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TOPIC 5DESIGN OF BIOCATALYTIC PROCESSESThe most common definition for immobilized enzymes is that proposed by Katchalski-Katzir in the 1960s:

Enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously.

According to this definition, three types of immobilized enzymes can be distinguished:1. Heterogenization of the soluble enzyme by coupling to an insoluble support by adsorption or covalent binding, by cross-linking of the enzyme or entrapment in a lattice or in microcapsules such as alginate beads

2. Retention of the enzyme by means of ultrafiltration membranes

3. Use of whole cells for biotransformations using their enzyme apparatusAdvantages over suspension cultures(1). Immobilisation provides high cell concentration(2). Immobilisation provides cell reuse and eliminates the costly processes of cell recovery and cell recycle(3).Immobilisation eliminates cell washout problems at high dilution rates(4). Combination of high cell concentrations and high flow rates allows high volumetric productivities(5).Favourable microenvironmental conditions(6). Improves genetic stability(7). Protects against shear damageAdvantages of immobilised cell reactorsBeing able to maintain high cell concentrations in the reactor at high dilution rates provides immobilised cell bioreactors with advantages over chemostats.

More biomass means that the fermenter contains more biocatalysts, thereby high bioconversion rates can be achieved. Immobilised cell bioreactors are also more stable than chemostats.

Inhibitor enters inlet feedImmobilised bioreactorChemostatTimeBiomassA higher cell concentration in the immobilised bioreactor prevents the microbial population from completely washing out.

cause a rapid drop in cell numberstake time for the cells numbers to build up againcells are not as easily washed out of an immobilized cell reactorrecovery time will be more quicker and fall in biomass concentration will be smallerLimitations(1). Often the product of interest has to be excreted from the cell

(2). Complications with diffusional limitations

(3). Control of microenvironment conditions is difficult due to heterogeneity in the system

(4). Growth and gas evolution can lead to mechanical disruption of the immobilised matrixActive immobilisationIs entrapment or binding of cells by physical or chemical forces

Physical entrapment within porous matrices is the most widely used method of cell immobilisation

Immobilised beads should be porous enough to allow transport of substrates and products in and out of the beadsActive immobilisationBeads can be prepared by 1) Gelation of polymers2) Precipitation of polymers3) Ion exchange gelation4) Polycondensation5) Polymerisation6) EncapsulationPassive immobilisationBiological films

The multilayered growth of cells on solid support surfaces

The support material can be inert or biologically active

Biofilm formation is common in natural and industrial fermentation systems, i.e biological wastewater treatment and mold fermentationsInactivation factors in designing immobilized cell reactorThe abrasion effect due to shear forces by stirrer or mixing mechanism, impeller type and particle-particle collisions in reactors can cause unfolding on enzymes must be considered.The bimolecular reactions of immobilized enzyme with substrates or products at high concentrations used in enzyme processes must be considered which can be controlled through flow rate. The substrate and product in enzyme reactor will affect reactor operation.Mass transfer limitations of reactor designed due to adsorption of molecules or microorganisms in or on support also need to be considered, where it can reduce the specific activity of enzymes from the immobilized cell.The temperature controlled in the reactor also must be considered wherein increasing and drecreasing in temperature which is not suitable can cause the unfolding of chemical modification and dissociation of oligomeric enzymes, respectively.The pH controlled in reactor must be considered due to increasing and decreasing of pH can cause unfolding and instability of enzyme from microorganism which might denatured the protein.Description of support materialThe HydrogelsNaturalCarrageenanAlginateAgarGelatin

SyntheticPolyvinyl alcoholPolyurethanePolyethylene glycolTypes 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 / packed bedFluidized bed reactors Flocculated cell systems

EffluentBiomass recycleFermenterFresh feedBiomass separation systemCell recycle systemIn a fermenter with cell recycle the cells are separated from the effluent and then recycled back to the fermenter; thus minimizing cell removal from the fermenter:Cell recycle systemsCell 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 would be used to separate the cells.

Biomass recycle systems can be easily modelled. Fixed bed reactorsIn fixed bed fermenters, the cells are immobilized by absorption on or entrapment in solid, non-moving solid surfaces.

Fixed bed reactorsIn 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 includewaste water treatment production of enzymes and amino acids steroid transformations

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

Fixed bed reactorsOne advantage 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.

Small moving particlesFluidised 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).

Fluidized bed reactorsThe 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 a2 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 reactorsFluidized 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 reactorsFluidised 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 reactorsLarge 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 reactorsCells form flocs which gently fall and rise with gases they produce Enzyme ReactorsMembrane reactors have been used quite recently as have applications of whole cell processes.

Retention of the cells within the reactor may be achieved by membrane separation or by the same immobilization methods that are used for isolated enzymes (34).

In principle, the cell itself can be regarded as a form of native immobilization of enzymes.

Biosensors are a very special form of carrier-fixed biocatalysts.1. Additional costs for support and chemicals performing the immobilization have to be balanced against the increase of stability.

2. Loss of activity during the immobilization step.

3. When the catalyst is immobilized only by adsorption or entrapment without covalent attachment, its leakage from the carrier support has to be examined and compared with the overall deactivation rate.When considering the use of soluble or carrier-fixed enzymes, the following topics we have to be addressed:4. Mass transfer limitations for enzymes on a support may cause problems when adjusting of the pH is necessary during the reaction.

5. With soluble enzymes, higher volumetric activities at high catalyst concentrations are possible, enabling conversion of poor substrates at reasonable rates.

6. Whereas membrane reactors can be easily sterilized before use, this is not possible for reactors with carrier-fixed enzymes.

To prevent microbial contamination, these processes are quite often operated at higher temperatures. 7. When enzymes are to be used together with organic solvents to increase reactant or product solubility or to alter their kinetics, it may become necessary to immobilize them on a support

The support will at the same time act as a water pool to maintain the enzymatic activity.

In such systems, water-insoluble organic solvents have less effect on the enzyme stability than water-soluble solvents.

Process design and operational strategies of immobilized enzyme reactorsThe final decision for a certain reactor design should be based on an optimization process covering all relevant factors contributing to the overall costs, including investment, catalyst consumption, or productivity.