Cell immobilization can be defined as physically confining or localizing cells in certain defined region or space while retaining their activities. This is usually achieved by attaching/confining them in certain relatively big and dense particles (carriers or support materials) such as beads, foams and glasses, retaining them in membranes, or even flocculating the cells so that they form big flocs that easily sediment and separate from the culture broth.

Advantages of immobilized cell systems

Immobilized microorganisms may display properties quite different from those of the freely suspended cells. Some of these altered properties offer advantages to biological processing that cannot be obtained with freely suspended cells. The specific advantages are, however, depended on the immobilization method employed, the type of cell, the type of bioreactor and the nature of the process. Some of such advantages are outlined below.

1. The productivity of immobilized systems is generally higher than that of freely suspended cells.

2. Immobilized cell reactors allow high cell densities by altering the rheological properties of the suspending medium. High densities result both in an increased productivity and decreased bioreactor volume for the same production rate, thereby reducing both the production and capital costs.

3. There is little cell washout even at very high dilution rates (short residence time). High productivities can be achieved by operating at hydraulic dilution rates higher than the maximum specific growth rate of the microorganism.

4. In batch processes, immobilization allows the re-use of the biocatalysts.

5. There is usually very low cell concentration in the effluent. This reduces the cost of removing the cells during product recovery.

6. Some immobilized bioreactors such as packed bed bioreactors, are operated without agitation. This saves energy, and thus reduces the cost of production. In freely suspended bioreactors, energy is used to agitate the culture in order to avoid cell sedimentation.

7. If the products can easily diffuse out of the immobilized cells, the cells are less subjected to the inhibitory effects of high concentrations of substrate and products.

8. The immobilized cells can be retained even when there are transit adverse conditions such as accidental shift in temperature and pH to inhibitory levels, depletion of nutrients, etc. The cells are retained until favourable condition is restored. In freely suspended cell reactors, such conditions can lead to cell death or cell washout.

9. At the same cell concentration, the fluid viscosity in immobilized bioreactor is usually lower than that of freely suspended cells. Lower viscosities contribute to better mixing and mass transfer properties in the bioreactor.

10. During active fermentation with freely suspended cells, there are at times losses of some metabolic intermediates and exo-enzymes. However, the presence of solid surfaces (such as in gel beads), may retard the diffusional loss of such materials, thus leading to higher productivity.

10. Immobilization may also improve mass transfer through an increase in the effective or apparent density of the microorganisms. The density of freely suspended cells is very similar to the density of surrounding medium. As a result, the differential velocity between the cell and the suspended medium is relatively small. This results in lower rates of diffusion of substrate from the medium into the cells. However, when a dense carrier is used for immobilization, the resultant increased differential velocities can lead to enhanced nutrient diffusion rates.

12 The contamination risk is reduced due to high cell concentration and high dilution rates. Even when immobilized cell bioreactor is contaminated, the contaminants can be washed out of the bioreactor while retaining the immobilized cells. Also the contaminants can easily be removed by addition of antibiotics which kills the suspended contaminants but does not diffuse into the entrapped cells.

Methods of cell immobilization

Methods used to immobilize cells include a) entrapment in a polymer matrix, b) immobilization in fibrous materials c) attachment to surfaces, d) retention behind membranes, e) microencapsulation, f) adhesion to a surface, and g) flocculation.

Entrapment in polymer materials

Carriers

Many polymer materials have been used to immobilize viable cells. Polyacrylamide gel was among the first polymers to be investigated. However, the polymerization conditions are severe and lead to extensive cell damage. This problem is avoided by using Pre-polymerized polyacrylamide. Other polymers include epoxy resins, gelatin beads, Calcium alginate, К-carrageenan gel, and agar gel and agarose. The method of immobilization depends on the type of gel matrix. It usually involves mixing the pre-cultured cells with the polymer solution and then inducing gelation either chemically (addition of appropriate ions) or thermally (by reducing the temperature. A few expemles are given below.

Immobilization in Calcium alginate gel beads

Alginates are produced from sea weeds and used as food additives. They are nontoxic to the cells. They are heteropolymer carboxylic acids coupled by 1-4 glycolitic bonds. They are composed of β-D-mamuronic acid and α-L-guluronic acid units. Gelation of sodium alginate is induced by addition of diavalent cations such as Ca²⁺, Sr²⁺, Ba²⁺, and Zn²⁺.

Typical procedure for immobilizing cells in cacium alginate gel beads

1. Pre-cultivate the cells to obtain active cells (Cells at log growth phase are preferred).

2. Prepare 2% sodium alginate solution by dissolving 2g of sodium alginate in 300 mL conical flask containing a magnetic bar and 100 ml of distilled water .

3. Prepare 0.2 M calcium chloride solution in a 300ml flask containing a magnetic bar.

4. Autoclave the sodium alginate and calcium chloride solutions at 121℃ for 15 minutes

5. Allow all to cool to room temperature.

 6. Centrifuge the pre-cultured cells at 3000 rpm for 10 minutes

7. Suspend the cell pellet in 10 ml of distilled water

8. Mix the cell suspension with the cooled sodium alginate

9. Put the flasks on magnetic stirrers and stir gently.

10. Add the mixture drop wise to the gently stirred calcium chloride solution. In a very small scale, this can be done using a sringe but production of large quantity of beads requires the use of a pump.

10. Allow the produced beads to cure for 2 hours (the stability of the beads increase with curing time up to 22 hours. However, when toxic gelling agents are used, it is better to reduce the curing time).

Immobilization in carrageenan gel beads

K-carrageenan is a naturally occurring polysaccharide isolated from sea weed. It is readily available and nontoxic. The polymer is composed of β-D-galactose sulfate and 3,6-anhydro-α-D-galactose units. The polymer gels when cooled to low temperatures of about 10℃ but the resulting gels are very soft. The mechanical stability can be improved by using gelling agents such as K⁺, Ca²⁺, and Al³⁺. Aliphatic amines, aromatic diamines, some amino acids, or water-miscible organic solvents such as ethanol and acetone can also be used. However, K⁺ is presently the best gelling agent. The method used to produce karrageenan gel beads, using K⁺ as a stabilized is outlined below.

1. Pre-cultivate the cells to obtain active cells (Cells at log growth phase are preferred).

2. Prepare 3% carrageenan solution by dissolving 3g of carrageenan in a flask containing 100 ml of distilled water and a magnetic bar.

3. Prepare 0.2 M potassium chloride solution in a flask containing a magnetic bar.

4. Autoclave the carrageenan and potassium chloride solutions at 121℃ for 15 minutes

5. Allow the potassium chloride to cool to room temperature but carrageenan should be maintained at about 45℃

6. Centrifuge the pre-cultured cells at 3000 rpm for 10 minutes

7. Suspend the cell pellet in 10 ml of distilled water

8. Mix the cell suspension with the carrageenan solution at 45℃.

9. When the potassium chloride has cooled to room temperature (better results are obtained if the solution is cooled to below 15℃), put the flasks on a magnetic stirrer and stir gently.

10. Add the mixture drop wise to the cold and gently stirred potassium chloride solution.

11. Allow the produced beads to cure for 2 about.

Advantages and disadvantages of immobilization by entrapment in polymer gels

Advantages

1. Particles of various shapes and sizes can be obtained

2. The porosity can be controlled to an extent by varying their concentrations and by the choice of the gelling agents.

3. They are relatively cheap

4. High cell density can be achieved

Disadvantages

1. They are mechanically unstable under high hydrodynamic stress.

2. Some of the polymers such as calcium alginate are chemical unstable in the presence of phosphates, citrate etc. This can be avoided by reducing the concentration of phosphates in the medium or by preteating the beads with some agents such as polyethylene imine.

3. During the cultivation of high concentration of active cells, pressure builds up inside the beads due to high rate of carbon dioxide evolution. This disrupts the beads, making them to float and increases cell leakage from the beads. This problem can be minimized by using very small particles, using very porous carriers, and using low initial cell concentrations.

4. There is an additional diffusional barrier to substrate and product transport imposed by the carrier. Depending on the bead diameter, and the cell concentration, the cells at the center of the gel beads may have little or no substrate. Thus cells grow preferentially at the periphery of the gel beads. This problem can be solved by reducing the diameter of the gel beads or by co-immobilizing anaerobic and aerobic microorganisms. In such co-immobilized cells, the anaerobic cells grow at the centre with very low oxygen concentration while the aerobic microorganisms grow preferentially at the periphery of the beads.

 

Immobilization of cells in microporous beads and fibrous materials

Carriers

Many micropororous beads and fibrous materials such as silica beads, ceramic beads, Porous glass, charcoal, resins, Loofa sponge, and polyurethane foams can be used as carriers for cell immobilization. In this case, the cells colonize and grow to high concentrations in the spores within the carrier.

Immobilization methods

i) Cut the carriers into desired sizes (in the case of fibrous materials) or wash the beads

ii) Pre-culture the cells, harvest by centrifugation, and re-suspend in sterilized distilled water.

iii) Add the carriers into a flask or bioreactor containing appropriate culture medium

iv)  Add the cell suspension and mix

v) Cell will move in and colonize the small pores in the carrier

vi) For carriers with rather large pore sizes such as loofa sponge, good cell loading can be achieved by using flocculating cells or by inducing flocculation by addition of some agents such as chitosan.

Advantages and disadvantages

Advantages

a) They are chemically inert.

b) They are very cheap

c) They are mechanically stable.

d) The immobilization method is very simple and cheap

e) The immobilization condition is very mild. Immobilization is done at room temperature and there is no chemical additives (except when floc induction is needed).

Disadvantages

a) The immobilized cell concentration is usually low when compared to cell entrapment method.

b) The cells are easily dislodged by vigorous aeration and agitation.

c) The leakage of cells into the culture broth can be high especially in mechanically agitated bioreactors, and in fluidized bed bioreactors

Immobilization by attachment of cells to surfaces

Carriers

Examples of carriers used for this type of immobilization include plane glass with rough surface, anion exchange resins, wood chips, wood shavings, and sugar cane baggasse.

Methods of immobilization

Immobilization can be achieved by simply mixing the cells and the carrier. In the case of positively charged carriers such as anion exchange resins, the negatively charged cells will naturally adsorb to the surface of the carrier. However, negatively charged cells also adhere to negatively charged inorganic carriers such as glass and ceramic. This may be due to partial co-valent bonding. The immobilized cell concentration can be increased by modifying the surface of the carrier. Also covalent binding by using some chemicals such as glutaraldehyde leads to reduced cell leakage.

Advantages and disadvantages

Advantages

a) Conditions for immobilization are usually very mild.

b) Mass transfer between the immobilized cells and the culture broth is usually higher than those immobilized by entrapment in polymer matrix

Disadvantages

1. Only low cell concentration can be immobilized.

2. The immobilized cells can easily be washed out of the carrier due to pH changes, ionic strength, high flow rates, culture agitation, carbon dioxide evolution etc. This limits the operational stability.

Cell immobilization by retention behind a membrane

Many bioreactor systems employ membranes to localize the cells within a certain part of the system. Examples of such systems include simple dialysis bioreactor, a rotating membrane dialysis reactor operating under pressure and hollow fiber bioreactors.

Advantages and disadvantages

Advantages

The membrane is often a part of the bioreactor and as such, can be used several times for various cells and processes.

Disadvantages

i) There is limitation to substrate and product diffusion.

ii) Membrane plugging can be a serious problem.

iii) They are mechanically complex.

iv) The operational stability is often low.

Immobilization of cells by microencapsulation

Microcapsules are spherical particles where a liquid or a suspension is enclosed by a dense semipermeable polymeric film.

Carriers

Many carriers such as polymers and cellulose derivatives have been used for encapsulation of cells.

Immobilization method

i) Entrap the cells in polymer materials such as calcium alginate as described before.

ii) Treat the resulting beads with a polycation solution such as polylysine (this leads to formation of insoluble polyelectrolyte complex).

iii) Re-dissolve the noncomplexed alginate by suspending in high phosphate or citrate buffer(this leaves the cells at the center in suspension).

Advantages and disadvantages

Advantages

a) They are suitable for immobilization of animal cells.

b) They have high potential for development of artificial organs.

c) Depending on the materials used, the rate of mass transfer is higher than that obtained in gel beads with hard cores.

Disadvantages

a) Depending on the type of materials used, transport of nutrients across the film may become rate-limiting

b) The capsules are relatively fragile and thus not suitable for use in mechanically agitated bioreactors. They are not stable for long term operation even in non-agitated systems.

Immobilization by cell flocculation

 Methods of immobilization

Cell flocculation is also a form of cell immobilization. When cells flocculate, they form large flocs which sediment and behave more or less as cells immobilized in gel beads. However, naturally flocculating strains are few. For non flocculating cells, flocculation can be induced by addition of cross linking chemicals such as glutaraldehyde. However, such chemicals are toxic to the cells. High molecular polymers such as chitosan can be used for physically cross linking the cells.

Advantages and disadvantages

Advantages

A main advangae of this method is that there is no need for support (carrier) materials.

Disadvantages

i) The stability depends on the cell age, ionic strength, pH and hydrodynamic stress inside the bioreactor. In other words, changes in these factors can lead to dispersion of the cells in the flocs.

ii) Carbon dioxide evolution can lead to disruption of flocs.

iii) In continuous processes, the flow rates should be slow to avoid floc disruption and subsequent cell washout.

Desired characteristics of carriers used for cell immobilization

1. It should be possible to control the size and porosity of the carrier.

2. Entrapment agents should form a suitable matrix in aqueous media under mild (suitable) temperatures and pH.

3. They should be cheap and easily available, and the cost of immobilization should be low.

4. They should possess mechanical stability so as to resist hydrodynamic stress generated by mixing and agitation of the culture over a long period of operation.

5. They should also possess chemical stability in the presence of various media components, products and by-products.

6. Carriers used for entrapment of cells should allow free diffusion of substrate, product and other metabolites in and out of the immobilized particles.

6. They should have high cell carrying capacity. It should be possible to immobilized high concentration of cells.