polymorpha with a high activity of AOX was chosen. Different approaches were used for monitoring FA-dependent cell response including analysis of their oxygen consumption rate by the use of a Clark electrode, as well as assay of oxidation of redox mediator at a screen-printed platinum electrode covered by cells entrapped in Ca-alginate gel. It was shown that oxygen consumption rate of permeabilized cells reached its saturation at 4 mM of FA (23 °C). The detection limit was found to be 0.27 mM. In the presence of redox mediator 2,6-dichlorophenolindophenol (DCIP), the screen-printed platinum band electrode covered by permeabilized cells did not show any current output to FA. In contrast, well-pronounced amperometric response to FA was observed in the case of intact yeast cells in the presence of DCIP. It was shown that current output reached its maximum at 7 mM concentration of FA. The detection limit was found to be 0.74 mM. Obviously, it is necessary to perform a directed genetic engineering of the yeast cells to improve their bioanalytical characteristics in the corresponding biosensors.

Co2+, Cu2+, and Zn2+ Accumulation by Cyanobacterium Spirulina platensis

The Spirulina platensis biomass was characterized for its metal accumulation as a function of pH, external metal concentration, equilibrium isotherms, kinetics, effect of co-ions under free (living cells, lyophilized, and oven-dried) and immobilized (Ca-alginate and polyacrylamide gel) conditions. The maximum metal biosorption by S. platensis biomass was observed at pH 6.0 with free and immobilized biomass. The studies on equilibrium isotherm experiments showed highest maximum metal loading by living cells (181.0 +/- 13.1 mg Co(2+)/g, 272.1 +/- 29.4 mg Cu(2+)/g and 250.3 +/- 26.4 mg Zn(2+)/g) followed by lyophilized (79.7 +/- 9.6 mg Co(2+)/g, 250.0 +/- 22.4 mg Cu(2+)/g and 111.2 +/- 9.8 mg Zn(2+)/g) and oven-dried (25.9 +/- 1.9 mg Co(2+)/g, 160.0 +/- 14.2 mg Cu(2+)/g and 35.1 +/- 2.7 mg Zn(2+)/g) biomass of S. platensis on a dry weight basis. The polyacrylamide gel (PAG) immobilization of lyophilized biomass found to be superior over Ca-alginate (Ca-Alg) and did not interfere with the S. platensis biomass biosorption capacity, yielding 25% of metal loading after PAG entrapment. The time-dependent metal biosorption in both the free and immobilized form revealed existence of two phases involving an initial rapid phase (which lasted for 1-2 min) contributing 63-77% of total biosorption, followed by a slower phase that continued for 2 h. The metal elution studies conducted using various reagents showed more than 90% elution with mineral acids, calcium salts, and Na(2)EDTA with free (lyophilized or oven-dried) as well as immobilized biomass. The experiments conducted to examine the suitability of PAG-immobilized S. platensis biomass over multiple cycles of Co(2+), Cu(2+), and Zn(2+) sorption and elution showed that the same PAG cubes can be reused for at least seven cycles with high efficiency.

Gas-Phase Silicon Alkoxide Reactivity vs. Na-Alginate Droplets for Conjugation of Alginate and Sol-Gel Technologies

Solutions with Na-alginate concentrations ranging from 0.5 to 2.5% w/v are processed to prepare Ca-alginate beads using a nozzle ejector under constant He-flow. Beads were spherical in shape and their size distributions were determined; in all samples the average diameter fell in the 120–140 μm interval. Volumetric yields were found to be linearly dependent of the original Na-alginate load whereas the bead diameters were almost constant, according to a constant hindrance of Ca-alginate macromolecular units in the final Ca-alginate gel. The rheology of Na-alginate solutions was studied, with determination of intrinsic viscosity; experimental evidence of microsphere formation, even at the lowest Na-alginate concentrations, indicated that ejection processing changes the rheological parameters controlling bead formation in ordinary dropping processing.

Kinetic Analysis of Beer Primary Fermentation Using Yeast Cells Immobilized by Ceramic Support Adsorption and Alginate Gel Entrapment

Yeast cells were immobilized by absorption onto porous ceramic support and evaluated for continuous beer primary fermentation using a bioreactor in comparison to yeast cells immobilized by entrapment in calcium alginate gel. The effects of temperature and flow rate as a function of reaction/fermentation time on fermentation rate were investigated. The fermentation reaction (in terms of loss of total soluble solids in the beer wort as a function of time) was first-order with half-lifes in the range of approximately 9-11 hours at approximately 10-12 degrees C at beer wort linear flow rates of approximately 0.8-1.6 cm/minute for ceramic support, compared with approximately 16 hours for Ca-alginate gel, the former support matrix being more efficient and demonstrating greater potential for future commercial application.

Similarities and differences between alginic acid gels and ionically crosslinked alginate gels

A Na-alginate solution can, under given conditions, undergo a sol–gel transition at pH below pKa for the uronides. These gels are presumably stabilised by intermolecular hydrogen bonds. This paper summarises the effect of alginate variables such as chemical composition and sequence and molecular weight on mechanical properties and network fine structure. Alginic acid gels resemble ionically crosslinked alginate gels in the sense that guluronic acid blocks are the building elements of most importance with respect to gel formation. The main difference is that mannuronic acid blocks, although with less efficiency compared polyguluronate, also support alginic acid gel formation. With the exception strictly alternating alginates made by treating mannuronan with the AlgE4 epimerase, the so-called alternating (random) sequence of the alginate molecule seems to destabilise alginic acid gel formation due to its lack of repeating structure. Small Angle X-ray Scattering (SAXS) reveal a scattering intensity for alginic acid gels being typically and order of magnitude higher than Ca-alginate gels, suggesting junction zones composed of random aggregates with a high degree of multiplicity. A molecular understanding of alginic acid gels is of importance in the general understanding of polyuronides at low pH as well as for the utilisation of alginates in drug delivery systems.

Optimization of lipase entrapment in Ca-alginate gel beads

Lipase from Candida rugosa was entrapped by drop-wise addition of an aqueous mixture of sodium alginate and the biocatalyst to a hardening solution of a Ca 2+ salt. Effects of immobilization conditions such as alginate concentration, CaCl 2 concentration, ratio by weight of enzyme to alginate (E/A) and bead size on loading efficiency (percent of total enzyme entrapped) and immobilization yield (specific activity ratio of entrapped lipase to free lipase) were investigated. An increase in alginate concentration raised loading efficiency, but decreased immobilization yield. CaCl 2 concentration was expected to have a similar effect on loading efficiency and immobilization yield, but the effect was small in the tested range of 50–300 mM. With increasing bead size, immobilization yield decreased due to mass transfer resistance, but loading efficiency was unchanged. In order to prevent enzyme from leaking out of the gel beads, beads were coated with chitosan and silicate. Compared to non-coated beads, coated alginate beads showed the higher re-usability, illustrating the effectiveness of the coating method. When coated with silicate, lipase-entrapped beads retained the highest catalytic activity.