Ca-alginate Gel Beads Research Articles

Monascus Yellow Pigment Production by Coupled Immobilized-Cell Fermentation and Extractive Fermentation in Nonionic Surfactant Micelle Aqueous Solution

Microbial fermentation with immobilized cells possesses many advantages. However, this fermentation mode is restricted to the production of extracellular products. Our previous study demonstrated that the extractive fermentation of Monascus spp. in nonionic surfactant micelle aqueous solution can export Monascus pigments that are supposed to be mainly intracellular products to extracellular culture broth and, in the meantime, extracellularly enhance the production of yellow pigments at a low pH condition; consequently, this makes the continuous production of yellow pigments with immobilized Monascus cells feasible. In this study, immobilized-cell fermentation and extractive fermentation in Triton X-100 micelle aqueous solution were successfully combined to continuously produce Monascus yellow pigments extracellularly. We examined the effects of cell immobilization and Triton X-100 on cell growth, pigment production, and pigment composition. In the repeated-batch extractive fermentation with immobilized cells, the biomass in Ca-alginate gel beads continued to grow and reached 21.2 g/L after seven batches, and dominant yellow pigments were produced extracellularly and stable for each batch. The mean productivity of the extracellular yellow pigments reached up to 22.31 AU410 nm/day within the first four batches (13 days) and 19.7 AU410 nm/day within the first seven batches (25 days). The results also provide a new strategy for producing such intracellular products continuously and extracellularly.

Inclusion of partly purified protease from Abrus precatorius Linn in Ca-alginate gel beads

Abrus precatorius is an edible endemic plant in Cameroon. In the northern regions of Cameroon, the leaves of this plant are used as traditional medications, for the liquefaction of wort obtained after mashing of sorghum or millets for processing the local fermented beer called bili-bili, and to sweeten gruels made from red millet. Abrus precatorius has also being explored as a potential source of proteolytic enzymes. In this work, a partially purified protease from the leaves and stems of the plant was immobilized in a calcium alginate gel beads and the protease activity (PA), investigated using a central composite design plan under the conditions: alginate content (1–5%) (w/v), enzyme/alginate ratio (10–30%) (v/v), and CaCl2 concentration (100–400 Mmol/L). Results showed that the optimum activity was obtained for an alginate content of 1% (p/v), an enzyme/alginate ratio of 10% (v/v) and a CaCl2 concentration of 400 mmol/L. At the optimum, kinetic parameters of the immobilized enzyme (KM: 6.83 mg mL−1; Vmax: 46.95 g.L−1. min−1) were comparable to those of free enzyme (KM: 1.26 mg mL−1; Vmax: 153.85 g.L−1. min−1). The beads have been preserved at room temperature and reused for four days before loosing 88% of its activity. This indicates that the protease activity of the leaves and stems of A. precatorius can be immobilized in calcium alginate beads and preserve four days at ambient temperature.

High rate algal systems for treating wastewater: A comparison

Algal systems can remove nitrogen (N) and phosphorus (P) from wastewater while producing valuable biomass. The microalga Chlorella vulgaris in three concentrated forms (suspended, entrapped in Ca-alginate gel beads and as a biofilm on supports and the macroalga Oedogonium cardiacum were compared for treating secondary effluent containing 15 mg/L of ammonium (N-NH4+), 6 mg/L of nitrate (N-NO3−), and 7 mg/L of total phosphorus (TP) with a hydraulic retention time of 12 h. Identical conditions and reaction vessels enabled a direct comparison of growth systems. The biofilm system was the most effective of the microalgal systems, decreasing concentrations to 1.9 mg/L TP and 0.5 mg/L N-NO3− on average from day 3 to 24, and like the other microalgal systems, was not as effective for N-NH4+ removal (average of 9.0 mg/L). The macroalgal system decreased TP to 1.3 mg/L and N-NH4+ to ≤0.5 mg/L on average from day 16 to 30 and operated for longer than the other systems, but was not effective for N-NO3− removal (average of 4.8 mg/L). Hence the minimum TN concentration of the effluent from the macroalgal system (7.1 mg/L) was lower than for the biofilm system (10.6 mg/L) from the feed of 24 mg/L. The biofilm system produced 56 mg/L/d and the macroalgae 102 mg/L/d of biomass. The production of the highest quality effluent for longer and of more biomass than the microalgal systems, combined with their larger cell size which facilitates reactor operation, demonstrates that macroalgae can compete with microalgae for wastewater remediation.

Inclusion of Partly Purified Protease from Abrus Precatorius Linn in Ca-Alginate Gel Beads

Inclusion of Partly Purified Protease from Abrus Precatorius Linn in Ca-Alginate Gel Beads

Adsorptive Removal of Se(IV) by Citrus Peels: Effect of Adsorbent Entrapment in Calcium Alginate Beads.

Selenium (Se) contamination in natural waters impacted by anthropogenic activities is becoming a prevalent and widespread problem. Investigation of novel, low-cost, and sustainable food-waste-sourced adsorbents for Se removal has largely been unexplored. Here, we report on the Se(IV) biosorption from a liquid solution using three waste-derived/low-cost biosorbents, namely citrus peels (bare), Ca-alginate gel beads, and Ca-alginate-citrus peels composite beads (Ca-alginate@citrus). The entrapment of citrus peels by Ca-alginate not only provided a structural framework for the citrus peel particles but also preserved the high-efficiency Se(IV) removal property of the citrus peels. From the modeling results, it was established that Se(IV) biosorption followed the fixed-film diffusion model, along with pseudo-second-order kinetics. Investigation of pH impacts along with initial dosing of sorbent/sorbate demonstrated that all of the three biosorbents exhibited optimum biosorption of Se(IV) at pH 6−8, 50–75 mg·L–1 of Se(IV), and 1–5 mg·L–1 of biosorbent. Overall, the maximum Se(IV) biosorption capacities were measured to be 116.2, 72.1, and 111.9 mg·g–1 for citrus peels, Ca-alginate, and Ca-alginate@citrus, respectively, with citrus peels (bare and immobilized) showing among the highest reported values in the literature for Se(IV) adsorption. This work provides a platform for the future development of an efficient filtration system using Ca-alginate@citrus as an inexpensive, novel, and sustainable biosorbent to treat Se(IV) contaminated water.

Efficient Grafting of Cyclodextrin to Alginate and Performance of the Hydrogel for Release of Model Drug

Controlling the rate of release of molecules from a hydrogel is of high interest for various drug delivery systems and medical devices. A strategy to alter the release profiles of soluble and poorly soluble active ingredients from hydrogels can be to combine the hydrogel forming ability of alginate with the inclusion forming ability of cyclodextrins (CyD). Here, β-CyD was grafted to alginate in a three-step synthesis using periodate oxidation, reductive amination and copper(I)-catalyzed azide-alkyne cycloaddition. A grafting degree of 4.7% mol β-CyD/mol sugar residues was obtained. The grafting degree was controlled by varying the reaction parameters where the amount of linker used in reductive amination was especially influential. Ca-alginate gel beads grafted with β-CyD showed increased uptake of the model molecule methyl orange. Release experiments showed that the grafted material had a prolonged release of methyl orange and an increased total amount of released methyl orange. These results show that the β-CyD grafted alginate is still able to form a hydrogel while the grafted cyclodextrins retain their ability to form inclusion complex with methyl orange. Further testing should be done with this system to investigate capability for drug delivery applications.

Biotransformation of acetophenone to R-1-phenylethanol with immobilized Pichia capsulata in batch reactor

Abstract Biotransformation of acetophenone to R-1-phenylethanol with Pichia capsulata immobilized in Caalginate gel beads was studied. Experiments were carried out in a batch reactor stirred on an orbital shaker (300 rpm) at 25 ℃. Based on experimental data, uncoupled parameters of reaction kinetics (measurements with free cells) and diffusion coefficients (sorption method) were estimated. The model expressing the conversion of acetophenone with immobilized cells in the batch reactor consisted of mass balance equations of acetophenone and R-1-phenylethanol in the liquid and solid phases. A satisfactory agreement between the experimental and predicted values of acetophenone and R-1-phenylethanol concentrations was achieved

Diffusion characteristics of different molecular weight solutes in Ca–alginate gel beads

Diffusion characteristics of cyanocobalamin (Vitamin B12) (VB12) and polyethylene glycol (PEG) in Calcium-alginate (Ca-alginate) hydrogel beads were critically investigated. Quasi-simultaneous and sequential gelation-and-release situations were conducted to determine the sorption and release kinetics of solutes with different molecular weights. In the first case, results were typical of a convection mechanism: high effective PEG diffusion coefficients and no effect of the PEG molecular weight on the overall transfer were observed. An increase in the sodium alginate concentration slightly decreased the diffusion coefficient of VB12, whereas, it had no effect on the PEG diffusion. On the other hand, the lower the calcium chloride concentration is, the higher the release of VB12. The opposite is true for PEG. In the second case, the sorption and release kinetics of VB12 or PEG was monitored after alginate gelation was completed. The diffusion coefficient was found to be strongly affected by the PEG molecular weight. For solute diffusion from the beads: when the alginate concentration increased, diffusivity found a significant decrease; calcium chloride concentration had a negligible effect on diffusivity. For solute diffusion into the beads: neither the alginate concentration nor the calcium chloride concentration had any effect on the diffusion of solutes with MW < 20,000.

Experimental and robust modeling approach for lead(II) uptake by alginate gel beads: Influence of the ionic strength and medium composition

Systematic kinetic and equilibrium studies on the lead ions removal ability by Ca-alginate gel beads have been performed by varying several internal parameters, namely, number of gel beads, nature and composition of the ionic medium and pH, which allowed us to model a wastewater in order to closely reproduce the composition of a real sample. Moreover, the effects brought about the different ionic species present in the reacting medium have been evaluated.Differential Pulse Anodic Stripping Voltammetry (DP-ASV), has been systematically used to perform kinetic and equilibrium measurements over continuous time in a wide range of concentration. Kinetic and equilibrium data have been quantitatively analyzed by means of robust approach both for the non-linear regression and the subsequent residuals analysis in order to significantly improve the results in terms of precision and accuracy.Alginate gel beads have been characterized by SEM and an investigation on their swelling behavior has also been made. Removal efficiency of the calcium-alginate gel beads has been calculated and results obtained have showed a relevant dependence on ionic strength, composition of ionic media, pH of solution and number of gel beads. The number of gel beads takes part as key crucial components, i.e., the higher the number of beads the greater the amount of Pb(II) species removed from the sample, the lower the time needed to reach the maximum removal efficiency of 90%.

Characterization of structure, physico-chemical properties and diffusion behavior of Ca-Alginate gel beads prepared by different gelation methods

Ca-Alginate beads were prepared with either external or internal calcium sources by dripping technique. It was found that beads synthesized with internal calcium source had a looser structure and bigger pore size than those produced with external calcium source. Consequently, a faster diffusion rate of Vitamin B12 (VB12) within the beads with an internal calcium source was observed. Furthermore, the concentration of calcium ion, ionic strength and pH of the external gel beads formation solution were investigated. Results showed that (a) the concentration of the calcium ion was found to be the determining factor in the gel formation phenomenon; (b) the weight and volume losses are in effect due to water removal; (c) NaCl acts as a competitor with calcium and a screen in the electrostatic repulsion; and (d) the pH controls the gel formation process by regulating the dissociation of alginate and the complexation of the calcium cations. These results are keys to understanding the behavior and performance of beads in their utilization medium.

Permeabilizing Escherichia coli for whole cell biocatalyst with enhanced biotransformation ability from l-glutamate to GABA

Abstract γ-Aminobutyric acid (GABA) is widely used as a pharmaceutical, nutraceutical, and as a precursor for synthesizing materials for industrial use. Bacterial cells can be exploited for use as biocatalysts with the potential to synthesize compounds such as GABA with greater efficiency, safety, and economy. However, efforts to use biocatalysts must overcome the permeability barrier of the cell envelope. Therefore, to produce a whole-cell biocatalyst with enhanced cell-associated glutamate decarboxylase (GAD) activity, we overexpressed the Glutamate (Glu)-GABA antiporter (GadC) in Escherichia coli (BL21(DE3)-pET28a- gadB ). The cell-associated GAD activity of the transformants was higher by a factor of 2.6 in comparison; however, expression of GadC inhibited growth. Therefore, we permeabilized the cells using either organic solvents, surfactants, or heat. Permeabilization with organic solvents increased cell-associated GAD activity as a function of their hydrophobicity, and hexane was the most effective, increasing cell-associated GAD activity by a factor of 9.65 (6.72 U/mg). The surfactants Triton X-100, CHAPS, NP-40, OGP, and Brij-35 enhanced cell-associated GAD activity, and Triton X-100 was the most effective, increasing cell-associated GAD activity by a factor of 10.8 (7.53 U/mg). Heating BL21(DE3)-pET28a- gadB at 70 °C for 30 min increased cell-associated GAD activity by a factor of 13.1. GAD did not leak from the permeabilized cells under optimum conditions. When the heat-permeabilized cells were immobilized on Ca-alginate gel beads, the biotransformation ability of beads maintained over 60% of their initial ability after 20 consecutive batches, and the beads retained 90% of their initial activity after 30 days of storage. These approaches for improving cell permeability to enhance cell-associated GAD activity show great promise for decreasing the cost of industrial production of GABA.

A novel biotransformation of astragalosides to astragaloside IV with the deacetylation of fungal endophyte Penicillium canescens

Under the deacetylation of fungal endophyte Penicillium canescens, which was isolated from pigeon pea, a novel and highly efficient biotransformation method of astragalosides to astragaloside IV in Radix Astragali was investigated. After single factor tests of the biotransformation procedure, the optimum biotransformation conditions were confirmed as the liquid solid ratio 20:1, the biotransformation temperature 30°C, time 36h and pH 7, respectively. Final content of astragaloside IV in Radix Astragali reached 7.66±0.44mg/g, which was 5.51-fold to that of untreated one and contents of astragaloside I and astragaloside II significantly decreased. The immobilized Ca-alginate gel beads with P. canescens could be reused at least for 13 runs. This is the first report that fungal endophyte was applied for the biotransformation of astragalosides to astragaloside IV in Radix Astragali and this novel high-efficiency biotransformation method will be an alternative to enhance the content of astragaloside IV in Radix Astragali in commercial process.

FABRICATION AND CHARACTERIZATION OF MACROPOROUS EPICHLOROHYDRIN CROSS-LINKED ALGINATE BEADS AS PROTEIN ADSORBENT

Porous epichlorohydrin cross-linked alginate beads (ECAB) were prepared by the following method. Na-alginate solution containing Na2SO4 was introduced dropwise into CaCl2 solution to simultaneously form CaSO4 precipitate and Ca-alginate gel beads. The resultant beads were cross-linked with epichlorohydrin and then thoroughly washed with ethylenediamine tetraacetic acid (EDTA) solution to remove CaSO4. The structural features of porous ECAB were assessed with scanning electron microscopy (SEM) and experiments on water content and adsorption of bovine serum albumin (BSA). The results showed that macroporous ECAB can be obtained when the mass ratio of sodium sulfate to sodium alginate is 4:1. The adsorption behavior of the macroporous ECAB was well described by the Langmuir isotherm with maximum adsorption capacity equal to 740 mg BSA/g dry weight in 50 mM Na2HPO4–citric acid buffer (pH 4.0). BSA was more effectively adsorbed by macroporous ECAB at around pH 3 and the mechanism of the adsorption of BSA to the ECAB was ion exchange. Finally, experiments of a concentration of 1 mg/mL BSA using macroporous ECAB were performed.

Ultrasound-assisted dextranase entrapment onto Ca-alginate gel beads

In this research work, dextranase has immobilized onto calcium alginate beads using a novel ultrasound method. The process of immobilization of the enzyme was carried out in a one-step ultrasound process. Effects of ultrasound conditions on loading efficiency and immobilization yield of the enzyme onto calcium alginate beads were investigated. Furthermore, the activity of the free and immobilized enzymes prepared with and without ultrasound treatment, as a function of pH, temperature, recyclability and enzyme kinetic parameters, was compared. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with an ultrasonic irradiation at 25kHz, 40W for 15min, under which the loading efficiency and the immobilization yield increased by 27.21% and 18.77%, respectively, compared with the immobilized enzymes prepared without ultrasonic irradiation. On the other hand, immobilized enzyme prepared with ultrasonic irradiation showed Vmax and KM value higher than that for the immobilized enzyme prepared without ultrasonic irradiation, likewise, both the catalytic and specificity constants of immobilized enzyme prepared with ultrasonic irradiation were higher than that for immobilized enzyme prepared without ultrasound, indicating that, this new ultrasonic method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared with immobilized enzyme prepared without ultrasound treatment, the immobilized enzymes prepared with ultrasound irradiation exhibited: a higher pH optimum, optimal reaction temperature, activation energy, and thermal stability, as well as, a higher recyclability, which, illustrating the effectiveness of the sonochemical method. To the best of our knowledge, this is the first report on the effect of ultrasound treatments on the immobilization of dextranase.

Ethanol fermentation of sugarcane molasses by Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and Ca-alginate matrices

Bio-ethanol production from cane molasses (diluted to 15 % sugar w/v) was studied using the bacterium, Zymomonas mobilis MTCC 92 entrapped in luffa (Luffa cylindrica L.) sponge discs and Ca-alginate gel beads as the immobilizing matrices. At the end of 96 h fermentation, the final ethanol concentrations were 58.7 ± 0.09 and 59.1 ± 0.08 g/l molasses with luffa and Ca-alginate entrapped Z. mobilis cells, respectively exhibiting 83.25 ± 0.03 and 84.6 ± 0.02 % sugar conversion. There was no statistical significant difference (Fischer's LSD) in sugar utilization (t = 0.254, p<0.801) and ethanol production (t =-0.663, p<0.513) between the two immobilization matrices used. Further, the immobilized cells in both the matrices were physiologically active for three more cycles of operation with less than 15 % decrease in ethanol yield in the 4(th) cycle, which was due to some leakage of cells. In conclusion, luffa sponge was found to be equally good as Ca-alginate as a carrier material for bacterial (Z. mobilis) cell immobilization for ethanol production. Further, it has added advantages such as it is cheap, non-corrosive and has no environmental hazard.

English

The production of bio-ethanol from cane molasses (diluted to 15% sugar, w/v) was studied, using the yeast, Saccharomyces cerevisiae immobilized in luffa ( Luffa cylindrica L .) sponge discs and in Ca-alginate gel beads. At the end of 96 h fermentation, the ethanol yields were 64.67 ± 0.016 and 65.21 ± 0.030 g/l molasses, with luffa and Ca-alginate entrapped S. cerevisiae cells exhibiting 89.90 ± 0.008 and 91.86 ± 0.072% sugar conversion, respectively. There was no statistically significant difference [Fisher’s least significance difference (LSD)] between the two immobilized systems in both sugar utilization (t = 0.254, p < 0.801) and in ethanol production (t = -0.663, p < 0.513). Key words : Bio-ethanol, cell immobilization, fermentation, molasses, Saccharomyces cerevisiae.

OPTIMIZATION OF LIPASE IMMOBILIZATION

Pseudomonas aeruginosa BBRC-10036 was used for lipase production. The organism secreted the enzyme extracellulary. In order to purify the enzyme, precipitation was done first, and then this lipase has been purified by Ion exchange Chromatography leading to 2.3-fold purification and 11.47% recovery. Lipase from P.aeruginosa was entrapped into Ca-alginate gel beads and effect of independent variables such as alginate concentration (%w/v), CaCl2 concentration (M) and enzyme load (%v/v) on immobilization yield and activity of immobilized enzyme were investigated. Media optimization for immobilization of lipase was carried out by Response Surface Methodology. The optimum conditions were as follows: sodium alginate concentration 2.5% (w/v), calcium chloride concentration 2.5(M) and enzyme load 50% (v/v). At those conditions, the highest immobilization yield and the optimum activity of immobilized enzyme, respectively, obtained were 93.65% and 2.64 unit/gr(IME).

Ethanol fermentation of sugarcane molasses by Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and Ca-alginate matrices

Bio-ethanol production from cane molasses (diluted to 15 % sugar w/v) was studied using the bacterium, Zymomonas mobilis MTCC 92 entrapped in luffa (Luffa cylindrica L.) sponge discs and Ca-alginate gel beads as the immobilizing matrices. At the end of 96 h fermentation, the final ethanol concentrations were 58.7 ± 0.09 and 59.1 ± 0.08 g/l molasses with luffa and Ca-alginate entrapped Z. mobilis cells, respectively exhibiting 83.25 ± 0.03 and 84.6 ± 0.02 % sugar conversion. There was no statistical significant difference (Fischer’s LSD) in sugar utilization (t = 0.254, p<0.801) and ethanol production (t =-0.663, p<0.513) between the two immobilization matrices used. Further, the immobilized cells in both the matrices were physiologically active for three more cycles of operation with less than 15 % decrease in ethanol yield in the 4th cycle, which was due to some leakage of cells. In conclusion, luffa sponge was found to be equally good as Ca-alginate as a carrier material for bacterial (Z. mobilis) cell immobilization for ethanol production. Further, it has added advantages such as it is cheap, non-corrosive and has no environmental hazard.

Esterification of Glucose by Coconut Oil Fatty Acids Using Immobilized CandidarugosaLipaseE.C.3.1.1.3. inCa-alginate Matrix

In this study, the synthesis of fatty acid - carbohydrate esters used glucose (G) and coconut oil fatty acid (FA). The esterification reaction was carried out enzymatically using Candida rugosalipase in organic solvent (n-hexane). Lipase was immobilized on Ca-alginate gel beads withthe optimum condition for immobilization was1 % of Na-alginate, resulting 61.66% immobilization yield. The optimum conditions for esterification using immobilized lipase in Ca-alginate were at 35 o C, the molar ratio 1:60 (G/FA), 16 hours incubation time and 0 g molecular sieve. Characterization of synthesized product with FT-IR showed that product exhibit the absorbtion of ester functional group at 1737 cm -1 , but with thegreater OH groupabsorption peakat3344.57cm -1 indicatedthat the degree ofesterificationwas still low. The synthesized product was then examined by simple emulsion test and was proved to be an emulsifier. Based on theemulsion test, it was known that theproducts ofthe reactionwereglucoseestersthat have propertiesampiphilicas anemulsifiersystem ofoil-in- water emulsion(o/w).

Simultaneous degradation of phenol and 3-chlorobenzoate by a mixture of separately immobilized cells of two strainsof Pseudomonas – A preliminary study

Simultaneous degradation of halogenated and non-halogenated aromatic compounds that are catabolised through different cleavage pathways is generally difficult due to biochemical incompatibility. However, free cells of a mixed culture of Pseudomonas sp. CP4 that degrades phenol through meta-pathway and Pseudomonas aeruginosa strain 3mT that degrades 3-chlorobenzoate through ortho-pathway was shown, earlier, to degrade mixtures of phenol/cresols and 3-chlorobenzoate in shake flasks. In the present study the degrading efficiency of these strains when immobilized (separately) in Ca-alginate gel beads was tested using a fluidized bed reactor. Complete mineralization of up to 5 mM equimolar mixture of phenol and 3-chlorobenzoate was observed when the beads were used at 1:1 ratio. From a 10 mM equimolar mixture although phenol was completely mineralized 3-chlorobenzoate degradation was not complete as evidenced by accumulation of some intermediary metabolites and the release of only 86% of inorganic chloride (Cl ¯ ). Degradation rates of both the compounds by the immobilized cells, in general, were far better than that by the free cells. With further studies to select suitable gel matrices and optimization of bioreactor conditions these strains can be effectively deployed for treatment of heterogeneous aromatic wastes.