Entrapment of Lavandula vera cells with synthetic resin prepolymers and its application to pigment production

Summary Cultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.Dedicated to Professor Dr. Georg Manecke on occasion of his 70th birthday     

Continuous production of oxytetracycline by immobilized growing Streptomyces rimosus cells

Summary Streptomyces rimosus cells were immobilized with urethane prepolymers and used in the production of oxytetracycline. Based on the criteria for oxytetracycline productivity, cell growth in gels, cell leakage from gels and mechanical strength of gel, a hydrophilic prepolymer, PU-1, the main chain of which was polyethylene glycol (molecular weight, approximately 1500) was employed as gel material among 11 kinds of urethane prepolymers. Use of glucose-free medium for cultivation of PU-1-entrapped cells increased the production rate of oxytetracycline and minimized cell leakage from the gels. When the gel-entrapped cells lost activity, treatment of the cell-entrapping gels with saline or 70% ethanol resulted in recovery of the oxytetracycline productivity. Continuous oxytetracycline fermentation using PU-1-entrapped growing cells was successfully achieved in air-bubbled reactor for at least 35 days with reactivation of the cells.     

Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3 cells immobilised in silica-coated magnetic alginate beads

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe₃O₄) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe₃O₄ nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)₂, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca²⁺ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.     

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.     

Production of indole alkaloids by gel-entrapped cells of Catharanthus roseus in a continuous flow reactor

Summary Calcium alginate gel-entrapped cells ofCatharanthus roseus were used to study the production of indole alkaloids in a flow through process. The bioreactor was functional for more than two months and product recovery was analyzed under various operating conditions.     

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.     

Immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase by a combination of heat treatment in the presence of minerals and entrapment in calcium alginate gels

A method of immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase was devised, based on techniques of heat fixation in the presence of minerals and, entrapment in calcium alginate gels. The optimum activity of the enzyme was obtained when the cells were heat-fixed at 60°C for 10 min in the presence of 50 mmol/L MgSO₄·7H₂O and 5 mmol/L CoCl₂·6H₂O and then cast into calcium alginate beads using 2% sodium alginate.     

Redirecting cellular metabolism by immobilization of cultured plant cells: A model study with Coffea arabica

Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible.     

An anomalous behavior of trypsin immobilized in alginate network

Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N _α-benzoyl-l-arginine-4-nitroanilide hydrochloride (l-BAPNA), and in the presence or absence of d-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of d-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with d-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus l-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.     

Effects of immobilization and environmental stress on growth and production of non-polar metabolites of Tagetes minuta cells

Tagetes minuta (marigold) cells were entrapped under sterile conditions in agarose, κ-carrageenan, agar and alginate. The effects of different supports on the growth rate of the entrapped cells during incubation for one week under standard conditions [I] were studied. In the second part (weeks 2 and 3) of the experiment the effects of low temperature (10°C) [II], intermittent N₂ gassing [III], and omission of carbohydrates from the medium [IV]—superimposed on that of entrapment—on growth rate and the production of non-polar secondary metabolites were investigated. Compared to free cells, the impact of agarose on growth during the first week was nil, while the inhibition of growth increased in the order κ-carrageenan, agar and alginate, probably as a result of increasing rigidity of the support. In the second period the plant cells clearly had reached the stationary phase of the growth cycle in all cases. Again the pattern of growth on agarose closely followed that of free cells, i.e. a small increase in cases I and III, and a small decrease under the other two conditions. Low temperature [II] had the greatest effect on cell growth and cell release, probably as a result of gel structure at this temperature. Similarly to the effects on growth, the impact on secondary metabolite production was most pronounced in the case of alginate combined with low temperature. Both the omission of carbohydrates, and Nin2 gassing resulted in low concentrations of non-polar compounds in the media. The major trend observed was a shift away from mainly intracellular compounds in the case of free cells to mainly extracellular compounds in the case of entrapped cells at 10°C.     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

Coenzyme Q<Subscript>10</Subscript> production directly from precursors by free and gel-entrapped <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. ZUTE03 in a water-organic solvent,two-phase conversion system

In a water-organic solvent, two-phase conversion system, CoQ₁₀ could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ₁₀ concentration in the organic solvent phase was significantly higher than that in the cell. CoQ₁₀ yield reached a maximal value of 60.8 mg l⁻¹ in the organic phase and 40.6 mg g⁻¹-DCW after 8 h. CoQ₁₀ also could be produced by gel-entrapped cells in the two-phase conversion system. Soybean oil and hexane were found to be key substances for CoQ₁₀ production by gel-entrapped cells of Sphingomonas sp. ZUTE03. Soybean oil might improve the release of CoQ10 from the gel-entrapped cells while hexane was the suitable solvent to extract CoQ₁₀ from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ₁₀ by a repeated-batch culture. After 15 repeats, the yield of CoQ₁₀ kept at a high level of more than 40 mg l⁻¹. After 8 h conversion under optimized precursor’s concentration, CoQ₁₀ yield of gel-trapped cells reached 52.2 mg l⁻¹ with a molar conversion rate of 91% and 89.6% (on PHB and solanesol, respectively). This is the first report on enhanced production of CoQ₁₀ in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.     

Photosynthetic production of fuels and chemicals in immobilized systems

Whole cells of algae, cyanobacteria and photosynthetic bacteria entrapped in alginate gels or polyurethane foams can retain their photo-synthetic activities for months and in some cases for years. Such immobilized cells can be used in bioreactors for the production of H₂, NH₃, NADPH₂, carbohydrates, hydrocarbons, etc., with sunlight as the energy source.     

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23°C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO₃ was added to the growth media. There were strong interactions between CaCO₃ and media, CaCO₃ and pH level, and CaCO₃, media, and pH level. In media with CaCO₃, all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO₃ increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 × 10¹⁰/g). In CaCO₃-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.     

Transformation of steroids by gel-entrapped Nocardia rhodocrous cells in organic solvent

Summary Whole cells of Nocardia rhodocrous were immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers of either hydrophilic or hydrophobic character, and their activity in converting 3- hydroxy-⁵ to corresponding 3-keto-⁴ in an appropriate organic solvent system (e.g., a water-saturated mixture of benzene and n-heptane (11 by volume)) was studied. Although both hydrophilic gel-entrapped cells and hydrophobic gel-entrapped cells could transform dehydroepiandrosterone to 4-androstene-3, 17-dione, the latter showed a significantly higher activity than the former. In the cases of the oxidation of cholesterol, -sitosterol and stigmasterol to the corresponding 3-keto-⁴-steroids, only hydrophobic gel-entrapped cells exhibited the catalytic activity.The cells entrapped in adequate hydrophobic gels had steroid transforming activity comparable to that of the free counterparts. The operational stability was improved by the entrapment, especially in the case of the transformation of dehydroepiandrosterone. The activity of the gel-entrapped cells was found to correspond closely to the partition coefficients of substrates between the gels and the external solvent.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

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.     

Use of immobilized cells of Rhizopus nigricans for the 11α-hydroxylation of progesterone

The filamentous fungus, Rhizopus nigricans, was immobilized in polyacrylamide, alginate, and agar gels and its ability to 11α-hydroxylate progesterone was examined. No activity was detected using polyacrylamide gel but both agar and alginate gels have proved capable of hydroxylation. Agar gels displayed faster rates and higher yields. It was possible to induce hydroxylase synthesis within agar and alginate gels, and microscopical examination provided evidence for hyphal growth within these gels. The concept of increased biomass was used to explain the observed increase in the rates of hydroxylase activity of the immobilized cells. Conversely, hyphal overcrowding was postulated for the rapid inactivation observed under some operating conditions.     

Bioconversion of lipophilic compounds by immobilized microbial cells in organic solvents

Microbial cells were gel-entrapped with photo-crosslinkable resin prepolymers or urethane prepolymers, respectively. The resulting gels have different tailor-made hydrophobic or hydrophilic character. They were used for successful bioconversion of hydrophobic steroids and terpenoids in watersaturated mixtures of organic solvents. The experiments show the influence of the hydrophobicity of the gels and the polarity of the solvent mixtures, respectively. Use of hydrophobic gels and less polar solvents is preferable for bioconversion of hydrophobic compounds. The selective formation of a desired product among diverse products from a single substrate by appropriate use of hydrophobic or hydrophilic gels is possible. In each case, tests should be made to select the appropriate gel and solvent mixture. Bioconversions tested are: dehydroepiandrosterone to 4-androstene-3,17-dione; cholesterol to cholestenone; β-sitosterol to β-sitostenone; stigmasterol to stigmastenone; pregnenolone to progesterone; testosterone to Δ¹-dehydrotestosterone or 4-androstene-3,17-dione, respectively; all with immobilized cells of Nocardia rhodocrous; and stereoselective hydrolysis of dl-menthyl-succinate to yield l-menthol with immobilized cells of Rhodotorula minuta var. texensis.