Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

An airlift loop reactor for the transformation of steroids by immobilized cells

Summary The flow behaviour of calcium alginate beads in an airlift reactor (ALR) with external loop was dependent on the airflow rate into and the amount of beads in the reactor. The performance of immobilizedArthrobacter simplex for the ¹-dehydrogenation of hydrocortisone in the ALR compared favourably to that in a stirred tank reactor. The physical stability of the calcium alginate beads was significantly greater in the ALR.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Production of ℓ-malic acid by Paecilomyces varioti

Summary Preliminary data for production of -malic acid from calcium acetate byPaecilomyces varioti is presented. Shake flask cultures with free cells and with cells immobilised in calcium alginate beads gave comparable results, acid concentrations of approximately 8 g/l being produced after 5 days from a medium containing 4% w/w of calcium acetate. A packed bed reactor, operated as an extended batch with product recycle, produced maximum acid concentrations of 32.6 g/l, equivalent to 73% of the maximum theoretical yield, after 3 days. Evidence obtained indicated that spores were more active than mycelia in the production of malic acid.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Immobilized Frankia spores remained viable on dry storage and on restoration to medium regenerated active colonies

Spores of Frankia strain ACN^1AG, immobilized in calcium alginate beads, germinated to produce colonies that increased in protein content and showed nitrogenase activity. Air dried immobilized spores remained viable for at least 15 days in dry condition, making the storage and transport of Frankia strains easy. This also opens the possibility of using beaded spores as inocula.     

Interactions between a genetically markedPseudomonas fluorescens strain and bacteriophage ΦR2f in soil: Effects of nutrients, alginate encapsulation, and the wheat rhizosphere

The introduction of bacteriophages could potentially be used as a control method to limit the population size of engineered bacteria that have been introduced into soil. Hence, the ability of a species-specific phage, R2f, to infect and lyse its host, a Pseudomonas fluorescens R2f transposon Tn5 derivative, in soil, was studied. Control experiments in liquid media revealed that productive lysis of host cells by phage R2f occurred when cells were freely suspended, whereas cells present in alginate beads resisted lysis. The presence of nutrients enhanced the degree of lysis as well as the production of phage progeny, both with the suspended cells and with cells escaped from the alginate beads. Experiments in which host cells and phage R2f were introduced into two soils of different texture revealed that host cells were primarily lysed in the presence of added nutrients, and phage reached highest titres in these nutrient-amended soils. Encapsulation of the host cells in alginate beads inhibited lysis by the phage in soil. Populations of free host cells introduced into soil that colonized the rhizosphere of wheat were not substantially lysed by phage R2f. However, P. fluorescens R2f populations colonizing the rhizosphere after introduction in alginate beads were reduced in size by a factor of 1,000. Cells migrating from the alginate beads towards the roots may have been in a state of enhanced metabolic activity, allowing for phage R2f infection and cell lysis. Correspondence to: J.D. van Elsas     

Growth and hydrocarbon production ofBotryococcus braunii immobilized in calcium alginate gel

Summary The colonial microalgaB. braunii, immobilized in calcium alginate beads, shows active photoautotrophic growth. Nevertheless, the rates of increase in cell number and, to a lesser extent, in biomass are substantially lower when compared to free cultures. Such features are related to steric contraints which occasion also the formation of large spherical colonies in the gel, showing an unsual mulberry organization. Some cracks due to the development of underlying colonies appear at the surface of the beads. Alga release remains low, however, during the cultures. EntrappedB. braunii retain the ability to produce extracellular hydrocarbons; the structure of the latter is not affected by immobilization but their relative abundances can undergo some variations. Entrapment leads to marked improvements in hydrocarbon production; decrease in growth rates is therefore associated, in alginate gel, with a still more pronounced diversion ofB. braunii metabolic activity towards hydrocarbon generation. It appears also that the improvements in hydrocarbon production, due to strain selection and to culture condition adjustment, obtained in free cultures, can be directly applied toB. braunii immobilized in alginate beads.     

Effect of gelation temperature and gel-dissolving solution on cell viability and recovery of two Pseudomonas putida strains co-immobilized within calcium alginate or κ-carrageenan gel beads

Summary Since a lethal effect of an increased temperature (42°C) on Pseudomonas putida strains PaW8 and PaW130 was demonstrated, strictly ionotropic gels such as calcium alginate or -carrageenan type X 0909 were used for cell co-immobilization, rather than a thermoionotropic -carrageenan gel. Among the variety of gel-dissolving solutions tested, only a 0.05M Na₂CO₃/0.02M citric acid solution was able to preserve around 100 % of the cell viability. A complete cell recovery was obtained from calcium alginate gel beads, while only 6 % of viable cells was recovered from the ionotropic -carrageenan gel.     

Alginate Inhibits Iron Absorption from Ferrous Gluconate in a Randomized Controlled Trial and Reduces Iron Uptake into Caco-2 Cells

Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003). Sub-group B (n = 9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009) and 35% (p = 0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p<0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01528644","term_id":"NCT01528644"}}NCT01528644     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

Fed-batch biotransformation of β-ionone by Aspergillus niger

Aspergillus niger IFO 8541 was found to be an efficient biocatalyst for the biotransformation of -ionone into hydroxy and oxo derivatives. The reaction had to be carried out with an inoculum made of about 4 × 10⁷ fresh spores/l and with a preliminary growth period giving at least 3 g/l biomass. The fungus developed in the form of pellets when cultivated as free mycelium; entrapment of the microorganism in calcium alginate beads was an efficient way to mimic this feature in an aerated, stirred bioreactor. The biotransformation was carried out using a fed-batch mode of operation involving sequential precursor addition. -Ionone stopped the fungal growth and was converted into metabolites only when the carbon source remained present in the medium; it was fully oxidized after sucrose exhaustion. These conditions allowed recovery of about 2.5 g/l aroma compounds after 230 h cultivation with a molar yield close to 100%.     

Production of alginate beads by emulsification/internal gelation. II. Physicochemistry

Alginate microspheres were produced by emulsification/internal gelation of alginate sol dispersed within vegetable oil. Gelification was initiated within the alginate sol by a reduction in pH (7.5 to 6.5), releasing calcium from an insoluble complex. Smooth, spherical beads with the narrowest size dispersion were obtained when using low-guluronic-acid and low-viscosity alginate and a carbonate complex as the calcium vector. A more finely dispersed form of the complexed calcium within the alginate sol promotes a more homogeneous gelification. Microsphere mean diameters ranging from 50 m to 1000 m were obtained with standard deviations ranging from 35% to 45% of the mean.     

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Summary Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 m long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is responsible for the bioconversion of citrate into flavour compounds (diacetyl and acetoin).     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

Influence of growth behaviour and physiology of alginate-entrapped microorganisms on the oxygen consumption

Summary The ability of alginate-entrapped microorganisms to supply oxygen was determined with regard to physiology and growth behavior of the cells. Oxygen diffusion through an alginate film containing different concentrations of Pseudomonas putida or Saccharomyces cerevisiae was measured. Oxygen diffusion decreased when the cell loading increased. Dependent on the physiological behavior of these organisms the course of the oxygen concentration under the gel film is quite different. In further experiments an Effectiveness-Factor of oxygen uptake of alginate beads with Saccharomyces cerevisiae or Aspergillus niger was determined in relation to the growth behavior of the organisms. The effectiveness factor is always higher when the biomass is concentrated in the outer region of the gel beads as if the microorganisms are distributed homogeneously in the alginate. Considering these results it is not possible to make a general statement on the ability of microorganisms in alginate to supply oxygen. The physiology and the growth behavior of the immobilized organisms have to be considered in any case.     

Enhanced β-lactam antibiotic production by coimmobilization of fungus and alga

Summary It was determined thatCephalosporium acremonium, an oxygenconsuming fungus, had a symbiotic relationship withChlorella pyrenoidosa, an oxygen-generating alga. The fungus and the alga were coimmobilized by entrapment in calcium alginate beads. The production rate of -lactam antibiotics was evaluated at various ratios of the fungus and the alga cells in the free and immobilized states. When the ratio of fungus to alga was one to eight in free mixed culture, the production rate of -lactam antibiotics was 240% of the rate in the presence of fungus alone in the free state. In coimmobilized cell system, the increased amount was 370% in comparison with immobilized fungus alone.     

Entrapment of biological control agents applied to entomopathogenic nematodes

Summary Hydrogels of alginate, phospho guar gum, carboxymethyl guar gum, k-carrageenan and cellulose sulphate, respectively were tested to find easily redissolvable gels. The entomopathogenic nematode, Heterorhabditis sp., was entrapped in calcium alginate beads, calcium alginate hollow spheres and foils made from different hydrogels. Emigration from calcium alginate beads after 7 days of storage was 100 % at room temperature and was lowered to 6 % at 6 °C, whereas no emigration from calcium alginate hollow spheres was found at either temperature. Highly concentrated polymer foils produced on gauze showed reduced emigration with a survival of 80 % after 24 h compared to foils produced on glass slides. Calcium alginate beads can be used for a controlled release of the nematode into the environment, while hollow spheres and foils are suitable for storage.Dedicated to Prof. Dr. F. Wagner on the occasion of his 65th birthday