“Diffusion” of microorganisms in calcium alginate beads

Summary The leakage of Serratia marcescens cells immobilised in Ca-alginate beads was quantified and the experimental values fitted to the pore diffusion model in order to obtain the biomass diffusion coefficient (D _x). This coefficient was then determined, resulting a value of 0.45 10^–7 cm² s^–1 at the beginning of the fermentation process. The variation of this coefficient, together with the porosity of the particles (_i), were also determined all along the process.     

微生物来源褐藻胶(alginate)生物合成及应用前景

褐藻胶及其制品在医药、食品及化工等领域具有重要价值。除褐藻以外,微生物是褐藻胶的另一重要来源。虽然微生物来源的褐藻胶未能够实现规模化生产,但是由于微生物合成褐藻胶具有发酵条件可控、产物单一、结构稳定并且易于纯化等优势,引起广泛的关注。并且利用生物工程技术已经实现了对微生物来源褐藻胶结构的调控和改造,促进了褐藻胶的高值化利用。此文综述了微生物褐藻胶生物合成的概况,并对利用基因工程改造褐藻胶的发展趋势和应用前景进行了论述。     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Study of phenol biodegradation using Bacillus amyloliquefaciens strain WJDB-1 immobilized in alginate–chitosan–alginate (ACA) microcapsules by electrochemical method

An aerobic microorganism with an ability to utilize phenol as sole carbon and energy source was isolated from phenol-contaminated wastewater samples. The isolate was identified as Bacillus amyloliquefaciens strain WJDB-1 based on morphological, physiological, and biochemical characteristics, and 16S rDNA sequence analysis. Strain WJDB-1 immobilized in alginate–chitosan–alginate (ACA) microcapsules could degrade 200 mg/l phenol completely within 36 h. The concentration of phenol was determined using differential pulse voltammetry (DPV) at glassy carbon electrode (GCE) with a linear relationship between peak current and phenol concentration ranging from 2.0 to 20.0 mg/l. Cells immobilized in ACA microcapsules were found to be superior to the free suspended ones in terms of improving the tolerance to the environmental loadings. The optimal conditions to prepare microcapsules for achieving higher phenol degradation rate were investigated by changing the concentrations of sodium alginate, calcium chloride, and chitosan. Furthermore, the efficiency of phenol degradation was optimized by adjusting various processing parameters, such as the number of microcapsules, pH value, temperature, and the initial concentration of phenol. This microorganism has the potential for the efficient treatment of organic pollutants in wastewater.     

Comparison of alginate and “Pesta” for formulation of Phanerochaete chrysosporium

Summary Alginate and wheat gluten (Pesta) matrices were compared for the encapsulation of the white rot fungus Phanerochaete chrysosporium. Pesta granules were not successful for formulating P. chrysosporium although control granules made with Alternaria alternata yielded viable fungal colonies; the gluten in wheat flour apparently inhibits growth of the white rot fungus. P. chrysosporium formulated in alginate with corncob grits or sawdust, and stored at room temperature, yielded over 50% viability of encapsulated mycelia after six months. Alginate encapsulation offers a promising technology for the delivery of white rot fungi to toxic waste sites.     

Heterologous β-phellandrene production by alginate immobilized Synechocystis sp. PCC 6803

Journal of Applied Phycology - The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and...     

Encapsulation of lactate dehydrogenase in carbon nanotube doped alginate–chitosan capsules

Alginate–chitosan shell–core (AC) capsules doped with carbon nanotubes (CNTs) were prepared for lactate dehydrogenase (LDH, EC 1.1.1.27) encapsulation to convert pyruvic acid to lactic acid coupling with the oxidation of NADH to NAD⁺. LDH was entrapped within the liquid core of the capsules and the CNTs were incorporated in the alginate or chitosan matrices or both. The physical properties of the capsules and the immobilized LDH activity were investigated. The AC capsules doped with CNTs showed better mechanical strength than that without CNTs. The LDH loading efficiency of the AC capsules with CNTs (10 mg/mL) doped in both the shell and the core was 30.7% higher than that without CNTs. The optimal pH value for the bioconversion catalyzed by immobilized LDH was 7.0, lower than that by free LDH (7.5). The optimal temperature was 35 °C for both immobilized and free LDH. Operational stability of the immobilized LDH was greatly improved by doping CNTs in AC capsules. The results showed that this method was efficient for enzyme encapsulation in the biotechnology applications.     

Culturing embryogenic protoplasts of ‘Hamlin’ sweet orange in calcium alginate beads

Two finely-dispersed, homogeneous and regenerable cell suspension cultures were established from embryogenic callus derived from immature and mature embryos in barley. The quality and viability of suspension cells obtained were determined using differential-interference-contrast microscope and fluorescence microscope. Cell suspension cultures, maintained in modified liquid CC medium, showed a 10-fold increase in dry weight after two weeks with a doubling time of about 3 days. Addition of l-proline and casein hydrolysate in the medium had positive effect on the growth of cell cultures. Subculture interval significantly affected mitotic index. Both cell lines established were able to regenerate plants by somatic embryogenesis, but cell line Z-IM showed much higher regeneration capacity than cell line Z-M. Comparatively high frequencies of variations in chromosome number and structure were found in both lines, and a correlation between karyotype and morphogenic capacity was noticed.Abbreviations KT kinetin - BAP benzylaminopurine - FDA-PI fluorescein diacetate and propidium iodide - 2,4-d 2,4-dichlorophenoxyacetic acid     

Synthesis of amidic alginate derivatives and their application in microencapsulation of λ-cyhalothrin

1-Octyl amine was covalently coupled to sodium alginate(NaAlg) in an aqueous-phase reaction via acidamide functions using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) as a coupling reagent to provide octyl-grafted amphiphilic alginate-amide derivative(OAAD) for subsequent use in λ-cyhalothrin (LCH) microcapsule application. The structure of OAAD was confirmed by FT-IR and (1)H NMR spectroscopies. The new alginate-amide derivative was used for fabricating microcapsule that can effectively encapsulate LCH by emulsification-gelation technique. The microcapsules were characterized by optical microscopy (OM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and laser particle size analysis. The encapsulation efficiency and drug release behavior of LCH from the microcapsules were investigated. Results showed that the microcapsules were in spherical form with diameter mostly in the range of 0.5-10 μm and possessed a structure with LCH as core and OAAD as shell. The encapsulation efficiency and the release performance of the microcapsules were influenced by DS of OAAD and amount of CaCl(2). The mechanism of LCH release was found to vary from anomalous to Fickian to quasi-Fickian transport with the DS of OAAD varied from 10.8 to 30.3 and the CaCl(2)/emulsion ratios varied from 0.09 to 0.03%.     

Bacterial cellulose–alginate composite sponge as a yeast cell carrier for ethanol production

A bacterial cellulose–alginate (BCA) sponge, fabricated by a freeze-drying process, was successfully used as a yeast cell carrier for ethanol fermentation. The BCA sponge exhibited several advantageous properties, such as high porosity, appropriate pore size, strong hydrophilicity and high mechanical, chemical and thermal stabilities. BCA has an asymmetric structure, with a thin, dense outer layer covering an interior of interconnected macropores that are distributed throughout the sponge, which is effective for yeast immobilization. At 48 h of the fermentation, the maximum ethanol concentration produced by the immobilized culture (IC) in the BCA carrier was about 100 g/L, which was approximately 13% and 45% higher than that from the suspended culture (SC) and from IC in Ca-alginate matrix, respectively. Repeated-batch ethanol productions using IC in BCA carriers were also more stable than those using SC or IC in Ca-alginate matrix. The results of a 15 cycle repeated batch operation demonstrated that the system with IC in BCA exhibited superior long-term stability for ethanol fermentation with the average ethanol productivity at 1.9 g/L h and the immobilized yield at 86%. The improved ethanol fermentation performance was mainly due to the water uptake ability and properly interconnected pore structure, which help to overcome limiting mass transfer.     

Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages?

The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected.     

The continuous hydrolysis of geniposide to genipin using immobilized β-glucosidase on calcium alginate gel

Summary The reaction velocity of immobilized -glucosidase was approximated by the first-order reaction kinetics. A plug flow reactor was used for continuous hydrolysis of geniposide with this immobilized enzyme. The activity of this immobilized enzyme was retained 100% for 600 h. The amount of genipin formed by using the immobilized enzyme was 17 fold that formed using the native enzyme without reuse. Using immobilized enzyme, purity and yield of genipin, which is a hydrolyzate of geniposide, was improved comparing with the native enzyme.     

Assay and properties of alginate lyase and 1, 3-β-glucanase in intertidal sands

Summary Alginate lyase and 1, 3--glucanase activity were detected in intertidal sands below decomposing seaweeds (Fucus sp. andLaminaria sp). Linear relationships between activity and sand weight; length of incubation and substrate concentration, were established for both enzymes. Other properties of these enzymes in intertidal sands are reported.     

Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant

Cannabis sativa L. (Cannabaceae) is an important medicinal plant well known for its pharmacologic and therapeutic potency. Because of allogamous nature of this species, it is difficult to maintain its potency and efficacy if grown from the seeds. Therefore, chemical profile-based screening, selection of high yielding elite clones and their propagation using biotechnological tools is the most suitable way to maintain their genetic lines. In this regard, we report a simple and efficient method for the in vitro propagation of a screened and selected high yielding drug type variety of Cannabis sativa, MX-1 using synthetic seed technology. Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 % sodium alginate with 50 mM CaCl₂.2H₂O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 %) under in vitro conditions. Under in vivo conditions, 100 % conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose and 0.5 % PPM. Plantlets regenerated from the encapsulated explants were hardened off and successfully transferred to the soil. These plants are selected to be used in mass cultivation for the production of biomass as a starting material for the isolation of THC as a bulk active pharmaceutical.Key words: Encapsulation, Nodal explants, Plant growth regulators, Plant regeneration, Synthetic seeds     

Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides

Thermostable α-galactosidase from Aspergillus terreus _GR was insolubilized using concanavalin A obtained from jack bean extract and in order to maintain the integrity of complex in the presence of its substrate or products, this complex was crosslinked with glutaraldehyde. Soluble α-galactosidase entrapped in calcium alginate retained 82% of enzyme activity whereas, Con A-α-galactosidase complex entrapped in calcium alginate and crosslinked Con A-α-galactosidase complex entrapped calcium alginate retained 74 and 61% activity, respectively. A fluidized bed reactor was constructed for continuous hydrolysis of galactooligosaccharides in soymilk using crosslinked Con A-α-galactosidase complex entrapped calcium alginate. Optimum conditions such as pH (5.0) and temperature (65°C) were the same for all immobilized enzyme preparations and soluble enzyme. Crosslinked Con A-α-galactosidase entrapped complex exhibited enhanced thermostability and showed 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Con A-α-galactosidase complex preparation was superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations. The entrapped crosslinked concanavalin A-α-galactosidase complex retained 95% activity after eight cycles of use.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Immobilization of urease by using chitosan–alginate and poly(acrylamide-co-acrylic acid)/κ-carrageenan supports

Jack bean urease (urea aminohydrolase, E.C. 3.5.1.5) was entrapped into chitosan–alginate polyelectrolyte complexes (C-A PEC) and poly(acrylamide-co-acrylic acid)/κ-carrageenan (P(AAm-co-AA)/carrageenan) hydrogels for the potential use in immobilization of urease, not previously reported. The effects of pH, temperature, storage stability, reuse number, and thermal stability on the free and immobilized urease were examined. For the free and immobilized urease into C-A PEC and P(AAm-co-AA)/carrageenan, the optimum pH was found to be 7.5 and 8, respectively. The optimum temperature of the free and immobilized enzymes was also observed to be 55 and 60 °C, respectively. Michaelis–Menten constant (K _m) values for both immobilized urease were also observed smaller than free enzyme. The storage stability values of immobilized enzyme systems were observed as 48 and 70%, respectively, after 70 days. In addition to this, it was observed that, after 20th use in 5 days, the retained activities for immobilized enzyme into C-A PEC and P(AAm-co-AA)/carrageenan matrixes were found as 55 and 89%, respectively. Thermal stability of the free urease was also increased by a result of immobilization.     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying β-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.