Strategies of Whole Cell Immobilisation

fi xing the cells, organelles or enzymes/other proteins (monoclonal antibodies) onto a solid support system, into a solid support matrix or retained by a membrane, in order to maintain stability and make possible their repeated or con-tinued use. The immobilised cell technologies comprise of modifi cations of the technique devel-oped for enzymes. However the microbial size has a signifi cant impact on these techniques.

The immobilisation of microbial cells occurs as a natural phenomenon or through artifi cial process.

The artifi cially immobilised cells are allowed restricted growth.

The early attempts of whole cell immobilisa-tion were developed from processes applied to the immobilisation of enzymes and generally involved non-viable cells, i.e. cells impaired by physical or chemical treatment to perform single step enzyme reactions. The obvious benefi t derived from using whole cell is to avoid enzyme extraction/purifi cation steps which consequently have an effect on enzyme activity, stability and cost. These techniques in due course were extended to viable cells as they were exploited in bioreactors and fermentation systems. The advantages of viable immobilised culture sys-tems are manifold. High cell densities are expected as viable immobilised cells multiply during the substrate metabolisation process while remaining confi ned with the immobilised matrix,

and thus high volumetric reaction rates are expected in the immobilised cell culture; further regeneration of immobilised cultures is possible even under hostile incubation conditions like low nutrients or the presence of toxic compounds. In continuous processes effi cient biomass retention is ensured minimising cell washout which nor-mally occurs at high dilution rates by whole cell immobilisation. Immobilisation of whole cells also facilitated cell/liquid separation thereby easing the downstream processing in fermentation processes using immobilised whole cells.

There are three basic methods which have been used for immobilisation of microorganisms, viz.

(1) attachment to a support, i.e. carrier binding, (2) entrapment and (3) self-aggregation (Fig. 12.1 ).

The overall composition of immobilised cell systems is less chemically defi ned as compared to the immobilised enzyme system. In this chap-ter the emphasis would be on various strategies which have been developed for immobilisation of the whole cells and their exploitation in different industrial settings.

12.2 Strategies of Whole Cell Immobilisation

12.2.1 Adsorption

This process relies on the tendencies of the cell to aggregate or adhere to particular surfaces or settle in the pores of the framework. This kind of

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cell immobilisation is usually achieved by keep-ing the support material and actively growkeep-ing cells in physical contact for a specifi c duration.

One of the classical examples is related to the process of vinegar production using woodchips as carriers of Acetobacter adsorption. A variety of substrates like zeolites, baked clay, glass beads, sponge rubber, cellulose acetate fi bre and activated carbon fi bre have been employed for the immobilisation of the whole cells’ adhesion or adsorption.

At times pretreatment of cells either by starva-tion or washing of the cells or activastarva-tion of the support material or cells may be benefi cial for the improvement of the adsorption characteristics.

Aluminium was used to neutralise the surface charge of Saccharomyces cerevisiae , and its absorption helped in its immobilisation on glass plates (Van Haecht et al. 1985 ). Erwinia rhapon-tici was immobilised on diethylaminoethyl (DEAE) cellulose by mixing 2 g of cells with 10 ml of thick DEAE slurry at pH 7 (Cheetham

et al. 1985 ). A variety of support material has been used for the immobilisation of the whole cells for different applications (Table 12.1 ).

The adhesive strength of adsorption-based immobilisation is not very strong until there exists an exceptional mechanism in the microbe for the surface-anchored growth. The major advantages of adsorption are simplicity and negligible changes on the cell physiology;

however, the drawbacks are limited cell loading and limited adhesion stability compared to cell entrapment.

12.2.2 Covalent Binding

Covalent binding is yet another way of attaching cells to the surface of the carrier. This method has been extensively exploited in enzyme immobilisation. It was realised that to achieve high effi -ciency binding, stability is to be enhanced, and this could be achieved by creating covalent link-Whole Cell Immobilization

Carrier binding Entrapment Self-aggregation

Physical adsorption

Ionic adsorption

Covalent binding

Lattice

Membrane

Microencapsulation

Flocculation

Chemical Crosslinking

Fig. 12.1 Broad classes of whole cell immobilisation

Table 12.1 Immobilisation of whole cells by covalent bonding

Microbe Covalent binding agent Product

Aspergillus niger Glycidylmethacrylate polymer + glutaraldehyde

Formation of gluconic acid from glucose Proteus rettgeri Carriers with epoxy

and halocarbonyl groups

Conversion of penicillin G to 6-APA

Acetobacter sp. Ti(IV) oxide Production of vinegar

Burkholderia cepacia Polyurethane foam Trichloroethylene degradation Saccharomyces cerevisiae Cellulose + cyanuric chloride Conversion of glucose to ethanol

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ages between the cell and the support surface.

Saccharomyces cerevisiae has been immobilised on silanised silica beads using α-aminopropyl triethoxy silane as a coupling agent (Navarro and Durand 1977 ). Pseudomonas stutzeri has been immobilised on polyethylene surface by chloro-sulphonic acid and chlorochloro-sulphonic acid com-bined with polyethyleneimine (Choi et al. 1999 ).

Various coupling agents which have been used for covalent immobilisation of cells are aminosi-lane, carbodiimide and glutaraldehyde, which introduce specifi c groups on the carrier surface and subsequently can interact with reactive groups on the cell surface (Table 12.1 ).

12.2.3 Cell to Cell Cross-Linking

Flocculation is one of the simplest methods of achieving cell aggregation in the form of larger particles with high cell densities. However the capacity of microorganisms to fl occulate natu-rally is limited. Hence chemical cross-linking is the most appropriate method to enhance fl occula-tion and stabilise the cell aggregates. Commonly used cross-linking agents are glutaraldehyde, polyamines, polyethyleneimine, polystyrene sul-phonates, polyvinyl alcohols, etc. Cross-linking reduces the chances of washout and improves the mechanical strength of the cell. Some common applications have been presented in Table 12.2 .

12.2.4 Encapsulation

The encapsulation technique generally employs the use of polymeric beads to immobilise the whole cells. This is broadly divided as macroencapsulation and microencapsulation.

Microspheres or microcapsules are usually spherical particles less than 1,000 μm in which liquid or suspension is enclosed by the dense but semipermeable polymeric fi lm. The major limitation of this technique is the transport of nutrients across the membrane. Probiotic microbes like Lactobacillus acidophilus , Lactobacillus casei and Bifi dobacterium bifi dum have been microencapsulated in substances like gelatine, carrageenan, etc. (Kailasapathy 2002 ).

The microencapsulation technique has applications in various fi elds like pharmaceuticals, agrochem-icals, nutrition and therapeutics. The different methods used for the process of microencapsula-tion are extrusion, spray drying, emulsifi camicroencapsula-tion and coacervation. The different methods adopted for immobilisation of microbial cells by microencapsulation are given in Table 12.3 .

Microspheres are mechanically stronger then macrospheres and exhibit effi cient diffusion of nutrients, oxygen and metabolites.

Microencapsulation is an advantageous method in the fermentation industry since it not only car-ries out the process effi ciently due to larger spe-cifi c area for nutrient utilisation and metabolite

Table 12.2 Immobilisation of whole cells by cell to cell cross-linking

Microbe Covalent binding agent Product

Saccharomyces cerevisiae 1 % albumin + 0.25 % glutaraldehyde Fructose-1,6 diphosphate production

Erwinia ariodea TSMPMV-2970 N′, N′- m- phenylene disasperimide (PDAI) Production of

L -aspartate-β-decarboxylase Bacillus subtilis TSMPMV-259 M N′, N′-m-phenylene disasperimide (PDAI) Production of

L -aspartate-β-decarboxylase Aspergillus niger Flocculation by polyelectrolytes Production of gluconic acid

from invert sugar Lactobacillus brevis Flocculation by chitosan Production of fructose

from glucose Saccharomyces formanensis Polymer of 2-hydroxyethcyrylate-and

methoxypolyethylene glycerol methacrylate using γ-rays and tetraethylene glycerol dimethacrylate as cross-linking agent

Production of ethanol 12.2 Strategies of Whole Cell Immobilisation

182

production but also allows easy separation of the cells thereby minimising the cell washout.

The technique also enhances the possibilities of the reuse of cells due to improved tolerance to undesirable processes like end product inhibition or contamination.

12.2.5 Entrapment

Physical entrapment of polymeric carriers of defi ned porosity is a widely used method for whole cell immobilisation. Different strategies of entrapment are gelation, precipitation, ionotropic gels and polycondensation. During the gelation process a temperature-controlled phase transition of the polymer solvent system is carried out wherein it is transformed into a homogenous sys-tem without change in the composition. Calcium alginate gels appear to be most compatible for immobilisation of living cells. Besides gelatine, agar and agarose have also been used for the process of gelation. The only concern with calcium alginate cells is the high affi nity for cal-cium which destabilises the cell. Other matrices which have been employed are agar, alginate, k- carrageenan, cellulose and its derivatives,

collagen, epoxy resin, polyacrylamide, polyester, polystyrene and polyurethane.

12.2.5.1 Precipitation of Polymers Polymer solution is primarily used as a dispersion medium of cells. However coagulation of the polymer is achieved by changing the physico-chemical parameters other than temperature, i.e. solvent and pH. The major drawback of this procedure is intensive contact of viable cells with non-physiological solvents thereby limiting its applications (Table 12.4 ).

12.2.5.2 Ionotropic Gelation of Polymers

Ionotropic gelation of polymers is based on the ability of a polyelectrolyte to cross-link in the presence of counterions to form hydrogels.

Ionotropic gelation is generally used to have highly water-swollen structures with controlled morphology. A well-known example is calcium alginate cells. Chitosan is a polycation which also fi nds application in cell immobilisation. This process is generally very simple and non-toxic;

various cells could be immobilised by complete preservation of viability (Table 12.5 ).

Table 12.3 Exploitation of encapsulation as a method of whole cell immobilisation

Microbe Covalent binding agent Encapsulation/microencapsulation Product Pantoea agglomerans E25 Sodium alginate +

calcium chloride

Modifi ed precision particle fabrication

Prevention against harsh environmental conditions

Lactobacillus delbruckeii subsp. bulgaricus NBRC13953

Sodium alginate + calcium chloride

Emulsifi cation Prevention against harsh environmental conditions

Saccharomyces cerevisiae Eudragit ^® Extension Production of ethanol from glucose Pseudomonas sp. SA01 Sodium alginate +

calcium chloride

Extrusion Phenol degradation

Bifi dobacterium lactis Gellan/xanthan gum blend, calcium chloride

Extrusion Protection against

harsh gastrointestinal conditions

Pseudomonas sp. Polyvinyl alcohol Extrusion Biodegradation of phthalic acid ester 12 Immobilisation and Biosensors

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12.3 Alginate Method of Whole