The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels.

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was deomonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.     

Inulase-secreting strain of Saccharomyces cerevisiae produces fructose

The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid.     

Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/ L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary-stirred-tank (CST) bioreactor using cells of K. marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66 h(-1) for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g/biomass/h and 1.21 g sugars/g biomass/h, respectively.     

Covalent stabilization of alginate gel for the entrapment of living whole cells

Summary Three methods were developed for preparing alginate gels containing cells that are stable in phosphate containing media. In Method I preformed alginate beads containing entrapped cells were treated with polyethyl eneimine followed by glutaraldehyde. In Method II alginate sol was treated with a carbodiimide and N-hydroxysuccinimide (to form active esters), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. In Method III alginate so] was treated with periodate (to form aldehyde groups), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. Saccharomyces cerevisiae cells, immobilized in such stabilized gels, exhibited almost the same fermentation activity as the standard preparation. The viability of the immobilized cells was retained during the stabilization procedure as judged from their ability to multiply in the presence of nutrients.The preparations remained stable in phosphate buffer for at least ten days without substantial release of cells. The extent of cross-linking was controlled by varying the time and the concentration of reactants, thus giving preparations ranging from beads with a thin stabilized shell to beads homogeneously stabilized.     

A new immobilization technique of whole cells and enzymes with colloidal silica and alginate

A mixed gel composed of colloidal silica and alginate (As gel) was prepared for the immobilization of enzymes or microorganisms. The physical strength of AS gel increased with the amount of colloidal silica. The ethanol production rate of Saccharomyces cerevisiae (IFO 0224) immobilized in AS gel was higher than in alginate gel (Al gel) in the early phase of growth. At a concentration of glucose of more than 10%, the ethanol production of immobilized yeast in AS gel was higher than in Al gel. Any difference was not recognized in the diffusion coefficient of glucose between AS and Al gels. The AS gel had an ability to retain proteins such as bovine serum albumin and gamma-globulin. The alkaline protease and beta-galactosidase in AS gel continued their function for a long time, but those immobilized in Al gel did not. Immobilized beta-galactosidase in AS gel had a higher thermal stability than in Al gel or free enzymes.     

Ethanol production at 45°C using preparations of Kluyveromyces marxianus IMB3 immobilized in calcium alginate and kissiris

The thermotolerant yeast, K. marxianus IMB3, was grown in free and immobilized states in batch-fed culture at 45°C and ethanol production was examined over a 61-day period. The organism was grown in the free state, in the free state with mineral kissiris, immobilized in calcium alginate and immobilized in calcium alginate together with kissiris. Initially, reactors were fed every two days with 10% (w/v) glucose-containing media and no significant difference in ethanol production was observed. In subsequent refeeding experiments, reactors were re-fed every two days with 15% (w/v) sucrose-containing media. Although overall ethanol concentrations decreased, production in the immobilized systems was higher. In the final stages fermentations were re-fed every 3 days and although overall ethanol production decreased further, production remained highest in the systems containing calcium alginate and kissiris.     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Viability, ultrastructure and cytokinin metabolism of free and immobilized tobacco chloroplasts

Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix.     

Alginate/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology

Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the enzymes’ diffusion and reached a plateau after 40 h for laccase and 70 h for glucose oxidase. Two-thirds of both enzymes was irreversibly retained inside the alginate beads. This proportion increased to 80% for laccase in combined alginate/carbon beads. Half-life of the adsorbed enzyme was enhanced to 74 days for laccase in carbon/alginate beads and 45 days for glucose oxidase in pure alginate as compared to 38 days and 23 days for free enzymes, respectively.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules

As a means of integrating cell growth and immobilization, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density. S. cerevisiae cells of SEY 2102 (MAT alpha ura3-52 leu2-3, 112 his4-519) harboring plasmid pRB58 with the SUC2 gene coding for invertase were grown to 83 g/L of liquid-core volume inside the capsule on a dry weight basis. The cloned invertase was expressed well in the immobilized cells with slightly higher activity than the free cells in a batch culture. Invertase in the immobilized cells showed slightly more improved thermal stability than in the free cells. Storage in a Na-acetate buffer at 4 degrees C and 10 degrees C for 1 month resulted in 7% and 8% loss in activity, respectively. The sucrose hydrolysis reaction was stably maintained for 25 repeated batches for 7 days at 30 degrees C. Continuous hydrolysis of 0.3 M sucrose was carried out in a packed bed reactor with a conversion of more than 90% at a maximum productivity of 55.5 g glucose/L per hour for 7 days. In a continuous stirred tank reactor, the maximum productivity of 80.8 g glucose/L per hour was achieved at a conversion of 59.1% using 1.0 M sucrose solution, and 0.5 M sucrose solution was hydrolyzed for 1 week with a 95% conversion at a productivity of 48.8 g/L per hour. (c) 1996 John Wiley & Sons, Inc.     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

New immobilization method of mammalian cells using alginate and polyacrylate

Mouse-mouse hybridoma cells were immobilized in polyacrylate-alginate gels. The immobilized hybridoma cells were cultured semi-continuously using a fluidized bed reactor, and allowed continuous antibody production without any gel destruction for one month. It has been proved that the polyacrylate-alginate gels were tolerant against physical stress. The composition of the gels suitable for cell growth and antibody production was given as follows; viscosity of alginate at 1% solution: 60–100 cP, alginate concentration: 0.8%, and polyacrylate concentration: 0.2%. In the semi-continuous culture using gels prepared under suitable conditions, the viable cell number was estimated as 2.5×10⁷ cells/ml-gel, and the antibody production rate was 2.2 mg/ml-gel/d, at maximum.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

Comparison between immobilized Kluyveromyces fragilis and Saccharomyces cerevisiae coimmobilized with beta-galactosidase, with respect to continuous ethanol production from concentrated whey permeate

Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with beta-galactosidase, for continuous ethanol production from whey permeate in packed-bed-type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15, and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than K. fragilis. The study went on for more than 5 weeks. K. fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stableduring the entrire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose-adapted immobilized S. cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/L ethanol was produced with a productivity of 2.5 g/L h. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/L with a productivity of 4.5 g/L h, whereas immobolized K. fragilis only produced 13 g/L ethanol with a productivity of 1.1 g/L h. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.