The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977)

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 demonstrated 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.     

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.     

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.     

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.     

The effect of calcium alginate entrapment on the physiology of Mycobacterium sp. strain E3

The influence of calcium alginate entrapment on the physiology of Mycobacterium sp. E3 is reported. As a model system the NADH-requiring conversion of propene to 1,2-epoxypropane in the presence and absence of glucose as co-substrate was selected. The co-factor-dependent reaction was used as a measure of the physiological status of the resting cells. Initial kinetic experiments established a system free from diffusional limitations. In the presence of glucose there were no differences between the physiology of the free and immobilized cells. The apparent differences observed in the absence of co-substrate were demonstrated to be caused by calcium ions and to a lesser degree alginate; the addition of calcium, alginate or calcium alginate beads containing no cells to the free cells gave similar data to that obtained with immobilized cells. The results presented highlight the high concentrations of calcium to which cells immobilized in calcium alginate beads can be exposed. Correspondence to: M. R. Smith     

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.     

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.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

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.     

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.     

On the elimination of artefactual effects in assessing the structure of calcium alginate cell immobilization gels

The structure of calcium alginate cell immobilization gels has been examined by scanning and transmission electron microscopy. The presence of an alginate coating around the extrude alginate fibres reduced the loss of cells from the immobilized state provided that strict nutrient controls were observed. The constraining alginate coating may be retained for > 500 h under normal operational conditions. The alginate structure within the extruded fibres (4% w/v alginate, 10% w/v Saccharomyces cerevisiae) remained unchanged during the course of a 500 h continuous ethanol fermentation and revealed a pore size within the alginate matrix, usually less than 90 nm. Inappropriate electron microscopy preparation prior to examination may erroneously indicately the alginate matrix to be a macroporous structure. To reduce artefactual effects the alginate structure should only be revealed prior to critical point drying and not at an earlier stage in the preparation for electron microscopy.     

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.     

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.     

Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells

Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.Abbreviations IAA indole-3-acetic acid - LMT S-adenosylmethionine: licodione 2-O-methyltransferase - CHS chalcone synthase     

Performance of rotating packed disk reactor with immobilized glucose oxidase

Summary Reactor performance was studied to investigate whether a rotating packed disk reactor (RPDR) can be used for the enzymatic oxidation of biochemicals. The disks were packed with calcium alginate beads with immobilized glucose oxidase and catalase, which catalyze the reaction of glucose and oxygen. The production rate of gluconic acid increased with the speed of rotation and the bulk flow rate. An optimum submergence for maximum productivity existed.     

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.     

Optical anisotropy of calcium-induced alginate gels

Alginate gels formed by diffusion of calcium ions into solutions of sodium alginate were found to exhibit optical anisotropy depending on preparation conditions. When observed under crossed nicols, the anisotropic alginate gels showed a birefringence pattern which is characteristic of radial orientation of polymer chains. Calcium alginate gels were prepared from different concentrations of sodium alginate and calcium ion, and the conditions for formation of the anisotropic gels were determined. The gel-formation process was measured by monitoring the development of the birefringent layer and was compared with the model in which the diffusion of calcium ions dominates gel formation.     

Proteomic analysis of calcium alginate-immobilized Saccharomyces cerevisiae under high-gravity fermentation conditions

Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight) (-1) h (-1) for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability.