Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

Production of l-glutamate by immobilized protoplasts

Summary Protoplasts of Brevibacterium flavum cultured in a medium containing 50 g·l^-1 of biotin were prepared with lysozyme and immobilized in matrices of agar-acetylcellulose filters. The immobilized protoplasts were applied to l-glutamate production from glucose and urea in a batch system. The productivity of l-glutamate by the immobilized protoplasts was 2.5 times higher than that by immobilized whole cells under optimal conditions. Maximal productivity initially reached 1.5 mg·ml^-1. The immobilized protoplasts of B. flavum could be used six times for l-glutamate production with retention of about 70% of the initial productivity.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

Production of cell wall accumulative enzymes using immobilized protoplasts of Catharanthus roseus in agarose gel

Catharanthus roseus cells and protoplasts were used for production of peroxidase and -galactosidase which are accumulated in the cell wall. Only 4% (0.026 U ml^–1) of the total peroxidase was secreted into the broth by cultured cells while in cultured protoplasts, 45% (0.12 U ml^–1) was secreted. Protoplasts were protected against the physical and osmotic stresses by immobilizing them in 3% agarose gel (high mass transfer, non-electric charge, low gelation temperature). In order to increase peroxidase production, the immobilized protoplasts were cultivated in shake cultures at low osmotic pressure (12.3 to 16.4 atm) without disruption. During batch peroxidase production, the total activities obtained with free cells at 4.9 atm, free protoplasts at 19.3 atm, and immobilized protoplasts at 12.3 atm were 0.17, 2.54, and 5.16 U, respectively. When four repeated-batch cultures of the immobilized protoplasts were performed at 16.4 atm, 11.8 U of peroxidase was obtained. This system was also useful for the production of -galactosidase.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Wastewater nutrient removal with microalgae immobilized in carrageenan

Two species of the green freshwater microalga Scenedesmus have been immobilized in beads of κ-carrageenan. Growth curves were similar to those observed with free cells. The uptake of nitrogen and phosphorus from wastewater was similar for free and entrapped microalgae. The reduction of N-NH⁺₄ was 90% within 4 h at pH 9.0 and 70% within 5 h at an adjusted pH of 7.7. From these experiments, we conclude that immobilized Scenedesmus microalgae can grow well within gel beads and that they are as physiologically active for wastewater nutrient removal as free cells.     

Ethylene production by apple protoplasts

Freshly prepared protoplasts from apple tissue that produced ethylene were obtained. Ethylene production was inhibited by osmotic shock, 0.01% Triton X-100, and aminoethoxyvinyl glycine. Protoplasts as well as the ethylene system were not greatly affected by protease treatment.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Comparison of the aspartase activity and its stability in Escherichia coli cells immobilized on polyacrylamide gel and carrageenan

The conditions for immobilization of Escherichia coli cells (Soviet strain 85) on the natural polysaccharide carrier carrageenan (Soviet-made) were investigated and kinetic regularities of the aspartase reaction catalysed by immobilized in carrageenan cells of E. coli 85 were established. The conditions for retaining a high aspartase activity and stability of biocatalysts based on the E. coli 85 cells immobilized in PAAG and carrageenan were determined using full-loaded tanks for continuous synthesis of L-aspartic acid. The time-stable aspartase activity of the biocatalyst can be increased by treating the beads of the catalyst with bifunctional reagents (hexamethylenediamine, glutaraldehyde), the most active catalyst for the biotechnological synthesis of L-aspartic acid being obtained when carrageenan is used.     

Palatinose production by free and Ca-alginate gel immobilized cells of Erwinia sp.

Palatinose is a non-cariogenic disaccharide obtained from the enzymatic conversion of sucrose, used in food industries as a sugar substitute. Free and Ca-alginate immobilized cells of Erwinia sp. D12 were used to produce palatinose from sucrose. Palatinose production was studied in a repeated-batch process using different immobilized biocatalysts: whole cells, disrupted cells and glucosyltransferase. Successive batches were treated with the immobilized biocatalyst, but a decrease in palatinose production was observed. A continuous process using a packed-bed reactor was investigated, and found to produce 55–66% of palatinose during 17 days using immobilized cells treated with glutaraldehyde and a substrate flow speed of 0.56 ml min⁻¹. However, immobilized cells in a packed-bed reactor failed to maintain the palatinose production for a prolonged period. The free cells showed a high conversion rate using batch fermentation, obtaining a palatinose yield of 77%. The cells remained viable for 16 cycles with high palatinose yields (65–77%). Free Erwinia sp. D12 cells supported high production levels in repeated-batch operations, and the results showed the potential for repeated reuse.     

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.     

Diacetyl production by immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 in the fibrous Ca-alginate gel

Summary Diacetyl production by (Citr^*)Lactococcus lactis subsp.lactis 3022 was found to be an oxygen-dependent reaction. The diacetyl production by the cells immobilized in conventional Ca-alginate gel beads (Diameter: 3 mm) was lower than that of the cells immobilized in Ca-alginate gel fibers (Diameter: 0.2 mm), probably because oxygen transfer to the immobilized cells is better in gel fibers than in gel beads.     

Production of gibberellins and bikaverin by cells of Gibberella fujikuroi immobilized in carrageenan

The production of gibberellins and bikaverin by immobilized and free cells of Gibberella fujikuroi strains was followed. Both types of cells, free and immobilized, produced similar titers of the secondary metabolites during the normal growth cycle. The kinetics of nutrient use and product formation by the immobilized cells lagged behind that of the free cells and this was assumed to be the result of diffusional limitations imposed on the immobilized cells. A noticeable difference was that in the immobilized cells, all of the bikaverin was excreted into the medium for both strains of G. fujikuroi tested but in the free cell fermentation 44% was excreted for strain ACC 917 and only 10% for strain GF1a. Gibberellin and bikaverin could be produced in a semi-continuous fashion with both free and immobilized cells for a period of 16 d in a resuspension medium containing 0.12 mM or 0.60 mM ammonium chloride. No definite advantage, on a productivity basis, for using immobilized cells over free cells could be seen.     

Improving immobilized biocatalysts by gel phase polymerization

A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and beta-dimethylaminopropionitrile (or N,N,N',N'-tetramethyl-ethylenediamine) into the performed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months. During this operation, the gel beads maintained their rigidity, and the maximum productivity was as high as 50 g h(-1) L(-1) gel. There was no appreciable decay of cell activity.     

Benzene degradation by bacterial cells immobilized in polyacrylamide gel

Summary Whole cells ofPseudomonas putida were immobilized in polyacrylamide gel and their ability to utilise benzene was examined. On initial immobilization cells were found to lose 40–70% of their activity. This activity could be restored by incubation in a medium containing benzene and succinate. It was also found that partial activation could be achieved by incubation with iron salts, in the absence of a carbon source. Electron microscopy showed this activation to be accompanied by an increase in cell numbers, with the formation of cell conglomerates within gel interstices. However, under some conditions, prolonged elution with substrate resulted in cell disruption and loss of activity.     

Hydrogen production by Rhodopseudomonas capsulata cells entrapped in carrageenan beads

Summary The photosynthetic bacteria Rhodopseudomonas capsulata strain B10 were immobilized in agar or in carrageenan beads (Ø = 1–3 mm). Beads containing 5.8 mg cell dry weight/mL of gel produced hydrogen from lactate at a rate of 54 mL/h.g dry weight; the efficiency of H₂ production by immobilized cells was comparable to that of free cells and was 60 to 65% that of the theoretical maximum from lactate. Carrageenan-entrapped cells produced H₂ steadily over a 16-day period.     

Suberin production by isolated tomato fruit protoplasts

The multilamellar wall secreted by protoplasts isolated from locule tissue of tomato (Lycopersicon esculentum L.) fruit was purified, and an extract was obtained after depolymerization with BF₃-methanol. Analysis of this extract using thin layer chromatography demonstrated the presence of fatty acid methyl esters, fatty alcohols, dicarboxylic acid dimethyl esters, and ω-hydroxy acid methyl esters. These components were quantified using an Iatroscan thin layer chromatography-flame ionization detection system. The different chain lengths in each group were identified and quantified using gas chromatography. The results clearly indicated the presence of suberin.     

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.     

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.