Ca-alginate Beads Research Articles

Numerical modelling of insulin and amyloglucosidase release from swelling Ca–alginate beads

The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca–alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 ( r m/ r m) larger AMG protein, where r m is the Stokes–Einstein effective molecular radius. Because the alginate bead size was not constant during the release experiments, it was hypothesised that in addition to the diffusional mass transfer, a non-negligible convective flow of liquid in or out of the beads was present due to swelling or shrinkage, respectively. Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting convective flow in the numerical model, release could be described more accurately. The numerical model was able to describe the release characteristics from both shrinking, swelling, and non-swelling alginate beads. To evaluate the effect of bead swelling on the protein release rate, a swelling modulus and a release index was defined, describing the different effects on release of smaller and larger proteins.

Hydrodynamic considerations on optimal design of a three‐phase airlift bioreactor with high solids loading

AbstractThe hydrodynamic study of a three‐phase airlift (TPAL) bioreactor with an enlarged gas–liquid dual separator was carried out. Different lengths and diameters of the draft tube were tested to show how the design of the separator zone affects the hydrodynamic performance of the TPAL reactor. Ca‐alginate beads with entrapped yeast biomass at different loadings (0, 7, 14 and 21% v/v) were used in order to mimic the solid phase of conventional high cell density systems, such as those with cells immobilized on carriers or flocculating cells. Important information on multiphase flow and distribution of gas and solid phases in the internal‐loop airlift reactor (ALR) with high solids loading was obtained, which can be used for suggesting optimal hydrodynamic conditions in a TPAL bioreactor with high solids loading. It is finally suggested that the ALR with a dual separator and a downcomer to riser cross‐sectional area ratio (AD/AR) ranging from 1.2 to 2.0 can be successfully applied to batch/continuous high cell density systems, where the uniform distribution of solid phase, its efficient separation of particles from the liquid phase, and an improved residence time of air bubbles inside the reactor are desirable. Copyright © 2003 Society of Chemical Industry

Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates

Cells of Candida krusei capable of producing phytase were immobilized in Ca-alginate gel beads and used for the preparation of myo-inositol phosphates. The immobilization yield was increased about 5-fold after the beads were treated for 96 h at pH 4.0, 4 degrees C. The increased yield was retained, even after 1 month, when the cells were kept at this temperature and pH. No shift in the pH optima of phytase of the immobilized cells was observed, compared with that of free cells. However, the optimum temperature for the enzyme of the immobilized cells was 55 degrees C, which was 15 degrees C higher than that of free cells. The degradation characteristics of the phytate in immobilized cells packed in a glass column (i.d. 1.2 cm, length 20 cm) were investigated. The variation in the composition of the products results from a change in the flow rate of phytate solution (5 mM). At a flow rate of 1.30 ml/min, a mixture of myo-inositol-2-monophosphate, myo-inositol-1,2,5-triphosphate and myo-inositol-1,2,5,6-tetrakisphosphate was produced, in which the latter two were physiologically active. Also, it was found by NMR analysis that the enzyme of this strain produced only one isomer of each of the inositol phosphates, with the exception of myo-inositol pentakisphosphate. Therefore, the pure isomers were easily isolated using ion-exchange chromatography.

Comparative biosorption of mercuric ions from aquatic systems by immobilized live and heat-inactivated Trametes versicolor and Pleurotus sajur-caju

Trametes versicolor and Pleurotus sajur-caju mycelia immobilized in Ca-alginate beads were used for the removal of mercuric ions from aqueous solutions. The sorption of Hg(II) ions by alginate beads and both immobilized live and heat-killed fungal mycelia of T. versicolor and P. sajur-caju was studied in the concentration range of 0.150–3.00 mmol dm −3. The biosorption of Hg(II) increased as the initial concentration of Hg(II) ions increased in the medium. Maximum biosorption capacities for plain alginate beads were 0.144 ± 0.005 mmol Hg(II)/g; for immobilized live and heat-killed fungal mycelia of T. versicolor were 0.171 ± 0.007 mmol Hg(II)/g and 0.383 ± 0.012 mmol Hg(II)/g respectively; whereas for live and heat-killed P. sajur-caju, the values were 0.450 ± 0.014 mmol Hg(II)/g and 0.660 ± 0.019 mmol Hg(II)/g respectively. Biosorption equilibrium was established in about 1 h and the equilibrium adsorption was well described by Langmuir and Freundlich adsorption isotherms. Between 15 and 45 °C the biosorption capacity was not affected and maximum adsorption was observed between pH 4.0 and 6.0. The alginate-fungus beads could be regenerated using 10 mmol dm −3 HCl solution, with up to 97% recovery. The biosorbents were reused in five biosorption–desorption cycles without a significant loss in biosorption capacity. Heat-killed T. versicolor and P. sajur-caju removed 73% and 81% of the Hg(II) ions, respectively, from synthetic wastewater samples.

English

  The conditions for immobilizing the new alkaline protease-producing bacteria strainTeredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline protease improved significantly with increasing alginate concentration and reached a maximum enzyme yield of 8000 U/ml at 25 g/l alginate concentration. This was about 176.8% higher than that obtained by free cells (2890 U/ml). The immobilized cells produced alkaline protease consistently over 5 repeated cycles and reached a maximal value of 9000 U/ml on the third cycle. This was 311.4% (3.11-fold) as compared with the control (free cells). Simple mass balance analysis was applied to describe the growth and the protease production behaviour of both fractions the cells in free form and the entrapped in Ca-alginate beads. Scanning electron microscope studies indicated the internal distribution pattern of the cells encapsulated in Ca-alginate beads. The results presented in this paper show the potential for using immobilized T. turnirae cells in Ca-alginate for the production of a novel alkaline protease.   Key words: Alkaline protease, Ca-alginate, immobilization, Teredinobacter turnirae, repeated batch.

Xylitol production by Ca-alginate entrapped cells: comparison of different fermentation systems

Candida guilliermondii cells were entrapped in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hemicellulosic hydrolysate in three different fermentation systems, namely 125-ml Erlenmeyer flasks (EF), 2.4-l stirred tank reactor (STR) and 2.4-l basket-type stirred tank reactor (BSTR). The EF system provided a xylitol production of 21.0 g P l −1, a product yield based on xylose consumption of 0.54 g P g S −1, and an overall production rate of 0.44 g P l −1 h −1 after 48 h of fermentation. By the STR system, 23.5 g P l −1 was produced after 60 h of fermentation, corresponding to a yield of 0.58 g P g S −1 and a production rate of 0.39 g P l −1 h −1. As the average volume of the beads decreased by 10.7% ( p<0.05) during this fermentation, a basket was fitted into the reactor vessel to prevent the beads abrasion and thus maintain their integrity. However, the xylitol yield (0.46 g P g S −1), production rate (0.21 g P l −1 h −1) and concentration (15.0 g P l −1) attained by this system were affected by mass-transfer limitations.

Growth of micropropagated bananas colonized by root-organ culture produced arbuscular mycorrhizal fungi entrapped in Ca-alginate beads

The effect of root-organ culture (ROC) produced arbuscular mycorrhizal fungi (AMF), i.e. Glomus proliferum, Glomus versiforme and Glomus intraradices, entrapped in Ca-alginate beads on the first stages development of micropropagated bananas ( Musa spp. cv. Grande Naine) was investigated. The experimental design consisted of banana plants inoculated with one of the three AMF and two controls, i.e. Control-AL ( with empty alginate beads), and Control ( no beads). Forty plants were considered per treatment and cultured under greenhouse conditions in a completely randomized design. Eight plants per treatment were harvested 40, 80, 120, 160 and 200 days after inoculation and analysed for root colonization, growth parameters and nutrient concentration. In addition, spores were enumerated in the substrate at the same intervals. Ca-alginate entrapped ROC-produced AMF spores were able ( 1) to colonize the root system of a micropropagated banana cultivar under nursery conditions, ( 2) to increase plant P nutrition and biomass, and ( 3) to proliferate in the commercial nursery substrate, therefore increasing the fungal inoculum biomass. The entrapment of ROC-propagated spores, adaptable to a wide range of Glomeromycetes, represents thus a forthcoming alternative pathogen-free inoculum.

Proliferation and Insulin Secretion Function of Mouse Insulinoma Cells Encapsulated in Alginate/Sol-Gel Synthesized Aminopropyl-Silicate/Alginate Microcapsule

Alginate/aminopropyl-silicate/alginate microcapsules, ca. 15 μm in membrane thickness and ca. 500 μm in diameter, were prepared via sol-gel process. The aminopropyl-silicate membrane was derived from two silicone alkoxide precursors, tetramethoxysilane and 3-aminopropyl-trimethoxysilane on Ca-alginate micro gel beads. Pancreatic β-cell line (MIN6) cells were encapsulated in the microcapsule. The encapsulated MIN6 cells proliferated and formed spheroidal tissues in vitro. The diameter of the MIN6 spheroids increased to approximately 250 μm with an increase in the incubation period until the day 35. Storeptozotocin-induced diabetic mice became normoglycemia after implantation of the MIN6-enclosing microcapsules. The normoglycemic state remained until the retrieval of the implanted microcapsules for 1 month. These results indicate that the potential use of the alginate/aminopropyl-silicate/alginate microcapsule as a vehicle for a genetically engineered cell-enclosing therapeutic material delivery system.

Effect of support materials on antibiotic MSW2000 production by immobilized Streptomyces violatus.

The production of an antibiotic by free and immobilized cells of Streptomyces violatus through entrapment or adsorption on different materials was investigated. S. violatus entrapped in Ca-alginate beads gave low antibiotic activity compared to the free cell or adsorbed cell, while the adsorption of S. violatus on sponge cubes yielded the highest antibiotic concentration after 4 days of incubation in static cultures. A starch concentration of 10 g/L was optimum for the production of the antibiotic by adsorbed cells. The weight and size of the sponge cubes used for immobilization influenced production of the antibiotic and the optimum weight and size of the sponge were 0.8 g and 1.0 cm(3), respectively, yielding a maximum antibiotic production of 280 mg/ml. Maximum antibiotic production was obtained at an initial pH value of 7.5 and in an inoculum size of 3 ml (spore suspension) per 50 ml. The production of the antibiotic in a fixed-bed bioreactor reached a maximum value after 2 days of incubation at a circulation rate of 30 ml/h. The immobilized cells in the bioreactor were reused seven successive times over a period of 14 days.

Use of free and immobilized Pseudomonas putida cells for the reduction of a thiophene derivative in organic media

An organic-aqueous two-liquid phase system was developed in order to perform the reduction of 3-thiophenecarboxaldeyde (3-TC) to 3-thiophenemethanol (3-TM), two intermediate logP compounds, using Pseudomonas putida S12 cells. Although, high bioconversion yields were observed in a bioconversion medium composed by Tris–HCl pH 8.5 buffer and n-octanol, adverse phase toxicity effects were observed following incubation periods in excess of 24h. Furthermore, a stable emulsion was formed in two-liquid phase system in the presence of free cells. This prevented effective separation of the biocatalyst and of the two-liquid phases. Whole cells were effectively entrapped in Ca-alginate beads, thus enhancing biocatalyst recovery. No shifts in the pH versus activity profile resulted from the immobilization. Successive batches were carried out with the immobilized biocatalyst, but a decrease in the specific activity was observed.

In vitro and in vivo evaluation of alginate/sol–gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol–gel process. The membrane has ≡Si–O–Si≡ bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499±32 μm in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly- l-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4±0.5 (mean±SEM)% and 88.0±1.5% ( p<0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.

Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate.

Candida guilliermondii cells, immobilized in Ca-alginate beads, were used for batch xylitol production from concentrated sugarcane bagasse hydrolyzate. Maximum xylitol concentration (20.6 g/L), volumetric productivity (0.43 g/L. h), and yield (0.47 g/g) obtained after 48 h of fermentation were higher than similar immobilized-cell systems but lower than free-cell cultivation systems. Substrates, products, and biomass concentrations were used in material balances to study the ways in which the different carbon sources were utilized by the yeast cells under microaerobic conditions. The fraction of xylose consumed to produce xylitol reached a maximum value (0.70) after glucose and oxygen depletion while alternative metabolic routes were favored by sub-optimal conditions.

Entrapment of Lentinus sajor-caju into Ca-alginate gel beads for removal of Cd(II) ions from aqueous solution: preparation and biosorption kinetics analysis

A white rot fungus species Lentinus sajor-caju biomass was entrapped into alginate gel via a liquid curing method in the presence of Ca(II) ions. The biosorption of cadmium(II) by the entrapped live and dead fungal biomass has been studied in a batch system. The heat-treatment process enhanced the biosorption capacity of the immobilized fungal biomass. The effect of initial cadmium concentration, pH and temperature on cadmium removal has been investigated. The maximum experimental biosorption capacities for entrapped live and dead fungal mycelia of L. sajur-caju were found to be 104.8±2.7 mg Cd(II) g −1 and 123.5±4.3 mg Cd(II) g −1, respectively. The kinetics of cadmium biosorption was fast, approximately 85% of biosorption taking place within 30 min. The biosorption equilibrium was well described by Langmuir and Freundlich adsorption isotherms. The change in the biosorption capacity with time is found to fit pseudo-second-order equations. Cadmium binding properties of entrapped fungal preparations have been determined applying the Ruzic equations. Since the biosorption capacities are relatively high for both entrapped live and dead forms, they could be considered as suitable biosorbents for the removal of cadmium in wastewater treatment systems. The biosorbents were reused in three consecutive adsorption/desorption cycles without significant loss in the biosorption capacity.

Hydrogen sulfide removal utilizing immobilized Thiobacillus sp. IW with Ca-alginate bead

H 2S removal by immobilized cell bioreactor system was investigated using continuous glass column bioreactor with diameter of 90 mm and with a height of 1000 mm. Thiobacillus sp. IW was encapsulated inside the Ca-alginate bead in a continuous H 2S removal bioreactor system and an optimum bioreactor performance was achieved at 1.5 l media volume, under the following conditions: operating temperature of 30 °C, pH 8, bead volume of 30%, and bead diameter of 2 mm. The most active cell-immobilized bead was made in a continuous bead maker at 3% Na-alginate, 3% CaCl 2, and 2.0 g/l of cell concentration. We could remove H 2S in the inlet stream completely up to 600 ppm of influent concentration. It could be confirmed by scanning electron microscope (SEM) that not only did the cells grow well inside the pores of the bead but they also grew out to the surface of the bead after the H 2S removal reaction. It has so far successfully run the continuous H 2S removal bioreactor for up to 240 h without pH control.

Permeability of a sol-gel synthesized aminopropyl-silicate-titanate hybrid membrane for the microcapsule-shaped bioartificial pancreas

The aminopropyl-silicate-titanate hybrid membrane was successfully synthesized onto a Ca-alginate gel bead by the sol-gel method, from two kinds of organometallic compound precursors, tetraethyl orthotitanate (TEOTi) [Ti(OC2H5)4] and 3-aminopropyltrimethyl orthosilicate (APTrMOS) [(CH3O)3Si(CH2)3NH2]. APTrMOS acted as a binding agent between inorganic titanate and alginate polymer via electrostatic interaction between amino groups of APTrMOS and carboxyl groups of alginate. From the measurements of the diffusion of ovalbumin and γ-globulin through the aminopropyl-silicate-titanate hybrid membrane, it was found that the molecular permeability of the membrane could be controlled by changing the amount of each precursor. The molecular weight cutoff point required for immunoisolation, less than 150 kDa, was achieved under the condition that the amount of APTrMOS was 3.40 mmol/(10 ml of Ca-alginate) and TEOTi was more than 1.42 mmol. The islets encapsulated in the membrane showed almost the same insulin secretion viability as the free islets.

Improvement in xylitol production from sugarcane bagasse hydrolysate achieved by the use of a repeated-batch immobilized cell system.

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate during five successive fermentation batches, each lasting 48 hours. The bioconversion efficiency of 53.2%, the productivity of 0.50 g/l x h and the final xylitol concentration of 23.8 g/l obtained in the first batch increased to 61.5%, 0.59 g/l x h and 28.4 g/l, respectively, in the other four batches (mean values), with variation coefficients of up to 2.3%.

Characterization of structure and diffusion behaviour of Ca-alginate beads prepared with external or internal calcium sources

Ca-alginate beads were prepared with either external or internal calcium sources. The structures of both beads were investigated with the aid of scanning electron microscopy (SEM) and confocal microscopy. It was shown that the beads with internal calcium source had a looser structure and bigger pore size than those with external calcium source. The attempts to interpret the difference were carried out by determining the Ca content within the beads at various times, which indicated that it was the different gelation mechanisms that caused the difference of structures of both beads. Furthermore, it was also found that the diffusion rate of haemoglobin (Hb) within the beads with an internal calcium source was faster than that of the beads with an external one, which was consistent with the observation of their structures.

Use of Immobilized Candida Yeast Cells for Xylitol Production from Sugarcane Bagasse Hydrolysate

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate. A full factorial design was employed to determine whether variations in the immobilization conditions would have any effects on the beads, chemical stability and on the xylitol production rates. Duplicate fermentation runs were carried out in 125-mL Erlenmeyer flasks maintained in a rotatory shaker at 30 degrees C and 200 rpm for 72 h. Samples were periodically analyzed to monitor xylose and acetic acid consumption, xylitol production, free cell growth, and bead solubilization. Concentrations of sodium alginate at 20.0 g/L and calcium chloride at 11.0 g/L and bead curing time of 24 h represented the most appropriate immobilization conditions within the range of conditions tested.

Alginate-polylysine-alginate microcapsules: effect of size reduction on capsule properties

Alginate-polylysine-alginate capsules containing insulin-producing cells have been used as a bio-artificial pancreas in the treatment of diabetes mellitus. In a search for microcapsules with improved diffusion characteristics, a high voltage system was developed that produces 250 000 beads/min with a diameter of 160 #181;m #45 3-5%. The diameter of the beads could be varied between 160-700 #181;m depending on the needle diameter and construction, the voltage, the distance between the electrodes and the flow of alginate solution. Ca-alginate beads with diameters of 200 and 500 #181;m were produced by the high voltage electrostatic system. The 200 #181;m beads were sensitive to poly-L-lysine (PLL) exposure and had to be washed in ion-free solution to avoid collapse. The 200 #181;m beads swelled more than the 500 #181;m beads in the washing and PLL treatment. Also, the porosity of the capsules changed with size, but capsules impermeable to tumour necrosis factor (TNF) could be made by exchanging PLL with poly-D-lysine (PDL) for the 500 #181;m beads. The 200 #181;m beads were impermeable to IgG after PLL exposure. Islets of Langerhans were encapsulated in alginate-PLL-alginate capsules and evaluated by measuring protruding islets and insulin production. Islets in microcapsules made by the high voltage electrostatic system did not function differently from islets in larger microcapsules made by an air jet system. In conclusion, alginate capsules made by a high voltage electrostatic system enable large-scale production of small capsules with a narrow size distribution that can meet the functional properties of larger capsules by small changes in the encapsulation procedure.

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO2 of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.