Effect of serum concentration on retroviral vector production from the amphotropic ψCRIP murine producer cells

The production of ethanol by Saccharomyces cerevisiae immobilized cells and its esterification with oleic acid, catalysed by a lipase from Rhizomucor miehei, was the biochemical process considered as model to illustrate the concept of extractive biocatalysis. The selection of the most suitable support for lipase immobilization was carried out. The best results for the ethanol/oleic acid esterification reaction were obtained with the lipase adsorbed on a polyamide type support, Accurel EP 700. The immobilization method was optimized in terms of immobilization pH, contact time and protein/support ratio. The better performances of the extractive fermentations of ethanol were obtained when entrapped k-carrageenan Saccharomyces cerevisiae cells and a lipase from Rhizomucor miehei, free or immobilized in Accurel EP 700, were used simultaneously. The observed reutilization capacity of the immobilized enzyme could be advantageous for its application in a continuous reactor.     

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.     

Dehydrogenation of cortisol by Arthrobacter simplex immobilized as supported monolayer

Arthrobacter simplex has been successfully immobilized by adhesion on glass, either by coating the support with colloidal particles of hydrous alumina or by pretreating the cells with aluminium ions. The use of glass slides as a model support has shown that a single, dense and regular layer of immobilized cells is achieved. The quantity of immobilized cells is about 7 × 10⁷ cells cm^?2. Immobilization on glass beads or glass wool packed as a bed in a column has also been successful. Transformation of cortisol to prednisolone has been tested under no-growth conditions in the absence of nutrients. The specific activity of immobilized cells is not significantly different from that of free cells. The use of a microreactor with the immobilized bacteria as biocatalyst demonstrated the feasibility of repeated use of the microorganisms.     

Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe₃O₄ nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g⁻¹ saturation magnetization. When the mixture of gellan gel and the Fe₃O₄ nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe₃O₄ nanoparticles was 9 mg ml⁻¹ and the saturation magnetization of magnetically immobilized cells was 11.08 emu g⁻¹. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

A simple method of cellulase immobilization on a modified silica support

Two types of cellulase (Robillarda sp. Y-20 and Trichoderma reesei) were immobilized on Aminosilica-1 by physical adsorption. Enzymes were quickly immobilized and stable on the support. Specific activities of two types of immobilized cellulase were 0.24 U/mg support with Robillarda cellulase and 0.15 U/mg support with the Trichoderma one as CMCase. The pH-activity curves of both cellulase shifted slightly to lower pH on immobilization. Both immobilized cellulases showed essentially the same pH stabilities as their free forms. However, the immobilized enzymes were less stable than the free forms at temperatures higher than 50°C.     

Immobilization of yeast cells by entrapment and adhesion using siliceous materials

Yeast cells (Saccharomyces cerevisiae) have been immobilized by entrapment in silica hydrogel, without significantly changing their biological activity; a simple model describes the rate of oxygen uptake by a film of immobilized cells. The cells have also been immobilized by direct adhesion to a glass surface; this is achieved by a well-controlled drying procedure, sufficient to bring the cells into close contact with the support, but without cell dehydration. The immobilized cells consume glucose at a rate which is about half of the rate obtained in suspension and they are resistant to strong mechanical strains.     

Transformation of cortisol to prednisolone by viable cells of Arthrobacter simplex covalently immobilized on cellulose granules

Arthrobacter simplex cells have been covalently immobilized to granules of microcrystallized regenerated cellulose by means of N-hydroxymethyl, N′-glucosylurea groups at pH 8.5, 18°C and cell suspension concentration of 60 mg/ml. The immobilization yield was found to exceed 100%. The maximum initial rate of Cortisol transformation to Prednisolone remained almost constant after 20-fold transformation in a nutrient medium containing 0.5% peptone at pH 8.0, 32°C and aeration with oxygen. The effect of the substrate concentration on the activity of the immobilized cells, as well as of the ratio between substrate and immobilized cells on the degree of transformation, was investigated. The immobilized cells were characterized by means of electronmicroscopic studies. Microbiological observations have shown that immobilized cells can proliferate and the free cells obtained are accumulated in the nutrient medium. The immobilized cells preserve their viability for a long time when they are stored at 4°C.     

Continuous interesterification of oils and fats using dried fungus immobilized in biomass support particles

The industrial feasibility of an interesterification process using acetone-dried fungus (as a lipase catalyst) immobilized in biomass support particles (BSPs) was examined by continuous interesterification between olive oil and methyl stearate, where the water content of the reaction mixture (C_w) was controlled at a given value. The C_w affected not only the inactivation rate of lipase in the cells but also the production rate of the by-product (diglycerides). The optimal C_w was determined as about 100 ppm. The half-life of lipase in the cells was about 1200 h at the optimal C_w, suggesting that the interesterification process using the immobilized fungus is industrially feasible.     

Immobilization of Paracoccus denitrificans cells with polyelectrolyte complex and denitrifying activity of the immobilized cells

Whole cells from Paracoccus denitrificans IFO 12442 were immobilized with a polyelectrolyte complex composed of potassium poly(vinyl alcohol) sulfate (KPVS) and poly(diallyldimethylammonium chloride) (PDDA) by the following procedures: An excess of PDDA was first mixed with a cell suspension to aggregate cells, then KPVS was added to form a complex with excess PDDA and to entrap the aggregated cells. Electron microscopic analysis showed that the aggregated cells were entrapped or surrounded by an amorphous complex support. The rate of nitrate reduction or carbon consumption by the immobilized cells was almost the same as that by the free cells, as determined by anaerobic incubation using a non-growth medium containing KNO₃ as a substrate and potassium aspartate as a carbon source. The immobilized cells exhibited activity at pH 4, at which the free cells lost their activity. The initial activity of the immobilized cells remained stable for at least one month in a phosphate buffer with gentle stirring.     

Operational stability of immobilized d-glucose isomerase in a continuous feed stirred tank reactor

d-Glucose isomerization has been studied using immobilized cells of Streptomyces phaeochromogenes in a continuous feed stirred tank reactor (CSTR) where the external film diffusion resistance was negligible. Experiments conducted with various sizes of enzyme particles indicated that a strong internal diffusion resistance improved the apparent stability of these particles. The performance equations of the CSTR were constructed by associating the material balances for the inside porous support matrix with the bulk liquid phase, and enzyme deactivation was also taken into consideration. An iterative method together with the orthogonal collocation method is proposed for the evaluation of effectiveness factor and the substrate concentration profile within the enzyme particles. The numerical results offer an alternative analytical proof for the observation that under strong internal diffusion control the apparent operational stability of immobilized enzyme is improved.     

Continuous production of 4-ethylguaiacol by immobilized cells of salt-tolerant Candida versatilis in an airlift reactor

The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption–desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h^?1 and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis–Menten constant (K _m) of 49.10 mM and a maximum reaction velocity (V _max) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Nitrate removal by immobilized cells of Phormidium uncinatum in batch culture and a continuous-flow photobioreactor

Cells of the non-N₂-fixing cyanobacterium Phormidium uncinatum were immobilized by adsorption into polyvinyl (PV-50) foam pieces. The effect of inoculum size as well as the initial inoculum/support ratio on the cell immobilization process was investigated. After 2 months of immobilization similar net O₂-exchange activity was measured in immobilized and free-living cells. Polyvinyl-adsorbed cells also showed similar nitrate uptake capacity to free-living cells. Nitrogen starvation promoted a remarkable increase in nitrate uptake rate of both free-living and immobilized cells. A lab-scale photobioreactor packed with polyvinyl foam pieces colonized in situ by cells was used for nitrate removal in a continuous mode. In the best working conditions found, nearly 90% of nitrate supplied in the influent (50 mg l^–1) was removed by cells having a residence time of 3–4 h. Correspondence to: J. L. Serra     

Determination of free sulphite in wine using a microbial sensor

Summary A microbial sensor of immobilized Thiobacillus thiooxidans S3 cells was assembled to determine free sulphite in wine. Sulphite oxidation activity of the immobilized cells was sufficiently high for use even after 3 months storage at 4° C. The sensitivity of this sensor was 116 nA·1·mg^-1 for sulphur dioxide. The relationship between the current decrease and the sulphur dioxide concentration was linear up to 17 mg·1^-1. The sampling rate achieved was 10 min per sample including washing time. This sensor method needed no pretreatment of wine samples, and wines diluted with 5 mM sulphuric acid solution could be directly introduced in the computer-aided analysis system. The pigments in red wine did not disturbed the analysis.     

Functional Display of a Heterologous Protein on the Surface of Lactococcus lactis by Means of the Cell Wall Anchor of Staphylococcus aureus Protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

Pigment Production from Immobilized Monascus sp. Utilizing Polymeric Resin Adsorption

Pigment production by the fungus Monascus sp. was studied to determine why Monascus sp. provides more pigment in solid culture than in submerged culture. Adding a sterilized nonionic polymeric adsorbent resin directly to the growing submerged culture did not enhance the pigment production, thus indicating that pigment extraction is probably not a factor. Monascus cells immobilized in hydrogel were studied and exhibited decreased pigment production as a result of immobilization. This result is thought to be due to diffusional resistance of the pigment through the hydrogel beads. Addition of the adsorbent resin to the immobilized Monascus culture increased both the maximum pigment yield and the production rate above those of the free-cell fermentations. The provision of a support for the mycelium may explain enhanced pigment production by the solid-state culture. These results indicate that product diffusion from immobilized cell systems can be the limiting factor and that in situ extraction is one possible way to circumvent this problem.     

Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil

The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g^?1). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.