Immobilization viable cells

In other applications there is now an emerging use of immobilized viable cells in the wine and beer industry. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

The method is widely used for immobilization of viable cells, where the open gel makes transport of nutrients/metabolites to and from the cell possible, but it has not gained any industrial importance in immobilizing enzymes. The reasons for this are the... 

Ariff and Webb studied production of glucoamylase using freely suspended cells of Aspergillus awamori in batch and continuous fermentations. Glucoamylase yields based on glucose consumed were 900 and 1080 U/g for batch and continuous fermentations, respectively. The immobilization of viable cells was achieved by adsorption to cubes of reticulated polyurethane foam. In comp uison with freely suspended cell fermentations, neither batch nor continuous fermentations of immobilized cells improved glucoamylase production significantly in tenns of yield or productivity. 

If sufficient nutrients (e.g., 0.3 g l 1 glucose) are present in a sample solution, a constant current is obtained from the electrode. The current depends on the total respiration activity of immobilized cells. Therefore, the total respiration activity of bacteria, the current, depends on the number of viable cells immobilized onto the acetylcellulose membrane. The relationship between the current and the cell numbers on the acetylcellulose... 

The equations in this section are applicable to any system in which the viable cell density can be determined. This includes all of the systems described above except for immobilized cell reactors. 

Because apparent metabolic yields can be determined without knowledge of n, Y values can be monitored to detect changes in metabohc patterns of cells in immobilized-cell bioreactors. For example, yj[ac,gic is easy to obtain and provides a good measure of the fate of the primary nutrient glucose. Relatively constant values for F[ac,gic NH3,gic Oz.gic other )delds provide a good indication of a stable culture environment. Under these conditions. Equation 4.2.44 may be used with confidence to estimate changes in the viable cell density. 

Gemeiner P, Rexova-Benkova L, Svec F, Norrlow O (1994) Natural and synthetic carriers suitable for immobilization of viable cells, active organ alles and molecules. In Veliky IA, McLean RJC (eds) Immobilized biosystems. Theory and practical applications. Blackie Academic and Professional Chapman and Hall, London p 1... 

Fig. 7 Cell mass and viable cell number of immobilized recombinant yeast cells during repeated batch fermentation. Fermentation time for each batch=7 days. Initial cell density=50 g/L. Cells were added at day 35. CDW Cell dry weight, CFU colony-forming unit...

Cellular Viability and Concentration Determined by counting cells in nine squares of a Neubauer chamber after dying them with methylene blue solution. Viable cells were not colored, and dead cells were blue free yeast concentration was also obtained by filtering a known volume of cell suspension and drying the wet mass until the constant weight was achieved viability and concentration of immobilized yeast were determined as already described, after dissolution of the pectin gel [1.0 g of cured pellets was dissolved in 20 ml of 5% ethylenediaminetetraacetic acid (EDTA) solution at constant agitation]. The cell concentration was calculated as ... 

Once immobilized, the cells must be kept in a viable state in the membrane without further excessive reproduction. This is necessary to channel the substrate into the desired product rather than to additional cell mass. Besides, it would minimize cell elution from the carrier matrix as well as preserve the mechanical integrity of the carrier. We have found that one way to accomplish this is by limiting the concentration of one of the essential nutrients in the medium for example, nitrogen concentration. An indirect benefit of this approach is lowering the growth of contaminating organisms. 

Catalyst Reincubation. It is one of the unique features of using whole cells as immobilized catalysts that the catalyst can be activated by reincubation as long as viable cells are present in the entrapped state. Reincubation of the catalyst beads with the appropriate nutrients facilitates the growth of new cells inside the polymer bead. This technique can be used either to improve the catalytic activity directly following immobilization or... 

Furuya et al. (468) reported the immobilization of P. somniferum cells in calcium alginate. CeUs remained viable for 6 months after immobilization. The cells were used in shake flasks and column bioreactors for the biotransformation of codeinone to codeine. The immobilized cells had a higher biotransformation ratio (70%) than suspended cells (61%). Most of the codeine formed was excreted into the medium (88%). The column bioreactor had a lower biotransformation ratio (42%). The cells in the bioreactor operated at 20°C and an aeration rate of 3.75 vvm (volume gas/volume broth/min) remained catalytically active for 30 days. In a more detailed study on the influence of substrate transport in immobilized cells, it was concluded that limitation of oxygen inside the beads caused deactivation of the cells. However, the reaction rate of the system was not affected by the limitation of oxygen transfer (469). Immobilization of P. 

Operation of various types of bioreactors in a perfusion mode (21, 22) enables the design engineer to combine several advantages of traditional modes of operation of well-stirred bioreactors (e.g., semibatch operation in a fed batch mode or use of recycle with a chemostat). Perfusion consists of operation in a mode in which a fresh growth medium (possibly together with a recycled growth medium) is fed to a bioreactor containing viable cells that are retained within the bioreactor by permselective membranes, microfllters, immobilization, or by partial separation and recovery from the reactor effluent followed by recycle to the entrance of the reactor. 

Nevertheless, industrial use of immobilized cells is still limited and it will depend on the development of processes that can be readily scaled up. Data available in the literature on materials and techniques used for viable cell immobilization for application in alcoholic beverages and food-grade ethanol production are highlighted and discussed. 

Freeman A. and Lilly M.D. Effect of processing parameters on the feasibility and operational stability of immobilized viable microbial cells. Enzyme and Microbial Technology 23 (5) (1998) 335-345. 

Junter G.-A. and Jouenne T. Immobilized viable microbial cells From the process to the proteome... or the cart before the horse. Biotechnology Advances 22 (8) (2004) 633-658. 

Adsorption of a biocatalyst onto a water-insoluble macroscopic carrier is the easiest and oldest method of immobilization. It may be equally well applied to isolated enzymes as well as to whole viable cells. For example, adsorption of whole cells of Acetobacter onto wood chips for the fermentation of vinegar from ethanol was first used in 1815 Adsorbing forces are of different types, such as van der Waals (London) forces, ionic interactions, and hydrogen bonding, and are all relatively weak. The appealing feature of immobilization by adsorption is the simplicity of the procedure. As a result of the weak binding forces, losses in enzyme activity are usually low, but desorption (leakage) from the carrier may be caused by even minor... 

In case an enzyme does not tolerate direct binding, it may be physically encaged in a macroscopic matrix. To ensure catalytic activity, it is necessary that substrate and product molecules can freely pass into and out of the macroscopic stracture. Due to the lack of covalent binding, entrapment is a mild immobiUzatimi method which is also applicable to the immobilization of viable cells. 

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