Ca-alginate Beads Research Articles

Enhanced degradation of phenol by Pseudomonas sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol(-1) vol(-1) min(-1), at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l(-1) of phenol as compared to 1500 mg l(-1) by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l(-1). In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l(-1) h(-1) was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l(-1) in the feed with a maximum degradation rate of 400 mg phenol l(-1) h(-1). The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.

Size-controlled microbeads through the influence of the coalescence effect in the emulsification solvent evaporation method

This paper reports a novel, simple, repeatable and cost-effective protocol for producing Ca-alginate beads with controlled sizes ranging from 50–250 μm with a narrow size distribution. The characteristics of the beads depend on the emulsion droplets formed, and the size of the beads can be controlled by manipulating the coalescence factor and the applied mechanical energy, which will also reduce the cost and overall time of the procedure. These results suggest that beads with diameters of 58±5, 69±7, 80±8, 145±11, 195±12 and 225±15 μm (mean diameter ± standard deviation) were easily produced. This was achieved simply by adding a minor amount of Pluronic F-127 (i.e., 0.03%) and controlling the coalescence effect to reduce the stabilization of the emulsion. Therefore, the method has strong potential for mass production on an industrial scale. Furthermore, the solvent evaporation technique successfully evaporated the volatile organic solvent used for emulsification. The beads were proven to be safe via a cell culture study and might be suitable for use in the medical, pharmaceutical and bioengineering field.

Biodegradation of Methyl Tert-butyl Ether in a Bioreactor using Immobilized Methylibium petroleiphilum PM1 Cells

Methylibium petroleiphilum PM1, which is capable of degrading of methyl tert-butyl ether (MTBE), was immobilized in calcium alginate gel beads. Various applications were explored to increase the mechanical strength of these gel beads. The introduction of 0.3 mol/L calcium chloride into the crosslinking solution, 0.002 mol/L calcium chloride into the growth medium, and 0.2% polyethyleneimine (PEI) as chemical crosslinking agent increased the stability of the Ca-alginate gel beads under the operation conditions of the bioreactor. The degradation rates of MTBE by the immobilized cells in the bioreactor system operated in batch and continuous mode , respectively, were compared. A MTBE biodegradation rate of 5.79 mg/L·h was reached for over 400 h (50 batches), and the immobilized cells in the bioreactor removed >96% MTBE during 50 days of operation. Molecular analysis of the PM1 cells revealed that microbial growth occurred predominantly as microcolonies in the outer area of the beads during the first 20 days of operation. The results of this study show that a continuous-mode, fixed-bed bioreactor reactor coupled with PM1-immobilized cells is a promising technology for remediating MTBE-contaminated groundwater.

Effective adsorption of heavy metal ions (Cu2+, Pb2+, Zn2+) from aqueous solution by immobilization of adsorbents on Ca-alginate beads

The performance of adsorbent system consisting of orange peel cellulose (OPC), banana peel cellulose (BPC), orange peel cellulose immobilized Ca-alginate beads (OPCCA) and banana peel cellulose immobilized Ca-alginate beads (BPCCA) for the removal of Cu2+, Pb2+, and Zn2+ from an aqueous solution was tested. The widely used Langmuir isotherm model was utilized to describe the adsorption equilibrium process. OPC and BPC were characterized by SEM and Fourier transform infrared spectroscopy (FT–IR) techniques. The concentration of metal ions was varied in order to obtain optimum concentration level. The effect of pH was studied at different pH values. The role of cellulose in the case of OPC and BPC and the role of alginate in OPCCA and BPCCA were well understood leading to two different mechanisms in native and immobilized cellulose. OPC was found to be a better adsorbent among OPC and BPC, and Cu2+ was considered to be adsorbed more effectively than Pb2+ and Zn2+. The extent of enhancement of adsorption capacity in the case of BPC was greater than that of OPC after immobilization. For the same amount of adsorbents used, OPC and OPCCA showed enhanced activity than BPC and BPCCA.

A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L−1 Kinetin (Kn), 0.5 mg L−1 α-naphthalene acetic acid (NAA) and 0.5 mg L−1 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl2 solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS2 solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L−1, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m−2 s−1 provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L−1, NAA 0.5 mg L−1, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.

EFFICIENT SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION FROM PROTOPLAST CULTURE OF CROCUS SATIVUS L.

The present study reports an efficient protocol for isolation and culture of protoplasts directly from embryogenic calli derived from shoot meristem culture of Crocus sativus L. Embryogenic calli were induced on Linsmaier and Skoog (1965) medium containing 4 mg/L kinetin and 1 or 4 mg/L 2,4-D. Protoplasts were isolated, embedded in Ca-alginate beads and cultured with nurse cells in Murashige and Skoog medium containing 2 mg/L kinetin, 1 mg/L 2,4-D, 100 mg/L ascorbic acid and 0.3 M mannitol at 25°C in darkness. After 4-5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads. Transferring beads to 1/2 MS medium supplemented with 0.2 mg/L kinetin and 0.1 mg/L 2,4-D increased the growth of embryogenic calli. Somatic embryo development was observed on 1/2 MS medium with 1 mg/L ABA. Maturated embryos germinated on a medium containing 25 mg/L GA 3 . Transferring germinated embryos to a medium containing 0.1 mg/L NAA and 1 mg/L BA and incubation at 20°C in a 16/8 hour light/dark cycle promoted conversion efficiency up to 88%.

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.

Preparation of Microbiological Agents for Organic Pollutants Removal in Wastewater

The authors have investigated the biochemical aspects of degradation processes of persistent organic compound benzothiazole by immobilized Rhodococcus rhodochrous cells, such as entrapped in Ca-alginate beads, or as being immobilized on some solid carries. The mineralization of toxicant was complete and biodestructive capacity of entrapped in alginate bacteria increased with each new experimental cycle.

Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads

Encapsulation of herbal aqueous extract through absorption with ca-alginate hydrogel beads was studied. A model herbal aqueous extract, Piper sarmentosum, was used in this study. The effect of process variables (i.e. alginate M/G ratio, alginate concentration, extract concentration, bead size and bead water content) on encapsulation efficiency and biochemical compounds stability were studied. The stability of biochemical compounds was evaluated by using mass balance analysis and FT-IR spectroscopy. The results show that the encapsulation efficiency was mainly affected by alginate M/G ratio and bead water content. In general, ca-alginate beads made of higher alginate M/G ratio or dried to a lower water content were found to absorb significantly more aqueous extract. However, the beads made of higher M/G ratio were less rigid after the absorption process. Besides, the mass balance analysis reveals that the encapsulation process and material did not degrade the bioactive compounds, as the total antioxidant content remained unchanged. This is well supported by the FT-IR analysis where the characteristic bands of chemical groups remained unaltered. Interestingly, the beads made of lower alginate M/G ratio were found to have higher antioxidant affinity. In conclusion, this study demonstrates the potential of using absorption process and hydrogel material for encapsulation of herbal aqueous extract.

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.

Effects of Drying Methods on the Release Kinetics of Vitamin B12 in Calcium Alginate Beads

The aim of this article was to investigate the morphology, swelling properties, and respective drug release kinetics of vitamin B12–loaded calcium alginate beads prepared by oven (air), vacuum, and freeze drying. The initial particle size was 1 mm. The mean bead sizes of dried Ca-alginate beads were 0.7, 0.8, and 0.9 mm for oven-, vacuum-, and freeze-dried beads, respectively. The surface morphology of the dried beads was affected by the different drying methods applied. Oven- and vacuum-dried beads shrank in size, and more cracks appeared on the surface of oven-dried beads. Freeze-dried beads almost retained the same size prior to drying; however, the surface was rougher and highly porous. The swelling profiles were also affected by the drying methods, whereby freeze-dried beads showed the fastest solvent uptake at the start of the experiment. The release data of the dried Ca-alginate beads were treated with first-order, Higuchi, Korsmeyer, and Peppas kinetic models. The data for oven and vacuum seemed to follow a combination of diffusion and swelling controlled release, whereas data from freeze-dried beads seemed to follow more diffusion-dominated controlled release.

Contribution of response surface methodology to the modeling of naringin hydrolysis by naringinase Ca-alginate beads under high pressure

Naringin is the dominant flavonoid bitter principle in grapefruit juice. The aim of this work was the modeling of the enzymatic hydrolysis of naringin, with naringinase immobilised in Ca-alginate beads, under high pressure, in order to optimize this technique for removal of the bitter taste from juices, using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving two variables (pressure and temperature) at five levels (−√2, −1, 0, +1, +√2). The second-order polynomial equations with R 2 values above 0.9 showed good agreement between experimental and predicted, respectively, naringinase activity, naringin conversion and naringenin formation. The higher naringin conversion of 81% was obtained using 205 MPa and 60 °C, during a process time of 30 min. It was found that pressure and temperature, as well as their interactions, had significant effects ( p < 0.001) on naringin hydrolysis in model solutions (acetate buffer pH 4.0). Validation experiments carried out under selected conditions showed good correspondence between experimental and predicted naringinase activity, naringin conversion and naringenin formation, in model solutions and in grapefruit juice. These are promising results to optimize this technique for the removal of the bitter taste and, after future microbial studies to be addressed, as a sterilisation/pasteurization process of citrus juices.

The use of free and immobilized Cunninghamella elegans for removing cobalt ions from aqueous waste solutions

This paper discusses the possible application to use free and immobilized Cunninghamella elegans for the removal of cobalt from aqueous waste solutions. Results indicated that the maximum uptake occurred at; pH 4.0–5.5 ± 0.2, temperature range between 15 and 50°C and stirring rate 250 rpm. The uptake increased with the increase of metal ion concentration up to 40 ppm. Also, it was found that the best biomass weights used for biosorption were 0.25 and 0.5 g for both free and immobilized biomass. The reuse of control alginate beads, alive and dead immobilized Cunninghamella elegans beads was investigated for five cycles. Results showed that the percent uptake decreased slightly after the first cycle. While, in the case of alginate beads there was increase in the second cycle then returned to the same level of uptake. The uptake of cobalt in the presence of Cr(VI) and Cd(II) at different mixture concentrations 40, 50 and 60 ppm was investigated. The results showed that the uptake amount of Co(II) in the presence of other metal ions was lower than Co(II) alone except for Ca-alginate beads. SEM studies for control alginate beads, alive and dead immobilized Cunninghamella elegans beads were conducted to investigate the beads before and after the accumulation of cobalt ions.

Impacts of calcium-alginate density on equilibrium and kinetics of lead(II) sorption onto hydrogel beads

Chronic exposure to Pb2+ above the 15-μg/L US Environmental Protection Agency action level for drinking water has been shown to cause a host of health problems in humans. Thus, it is important to study new methods available for the treatment and removal of Pb2+ from drinking water and wastewater, where elevated levels of heavy metals are found. Alginate-based beads represent one such possible method for heavy metal removal. The impact of alginate density on the equilibrium and kinetics of Pb2+ sorption onto hydrogel beads was investigated using Ca-alginate beads ranging from 1% to 8% (w/v) and exposed to Pb2+ concentrations ranging from 100 to 1,000 mg/L. When Ca-alginate beads were characterized using Fourier transform infrared analysis, the carboxylic acid groups of the mannuronate and guluronate residues in alginate were the primary functional groups that interacted with Pb2+. Hydration of Ca-alginate beads was also examined and found to decrease as Ca-alginate density increased. A positive correlation was observed between Ca-alginate hydration and Pb2+ sorption. Sorption of Pb2+ was fast, reaching equilibrium after approximately 4 h, and is well described by the Langmuir adsorption isotherm. Maximum sorption capacities for 1%, 4%, and 8% beads were 500 ± 100, 360 ± 30, and 240 ± 20 mg/g (dry weight), respectively. The kinetics of sorption were best described by the pseudo-second-order Lagergren model, with rate constants determined as 3.2 ± 0.1 × 10−4, 1.0 ± 0.1 × 10−4, and 1.6 ± 0.1 × 10−4 g mg−1 min−1 for 1%, 4%, and 8% beads, respectively.

Cryopreservation of Dendrobium candidum Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l−1 α-naphthalene acetic acid and 0.5 mg l−1 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m−2 s−1) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl2 solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS2) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.

Prediction models for shape and size of ca-alginate macrobeads produced through extrusion–dripping method

The aim of this work was to develop prediction models for shape and size of ca-alginate macrobeads produced through extrusion–dripping method. The relationship between the process variables on the shape and size of the alginate drops before and after gelation was established with the aid of image analysis. The results show that a critical Ohnersorge number ( Oh) > 0.24 was required to form spherical beads. The shape transition of ca-alginate beads could be typically distinguished into three phases along the collecting distance and it was affected by the combined influence of the solution properties, the collecting distance and the drop size. Mathematical equations and a master shape diagram were developed to reveal a clear operating region and the overall process limits within which spherical ca-alginate beads could be formed. In terms of bead size, the overall size correction factor ( K) which accounted for the liquid loss factor ( k LF ) and the shrinkage factor ( k SF ), varied between 0.73 and 0.85 under the experimental conditions. The size prediction model correlated well with the experimental data. The approach and the outcome could be used as a model to develop prediction tools for similar bead production systems.

Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics

This work intended to study the relationship between diuron herbicide dissipation and the population dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation rate of diuron by 53% (0.141 mg ml −1 h −1). In that case, immobilization strongly modified the final equilibrium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inoculant formulation played a major role in the cell growth of each cultivated strain possibly increasing diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.

Removal of Cadmium Ions from Aqueous Samples by Synechocystis sp.

The biosorption of cadmium ions from aqueous solution by dried, immobilized dried and immobilized live Synechocystis sp. was investigated. Sorption of plain Ca-alginate beads, which were used as substrate for immobilization, was also studied for comparison. Removal efficiency of biosorbents was studied as a function of pH (2-8), temperature (20–40°C), initial cadmium ion concentration (50–300 mg/L), and contact time (0–120 min). The maximum biosorption capacities of the dried, immobilized dried, and immobilized live Synechocystis sp. and plain Ca-alginate beads were found as 75.7, 4.9, 4.3, and 3.9 mg/g, respectively at optimum conditions. Biosorption equilibrium was established in about 15 min. Dried biomass of Synechocystis sp. was found to be more suitable and an efficient biosorbent for the removal of cadmium ion from aqueous solution. Both of the isotherm models (Langmuir and Freundlich) were suitable for describing the biosorption of cadmium by the dried biomass of Synechocystis sp. All the tested cyanobacterial forms could be recovered more than 90% and reused in five biosorption–desorption cycles without any considerable loss in the biosorption capacity.

Biodegradation of wastewater pollutants by activated sludge encapsulated inside calcium-alginate beads in a tubular packed bed reactor

The wastewater treatment plants produce large quantities of biomass (sludge) that require about one-third of the total inversion and operation plant costs for their treatment. By the microorganisms immobilization it is possible to handle high cell concentration in the reactor, increasing its efficiency, reducing the loss of biomass and the wash out is avoided. Moreover, there is no cell growth then the sludge production is reduced. In this study, the COD removal and VSS variation were modeled in a tubular reactor with activated sludge immobilized in Ca-alginate. Moreover, two aspects that are commonly not considered in the performance of the actual reactors of this kind were introduced; the performance in non-steady state and the dispersion effect. The model was calibrated with an actual wastewater taken out from a Mexican wastewater treatment plant. The results of the performance of the tubular bioreactor at different scenarios (i.e., different residence time and VSS in the reactor) are presented. With longer residence times and higher VSS concentration in the Ca-alginate beads in the tubular bioreactor it is possible to increase the time operation of the bioreactor and to treat higher volumes of wastewater. During the process, the sludge generation was drastically reduced and it is possible to remove nitrogen form the wastewater making this process more attractive.

Use of Ca–alginate as a novel support for TiO2 immobilization in methylene blue decolorisation

This study provides a preliminary contribution to the development of an industrial process for the UV/TiO(2) water treatment by introducing a novel support for TiO(2) immobilization. For the following study, Methylene Blue (MB) was chosen as the model dye to evaluate this novel immobilization system. The results showed that TiO(2) immobilized in a Ca-alginate bead retained its photoactivity during all of the experiments and the TiO(2)-gel beads presented good stability in water for maintaining its shape after several uses. When a proportion of 10% (v/v) of these beads was used, the configuration system demonstrated an improved mass transfer and consequently enhanced degradation efficiency. Experiments were also performed using 'recycled' beads. The results showed an increase in the degradation efficiency when the beads were reused, with an eventual 'self-destructive' effect. These studies showed great promise regarding the recyclable reagents with a reduction in waste at no greater cost or reduction in efficiency. Therefore, the potential of TiO(2)-gel beads as a simple and environmentally friendly catalyst for continuous use was developed.