1.Inert. 2.Physically strong and stable. 3.Should be cheap enough to discard. 4.Better if it could be regenerated after the useful lifetime of the immobilised enzyme. 5.The surface available to the enzyme. 6.More desirable properties: * Ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst by means of magnetic fields). Catalytic surface (e.g. manganese dioxide, which catalytically removes the inactivating hydrogen peroxide produced by most oxidases).

Types of immobilized cell reactors There are many types of immobilized cell reactors either in use or under development. In this section we will look at four major classes of immobilized cell reactors:  Cell recycle systems  Fixed bed reactors  Fluidized bed reactors  Flocculated cell systems

Cell recycle systems In a fermenter with cell recycle the cells are separated from the effluent and then recycled back to the fermenter; thus minimising cell removal from the fermenter:

Effluent Biomass recycle Fermenter Fresh feed Biomass separation system Cell recycle system

Cell recycle systems Cell recycle is used in activated sludge systems. A portion of the cells are separated in a settling tank and returned to the activated sludge fermenter. Biomass recycling for product or biomass production is more difficult due to the need for maintaining sterility during cell separation. Centrifugation which is a faster process than settling may be used to separate the cells. Biomass recycle systems can be easily modeled.

Fixed bed reactors In fixed bed fermenters, the cells are immobilized by absorption on or entrapment in solid, non-moving solid surfaces.

Fixed bed reactors In one type of fixed bed fermenter, the cells are immobilized on the surfaces of immobile solid particles such as  plastic blocks  concrete blocks  wood shavings or  fibrous material such as plastic or glass wool. The liquid feed is either pumped through or allowed to trickle over the surface of the solids where the immobilized cells convert the substrates into products.

Fixed bed reactors Once steady state has been reached there will be a continuous cell loss from the solid surfaces. Fixed Bed types of fermenters are widely used in waste treatment In other types of fixed-bed fermenters, the cells are immobilized in solidified gels such as agar or carrageenin

Fixed bed reactors In these fermenters, the cells are physically trapped inside the pores of the gels and thus giving better cell retention and a large effective surface area for cell entrapment. In order to increase the surface area for cell immobilization, some researchers have investigated the use of hollow fibres and pleated membranes as immobilization surfaces. Industrial applications of fixed bed reactors include  waste water treatment  production of enzymes and amino acids  steroid transformations

Fixed bed reactors One advantage of fixed bed reactors is that non-growing cells can be used. In such systems, the cells enzymatically act on substrates in the feed. The cells can be either inactivated or not fed nutrients required for growth.

Fluidised bed reactors In fluidized-bed fermenters the cells are immobilized on or in small particles. The use of small particles increases the surface area for cell immobilization and mass transfer. Because the particles are small and light, they can be easily made to flow with the liquid (ie. fluidised).

Small moving particles Fluidised bed reactors

Fluidized bed reactors The fluidisation of the particles in the reactor leads to the surface of the particles being continuously turned over. This also increases the mass transfer rate. Fluidised beds are typically categorized as either being a  2 phase system which are not aerated and  3 phase system which is aerated by sparging Fluidized bed bioreactors are used widely in wastewater treatment.

Fluidized bed reactors Fluidized bed bioreactors are also used for animal cell culture. Animal cells are trapped in gels or on the surface of special particles known as "microcarriers". Fluidized bed reactors are one example of perfusion culture technology used for animal cell culture.

Comparing fluidised bed and fixed reactors Fluidised bed reactors are considerably more efficient than fixed bed reactors for the following reasons: 1) A high concentration of cells can be immobilized in the reactor due to the larger surface area for cell immobilization is available 2) Mass transfer rates are higher due to the larger surface area and the higher levels of mixing in the reactor. 3) Fluidised bed reactors do not clog as easily as fixed bed reactors.

Comparing fluidised bed and fixed reactors Fluidised bed reactors are however more difficult to design than fixed bed reactors. Design considerations include:  Setting the flow rate to achieve fluidisation  Ensuring that bubble size remains small during the fermentation.  Prevention of the cells from falling or "sloughing" off the particles.  Minimising particle damage.

Flocculated cell reactors In flocculated cell reactors, the cells are trapped in the reactor due to an induced or natural flocculation process. In flocculation cells tend to group together causing them to come out of solution and to sink towards the base of the reactor. Flocculated cell reactors are used widely in anaerobic waste treatment processes. In these reactors, the methanogenic and other bacteria form natural flocs. The flocs move due to the release of methane and carbon dioxide by the cells.

Flocculated cell reactors Large scale anaerobic flocculated cell systems, known as Upflow Anaerobic Sludge Blanket processes are widely used in Europe for the anaerobic digestion of high strength industrial wastewaters. The reactors are typically egg-shaped.

Flocculated cell reactors Cells form flocs which gently fall and rise with gases they produce

Kinetics of immobilized enzyme  Km and Vmax are changed

Biosensor Coating of immobilized enzymes encapsulated in biomaterial Species to be analyzed must diffuse towards encapsulated enzyme (diffusion path must be short as possible to minimize response time) Chemical response of enzymatic reaction is then transformed into measurable signal (electrochemical or optical)

Biosensor An enzyme specific for the substrate of interest is immobilized between two membrane layers, polycarbonate and cellulose acetate. The substrate is oxidized as it enters the enzyme layer, producing hydrogen peroxide, which passes through cellulose acetate to a platinum electrode where the hydrogen peroxide is oxidized. The resulting current is proportional to the concentration of the substrate.hydrogen peroxide is oxidized

Immobilized enzymes-Applications in food industry : Lactose removal in milk – Milk contains 5% lactose which must be removed before feeding babies deficient in β-galactosidase (lactose hydrolyzing enzyme). A column packed with β-galactosidase immobilized in polyacrylamide gel allows continuous lactose removal. Hydrogen peroxide removal in milk after sterilization – Immobilized catalase by covalent binding with carboxychloride resin Bitterness removal in orange juice – Naringinase immobilized on copolymer, used in both batch and column systems

Applications in waste treatment: Reduction of phenol content in wastewater using immobilized polyphenol oxidase Removal of cyanide: – Improve use of microorganisms to decompose cyanide by entrapping enzyme in polyacrylamide gel and packed into column Does not allow simultaneous removal of several different substances but allows removal of specific substances

Uses in medicine Detoxification: immobilization of phospholipase A 2 and heparinase in bioreactors – Urease-containing microcapsules injected intraveneously decrease urea content in blood Wound dressings: Immobilization of proteolytic enzymes (trypsin) onto textiles used as wound dressings to accelerate cleaning and healing of infected wounds and reduces amount of enzyme used as compared with use of enzyme solutions Low Molecular Weight Protamine (LMWP) Reverse haparin after surgery Some patients are sensitive to protamine

Immobilized enzymes for controlled release Two major applications: – Pharmaceutical (enzymes as drugs),food (release of enzymes from liposomes)

1.Higher stability against denaturing actions and prolonged circulation life-time without loss of activity due to degradation, inhibition or capture by cells of reticuloendothelial system 2.Where enzyme presence in other organs and tissues is undesired

Liposomes Carriers for immobilized enzymes

Examples in Food Industry Food additives: encapsulated β-galactosidase added to milk. The enzyme is only active during digestion process, when the enzyme is released from the lipid vesicles Acceleration of cheese ripening: hard cheeses take up to 2 yrs to reach optimum flavor and texture; need to reduce ripening costs. Immobilize ripening enzymes into liposomes. Method of release: many parameters in cheese can induce a destabilization of lipid bilayers. Sensitivity to pH, temperature, and interactions with other lipids could induce release.

Conclusion Immobilized enzymes are used in a variety of fields in industry Method of immobilization is crucial in determining how the enzyme will function More work is required to optimize usage of immobilized enzymes for improved use

REFERENCES Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),Prentice Hall, New York, James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986.