Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Continuous production of lacticin 3147 and nisin using cells immobilized in calcium alginate

Bacteriocinogenic strains, Lactococcus lactis subsp. lactis DPC 3147 and L. lactis DPC 496, producing lacticin 3147 and nisin, respectively, were immobilized in double-layered calcium alginate beads. These beads were inoculated into MRS broth at a ratio of 1:4 and continuously fermented for 180 h. Free cells were used to compare the effect of immobilization on bacteriocin production. After equilibrium was reached, a flow rate of 580 ml h(-1) was used in the immobilized cell (IC), and 240 ml h(-1) in free-cell (FC) bioreactors. Outgrowth from beads was observed after 18 h. Bacteriocin production peaked at 5120 AU ml(-1) in both IC and FC bioreactors. However, FC production declined after 80 h to 160 AU ml(-1) at the end of the fermentation. Results of this study indicate that immobilization offers the possibility of a more stable and long-term means of producing lacticin 3147 in laboratory media than with free cells.     

Decolorization of a kraft mill effluent with fungal mycelium immobilized in calcium alginate gel

A white-rot fungus Coriolus versicolor was immobilized by entrapment in calcium alginate beads. Treatment of a kraft mill effluent with the immobilized fungus in the presence of sucrose resulted in 80% loss of color of the effluent within 3 days. The minimal concentration of sucrose required for the decolorization was 10 mM. Other carbon sources (xylose, glucose, glycerol, and ethanol) could also be used.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

The production of ethanol by Saccharomyces cerevisiae immobilized in polycation-stabilized calcium alginate gels

Summary Polycation treatment of preformed calcium alginate beads produced a matrix with higher resistance to phosphate ions. The treatment of immobilized Saccharomyces cerevisiae in the calcium alginate beads inhibited respiration of the entrapped cells but did not reduce ethanol production.     

Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y_x/s of 0.295 g of cells/g sucrose and a specific growth rate (μ) of 0.192 h⁻¹. The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 °C an isomaltulose yield of 89–94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 °C ranged from 1.6 to 4.0 g isomaltulose g⁻¹ pellet h⁻¹, respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.     

Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Characterization of nitrifying bacteria immobilized in calcium alginate

Activated sludge has been fed with a medium containing ammonium ions as the sole nitrogen source. Biomass collected from this continuous culture was immobilized in calcium alginate. The influence of pH, temperature, and the size and cell load of the biocatalyst beads on the nitrifying activity was determined, as well as the storage and operational stability of the system. The results are compared with those obtained with Nitrosomonas europaea. It has been concluded that the mixed culture is more difficult to work with than the pure strain and that the reproducibility of the results is lower. The trends found, however, were largely similar, except for the operational stability which was poorer in the case of the immobilized mixed culture.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii

Botryococcus braunii is a green colonial fresh water microalga and it is recognized as one of the renewable resources for production of liquid hydrocarbons. CFTRI-Bb-1 and CFTRI-Bb-2 have been reported for the first time and their performance with regard to growth and biochemical profile is presented here. The present study focused on effect of carbon dioxide (CO2) on biomass, hydrocarbon, carbohydrate production, fatty acid profile, and carotenoid content in various species of B. braunii (LB-572, SAG 30.81, MCRC-Bb, N-836, CFTRI-Bb-1, and CFTRI-Bb-2) at 0.5, 1.0, and 2.0% (v/v) levels using a two-tier flask. CO2 at 2.0% (v/v) level enhanced growth of the organism, and a two-fold increase in biomass and carotenoid contents was observed in all the B. braunii strains studied compared with control culture (without CO2 supplementation). At 1% and 2% (v/v) CO2 concentrations, palmitic acid and oleic acid levels increased by 2.5 to 3 folds in one of the strains of B. braunii (LB-572). Hydrocarbon content was found to be above 20% at 2% CO2 level in the B. braunii LB-572, CFTRI-Bb-2, CFTRI-Bb-1, and N-836 strains, whereas it was less than 20% in the SAG 30.81 and MCRC-Bb strains compared with control culture. This culture methodology will provide information on CO2 requirement for growth of algae and metabolite production. B. braunii spp. can be grown at the tested levels of CO2 concentration without much influence on culture pH.     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

High frequency plant regeneration of garlic (Allium sativum L.) calli immobilized in calcium alginate gel

Calli obtained from a shoot-tip of garlic,Allium sativum L., were encapsulated using a calcium alginate gel. Some of the encapsulated calli were cultured on a 1/2 MS medium supplemented with 3% sucrose, 10⁻⁵ M kinetin, and 5×10⁻⁶ M NAA, whereas the remainder was stored for 40 days at 4°C. All the naked calli regenerated on the solid medium, while 95% of the encapsulated calli regenerated, and 88% of the encapsulated calli regenerated after 40 days of storage at 4°C. The capsule matrix delayed the germination time of the encapsulated calli, yet activated the shoot formation of the artificial garlic seeds. The shoot length of the encapsulated garlic calli was much longer than that of the naked garlic calli. The encapsulated garlic calli were dried in a laminar airflow cabinet and the conversion frequency of the dried artificial garlic seeds on a 1/2 MS medium remained at 93% with a water loss of less than 50%.     

Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel

Production of β-cyclodextrin (β-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on silica–titania (SiO₂/TiO₂) and silica–manganese dioxide (SiO₂/MnO₂) matrices, was optimized for temperature, substrate concentration and initial biomass. The immobilization process was most efficient at 60 °C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a β-CD production of 11.7 ± 0.1 mM for cells immobilized on SiO₂/TiO₂ and 11.2 ± 0.1 mM in SiO₂/MnO₂. Entrapment in alginate gel resulted in a maximum β-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. β-CD production by cells immobilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this same assay, cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, showed the best β-CD production results at the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. A study of the immobilization of beta-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. A retention of 96.5% of initial activity after ten sequential batch uses of the immobilized preparation was observed. The pH and temperature optima for free and immobilized cells were the same, i.e., 6.5 (0.05M Maleate buffer) and 50 degrees C, respectively. Even though the temperature optimum was found to be 50 degrees C, the enzyme exhibits a better thermal stability at 45 degrees C. Beads stored at 4 degrees C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells shown that glucose is a noncompetitive product inhibitor.The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45 degrees C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seventeen days at a temperature of 40 degrees C and decreasing to less than two days at 50 degrees C.