A new method for immobilization of microbial cells by cross-linking

A method for immobilization of microbial cells was designed. The method uses generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifuctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for use in biocatalysis with high-molecular-weight substrates.     

Vegetable sponge: A new immobilization medium for plant cells

Summary Immobilization of Saccharum officinarum cells within the vegetable sponge of Luffa cylindrica is reported as a new medium for the cells of higher plants. The use of biostructure of the sponge offers an alternative matrix to the ones used presently for immobilization.     

A new method for immobilizing microbial cells by crosslinking

A method for immobilization of microbial cells was developed designed. The method uses based on generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifunctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for the use in biocatalysis with high-molecular-weight substrates of high molecular weight.     

Several novel methods for immobilization of enzymes, microbial cells and organelles

Two novel methods--"photo-crosslinkable resin prepolymer method" and "urethane prepolymer method"--have been developed in our laboratory. These methods have the following advantages : 1) Prepolymers of desired properties, such as optional chain length, hydrophilicity or hydrophobicity, and ionic character etc., can be used for entrapment of biocatalysts : (2) preparation of gel-entrapped biocatalysts can be easily achieved under very mild conditions. Photo-crosslinked gels are conveniently obtained by several minutes illumination with near-UV light, of a mixture of liquid prepolymers having photo-sensitive functional groups, an appropriate sensitizer and the solution or suspension of biocatalyst. Formation of polyurethane gels is completed by only mixing water-miscible urethane prepolymers with the aqueous solution or suspension of biocatalyst. The biocatalysts entrapped by these methods are useful for a variety of purposes.     

A new mechanochecmical method of enzyme immobilization

A new mechanochemical method for enzyme immobilization has been elaborated. The principle of this method consists of the following precepts. Partially hydrolyzed nylon fiber, the surface of which is known to be strewn with micro-cracks, is reversibly stretched (～25%) and placed into an enzyme solution. Then, in the same solution, the fiber is made to relax and is taken out. The fiber retains considerable enzymatic activity even after numerous thorough washings (in a similar procedure without fiber stretching, equivalent washing removed all the enzymatic activity from the fiber). Immobilization on the fiber proceeds due to trapping of enzyme molecules by the microcavities on the surface of the support. The catalytic activity of mechanochemically immobilized chymotrypsin and trypsin is commensurable with their activity on covalent immobilization on nylon (calculated per unit of the macrosurface). A wide range of commercial polymers may be made of use as supports in the mechanochemical method of immobilization.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

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.     

A new method for cell immobilization.

A new method for producing particles and membranes containing immobilized bacteria is presented. These immobilized bacteria display good stability over time making them well suited for use in a packed-bed reactor. Such a reactor is tested as a function of the different parameters of the system. The results are qualitatively similar to those obtained with purified enzyme reactors, but some discrepancies with the plug-flow model are noted. It is necessary to use a more sophisticated model in order to fit the experimental data.     

Cell immobilization for microbial production of 1,3-propanediol

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.     

A new method for immobilization of pectic enzymes

Summary Polygalacturonase and pectinesterase have been successfully immobilized on gamma alumina by activation of the support with glutaraldehyde at pH 3.0. The half life of the enzymes increased by four and two fold compared to the immobilization on gamma alumina without activation.     

Calculation of microbial growth efficiency from15N immobilization

Microbial growth rate was estimated by multiplying¹⁵N immobilization by an estimated microbial C:N ratio. This growth rate, in combination with measurements of respiration, was used to calculate growth efficiency. Growth rates and efficiencies were calculated for grassland and cultivated soils of three textures. Calculated efficiencies (Y_c), assuming a microbial C: N ratio of 7, ranged from 32 to 54. Cultivated soils tended to have higher Y_c values than did grassland soils. This calculation depends on several hard-to-verify assumptions, but yields numbers that should be of great interest in comparative studies.     

Hydroxyapatite-pulp composite fiber sheet bed: A new material for the immobilization of CHO-K1 cells

A new immobilization material for cell culture, ahydroxyapatite-pulp composite fiber (HAPC) sheet bed, was usedto grow CHO-K1 cells. The sheet bed for cell culture wasprepared from HAPC fiber by paper-making techniques. Scanning electron microscopic analysis revealed that the HAPCsheet bed had a structure consisting of piled fibers with spaces 10–200 m in diameter and a pore surface area of 0.32 m² g^-1. Using a 25 × 25 mm² squareHAPC sheet bed 0.41 mm in thickness (85 g m^-2 basis weight) for cell culture, CHO-K1 cells grew to a cell densityof 3.7 × 10⁷ cells cm^-3 in a 60 mm plastic dish over a 6-day culture period. High-density culture of CHO-K1 cells was successfully performed using the HAPC sheet bed in a 500 ml spinner flask over a 21-day culture period. The HAPC sheet bed, wound around the stirrer paddle, was rotated in the spinner flask in order to supply nutrientsand remove waste products efficiently. The HAPC sheet bedhas a large surface area to support cell growth and there islarge diffusion space inside of the bed. This newautoclavable substrate for anchorage-dependent cells can be easily scaled-up.     

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels.

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 deomonstrated 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.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

A new route for chitosan immobilization onto polyethylene surface

Low-density polyethylene (LDPE) belongs to commodity polymer materials applied in biomedical applications due to its favorable mechanical and chemical properties. The main disadvantage of LDPE in biomedical applications is low resistance to bacterial infections. An antibacterial modification of LDPE appears to be a solution to this problem. In this paper, the chitosan and chitosan/pectin multilayer was immobilized via polyacrylic acid (PAA) brushes grafted on the LDPE surface. The grafting was initiated by a low-temperature plasma treatment of the LDPE surface. Surface and adhesive properties of the samples prepared were investigated by surface analysis techniques. An antibacterial effect was confirmed by inhibition zone measurements of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The chitosan treatment of LDPE led to the highest and most clear inhibition zones (35mm(2) for E. coli and 275mm(2) for S. aureus).     

A new rate law describing microbial respiration

The rate of microbial respiration can be described by a rate law that gives the respiration rate as the product of a rate constant, biomass concentration, and three terms: one describing the kinetics of the electron-donating reaction, one for the kinetics of the electron-accepting reaction, and a thermodynamic term accounting for the energy available in the microbe's environment. The rate law, derived on the basis of chemiosmotic theory and nonlinear thermodynamics, is unique in that it accounts for both forward and reverse fluxes through the electron transport chain. Our analysis demonstrates how a microbe's respiration rate depends on the thermodynamic driving force, i.e., the net difference between the energy available from the environment and energy conserved as ATP. The rate laws commonly applied in microbiology, such as the Monod equation, are specific simplifications of the general law presented. The new rate law is significant because it affords the possibility of extrapolating in a rigorous manner from laboratory experiment to a broad range of natural conditions, including microbial growth where only limited energy is available. The rate law also provides a new explanation of threshold phenomena, which may reflect a thermodynamic equilibrium where the energy released by electron transfer balances that conserved by ADP phosphorylation.     

Surface immobilization of plant cells

A novel technique has been developed to immobilize plant cells. The cells are deposited on a surface of manmade fibrous material that provides for strong binding of the plant tissue biomass growing in the submerged culture. The immobilized plant cells remain fully viable. Relatively uniform biomass loadings of up to 20 mg d.w. plant cells/cm(2) support material have been attained. All plant cells from the inoculum suspension became attached within the first 24-48 h depending on the support matrix configuration and hydraulic culture conditions. The advantages and scale-up potential of this technique are discussed and compared to other culturing modes.     

A new microbial degradation pathway of steroid alkaloids

In the degradation pathway of the steroid alkaloid tomatidine by Gymnoascus reesii the A-ring of tomatidine is opened with the formation of the 4-hydroxy-3,4-secotomatidine-3-oic acid, which was identified in the form of N-acetyl-3,4-tomatidine-carbolactone by mass, IR and 1H NMR spectra. Cleavage of the A-ring in the starting reaction indicates that an alternative pathway must be operating, instead of the general oxidative one.