Plant cell culture has been for sometime considered as an alternative method for the production of flavours, colors and pharmaceuticals to their extraction from plants. Characteristics of plant cell cultures, such as slow growth, the compounds produced should be of high value ($ 500-1000kg-1) and low volume. One of the major limiting factors in the development of a commercial production system using plant cell culture has been the production cost of phytopharmaceuticals. The use of high biomass levels for extended periods would be one method of increasing productivity and hence reducing the costs. This can be achieved by the immobilization of plant cells.
The immobilization of enzymes and cells has received increasing attention, and used to produce amino acids and carbohydrates. The immobilization of microbial cells is not new, it has known for sometime that cells will adhere to many surface in nature i.e. polymers.
Immobilization is the newest culture technology of plant cell, and considered as to be the most “natural”. It has been defined as a technique, which confines to a catalytically active enzyme or to a cell within a reactor system and prevents its entry into the mobile phase, which carries the substrate and product. The first successful immobilization of plant cells was reported by Brodellus et al. (1979) and they entrapped Catharathus roseus and daucus carota cells in alginate beds. Following success with enzymatic and microbial process, immobilization has been suggested as a strategy to enhance the overall productivity of secondary metabolite in plant cell culture. The ability to immobilize plant cells has been reported for a large number of plant cells and protoplasts by using a variety of polymers. Immobilization of plant cells has been used for a wide range of reactions, which can be divided into three groups. (1) Biotransformation or bioconversion, (2) synthesis from precursors and (3) the De- Novo synthesis of compounds.(16)
Advantages of Plant Cell Immobilization:
Retention of biomass enables its continuous reutilization as a production system, a definite advantage with slow growing plant cells e.g. Papaver somniferum have remained stable and active for up to six months.
High biomass levels: The immobilization of cells allows the use of a higher biomass level compared to cell suspension culture, because of the limitation of mass transfer and settling, e.g. bead densities of 110 g dry weight/L have been obtained with calcium alginate entrapped cells when 30 g dry weight/L in suspension cultures. The high cell density allows a reduction in contact in packed bed catalyst leading to an increased volumetric productivity.
Separation of cells from medium: the immobilization separates cells from medium and the product is extra cellular, which will simplify down stream processing compared to extract from tissue.
Continuous process: Immobilization allows a continuous process, which increase volumetric productivity and allows the removal of metabolic inhibitors.
Decoupling of growth and product formation: Immobilization is compatible with non-growth associated product formation.
Reduces problems such as aggregate, growth and foaming: the immobilization reduces some of the physical problems associated with the cultivation of plant cells such as the formation of aggregates, and susceptibility to mechanical damage (shear stress) are problems which do not affect immobilized system compared to cell culture.
Disadvantage of Plant Cell Immobilization:
Secretion of secondary metabolites requires cellular transport or artificially altered membrane permeability.
The efficiency of the production process depends on the rate of release of products rather than actual rate of biosynthesis. The immobilization process may reduce biosynthetic capacity. Products must be released from the cell into medium. Release of single cells from cell aggregate may make processing of the product more difficult. The microenvironment avouring optimal production can be unfavorable for released secondary metabolites and cause their degradation or metabolization.The prerequisites for a successful immobilization of plant cells are as follows:
1. Non-growing cells must produce products.
2. Products must be released from the cell into the medium.
Need for immobilization:
Plant cells are characterized by large size, sensitivity to shear and oxygen and need of a cell to cell contact for metabolic production. The secondary metabolites are triggered by short periods of stress in cultures. Immobilization can overcome many of the limiting factors of suspension cultures with the distinct advantages of easier operation of biocatalyst from the product and also being amenable for biotransformation of low value compounds to high value products.
Different types of immobilization:
1. Direct intracellular binding due to natural affinity (adsorption, adhesion and agglutination).
2. Covalent coupling on otherwise inert matrices.
3. Intracellular connection via bi or poly functional reagent (cross-linking).
4. Mixing with suitable materials, changing their consistency with temperature (embedding).
5. Physical retention within the framework of diverse pore size and permeability (entrapment, micro encapsulation).
Selection of immobilization system:
The choice of a suitable immobilization system is determined by the following requirements.
1. The polymer material used for immobilization must be available in large quantities; itmust be inert, non-toxic and cheap.
2. It must be able to carry large quantities of biomass and its fixing potential must be high.
3. The immobilization process must not diminish enzymatic activity of biological catalyst.
4. Manipulation of the biological catalyst must be as simple as possible (hall et al 1988).
Methods For Immobilization
Gel entrapment by polymerization:
A monomer or a mixture of monomers is polymerized in the presence of a cell suspension, which is entrapped inside the lattice of the polymer. The most common example is polyacrylamide. The method is based on the free radical polymerization of acrylamide in an aqueous solution. As the linear polymers are soluble in water, they have to be insolubilized with bifunctional compounds such as N, N, - Ethylene bisacrylamide. The free radical polymerization of acrylamide is conducted in an aqueous solution containing the cells and the cross-linking agent. Polymerization is commonly carried out in the absence of oxygen and at lower temperature (100C) to avoid damage to the cell during the operation. An initiator N,N,’ Tetramethylethylene diamine (TEMED) is used. Both the initiation and the cross-linking agents are toxic to the cells and therefore, their viability can be lost e.g. Chatharanthus. roseus and Silybum marianum.
Gel entrapment by ionic net work formation:
In this method, polymerization of polyelectrolyte is achieved by addition of multivalent ions. The most common method is the entrapment in calcium alginate. This is a non-toxic process in which sodium alginate solution containing the cell suspension is dropped into a mixture of counter ion solution such as calcium chloride. A uniform, spherical and highly microporus structure results, which retains the cell.
The method may be inconvenient over long run in media containing calcium chelating agents, such as phosphates and certain cations such as Mg+2 that may accelerate disruption of gel by solubilizing bound Ca+2.Carrageenan is also a suitable matrix for the immobilization of plant cells. The gels of calcium and potassium carrageenates are very stable at room temperature and PH above 4.5.
Gel entrapment formation by precipitation:
Gels may be formed by precipitation of some natural and synthetic polymers by changing one or more parameters in the solution, such as temperature, salinity or PH of solvent. Several materials can be used for entrapment. The examples include methods involving thermal treatment. Some disruption of viability can occur naturally.
Entrapment in preformed structures
Hollow fiber reactors can be used to immobilize plant cells by entrapment. The cells are placed on the shall side of the reactor and nutrient medium is rapidly re-circulated through the fibers. This may have important applications in large-scale.
In other examples, the cells are added to preform polymerized structures such as polyurethane foam. When cells and suspansion are mixed with these materials, they are rapidly incorporated into the net work and subsequently grown into the cavities of the mesh and are entrapped by physical restriction and attachment to the matrix material. The mechanism of this involvement is at first a mechanical entrapment and later, the fixation of the cells due to mechanisms of adsorption and adhesion or even due to their natural tendency for aggregation. These methods of immobilization have several advantages over other methods in that they are simple, cheap, gentle and rapid and maintain cellular functions.
Surface immobilization may occur on both natural and other matrices. Examples of natural matrices are deeper callus layers and cellulose, while synthetic one includes nets of steel and nylon. For e.g. cells of Solanum aviculare were covalently linked to beds of polyphenylene oxide, which had been achieved using glutaraldehyde. Archambault et al (1986) described the spontaneous and rapid binding of Chatharanthus. roseus cells to a man made material.(16)
Immobilization by embedding:
The temperature dependent solubility of macromolecules like agarose, agar and carrageenan or the differing solubility of the sodium and calcium salts in the case of alginate are utilized to form polymeric gels or gel combination. Insoluble are formed under cold conditions (Agar) or in aqueous CaCl2 solutions (Alginate). Their structure is non-uniform, with differing pore diameters at the surface and in deeper layers. The size and form of the beds can be determined in part by stirring speed and using alginate, by the viscosity of the solution and dropping aperature
Types of bioreactors used for immobilization of plant cells:
The following types of reactors are generally used for immobilized plant cell.
Packed bed reactors
In this reactor, cells can be immobilized either on the surface or through the support and fluid containing S flows past the support particles. When the cells are immobilized through the support, the packed bed can accommodate a large number of cells per reactor volume.
1. Low degree of mixing causes difficulties in transfers of oxygen, CO2 etc, when it is necessary.
2. The pressure required to pump fluid through the packed bed is inversely proportional to the support particle size, and thus reduces the energy requirements for flow, large incompressible support particles are needed.
3. The packed bed reactors are having filters. When particulate matters are included in the media or when the support particles fragment during operation, they will be trapped in the column and block the pathways for fluid flow.
Well mixed reactor
The advantages of a well-mixed reactor for immobilized cells are that gaseous substrates can be directly sparged, the temperature and PH can be carefully controlled, and mass transfer rates can be improved over packed bed reactors. The suspension culture with complete cell recycle is essentially a well-mixed “immobilized” cell reactor.
The disadvantages of well-mixed reactor for immobilized cell systems are the possibility of fragmenting the support due to particle collision and shear. Using gentle pneumatic agitation can reduce this problem.
Fluidized bed reactors
Typical fluidized bed reactors utilize the energy of the following fluid (liquid and/or gas) to suspend the particles. Because the energy required for fluidization increases with increased particle size, small immobilized particles are often employed. The mass transfer advantages of these small particles is one of the major benefits of the fluid must often be retained in the reactor for an extended period. This may be incompatible with the requirement that the fluid flow rate be sufficient to suspend the particles. To satisfy these two requirements, large gas volumes can be used to suspend the immobilized cells while maintaining low liquid flow rates, or the liquid can be rapidly recirculated through the bed. Both these conditions lead to a large degree of fluid mixing. Large recirculation rates were in the fluidized bed reactors.
The disadvantages of the fluidized bed reactor are that shear and particle collisions may damage the immobilized supports and the complex fluid dynamics of such reactors make scale-up difficult.
The most commonly considered membrane reactors are the hollow fiber and spiral wound reactors. In the hollow fiber reactor, the cells are retained either within the tubes or in the outer region. Preliminary studies to evaluate the use of these reactors have been reported by shuler et al., (1983); Jose et al., (1983) and prenosil and Pederson (1983). The spiral wound membrane reactor is essentially a flat plate reactor rolled into a cylindrical shape and although no reports have appeared on the use of spiral wound reactor. Hallsby and shuler (1986) have studied plate reactor with tobacco cells. The studies have described that in addition to the membrane, the thickness of the cell layer is also important. The inner portions of thick cell layers are generally characterized by substrate deficiencies and product accumulation and these conditions may prove beneficial for the morphological and chemical differentiation of plant cells.
The advantages of membrane reactors are the possibilities that these membranes can be reused. Thus, despite the high capital costs, the reusability may make this form of immobilization more economical. Also because plant cells are metabolically less active than microbes, thicker cell layers can be employed. Since reactor inoculation, or cell loading is easier with larger cell layer thicknesses it may be possible to develop plant cell reactors, which can be easily inoculated.
Application of plant cell immobilization:
Hydroxylation of cardiac glycosides has proved an interesting application of immobilized plant cells. Alfermann et al. (1982) used Digitalis lanata in alginate beds in a process aimed at converting beta- methyldigitoxin into clinically more beta-methyl-digoxin.(15) In batch process, the fluid bathing of the cells was changed every second day over a period of 59 days and resulted in a higher cell productivity than that of a free cell system. Brodelius (1981) successfully used a column of similar beds for upto 70 days.(16) Using alginate immobilized cells of Daucus carota; jones and Veliky (1981) achieved 5-Hydroxylation of digitoxigenin into periplogenin over a period of 12 days, but noted deterioration in biocatalytic activity due to accumulation of inhibitory levels of toxic substrate in the cells.(16)
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