Willaert R., Baron G. (1996) Gel entrapment and micro...

Gel Entrapment and Micro-encapsulation

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Willaert R., Baron G. (1996)Gel entrapment and micro-encapsulation:

methods, applications and engineeringprinciples.

Reviews in Chemical Engineering 12: 5-205.

R.G. Willaert and G.V. Baron Reviews in Chemical Engineering

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GEL ENTRAPMENT AND MICRO-ENCAPSULATION:METHODS, APPLICATIONS AND ENGINEERING PRINCIPLES

Ronnie G. Willaert

and

Gino V. Baron

Vrije Universiteit BrusselDepartment of Chemical EngineeringPleinlaan 2, B-1050 Brussel, Belgium


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CONTENTS

ABSTRACT...............................................................................................................................51. INTRODUCTION AND SCOPE...........................................................................................5

1.1 Classification of Immobilised Cell Systems...................................................................61.1.1 Attachment to a Surface........................................................................................91.1.2 Entrapment within Porous Matrices....................................................................10

1.1.2.1 Gel Entrapment.....................................................................................111.1.2.2 Preformed Support................................................................................12

1.1.3 Containment behind a Barrier.............................................................................121.1.4 Self aggregation of Cells.....................................................................................16

1.2 Scope..............................................................................................................................162. GEL ENTRAPMENT..........................................................................................................20

2.1 Gel Materials and Methods...........................................................................................202.1.1 Natural Polymers.................................................................................................21

2.1.1.1 Polysaccharides.....................................................................................212.1.1.2 Proteins.................................................................................................29

2.1.2 Synthetic Polymers..............................................................................................302.2 Physiology of Gel Immobilised Cells...........................................................................36

2.2.1 Physiology and Experimental Techniques..........................................................362.2.2 Additional Physiological Aspects.......................................................................43

2.2.2.1 Bacteria.................................................................................................432.2.2.2 Fungi.....................................................................................................452.2.2.3 Algae.....................................................................................................482.2.2.4 Plant Cells.............................................................................................492.2.2.5 Animal Cells.........................................................................................50

2.3 Applications...................................................................................................................512.3.1 Gel Immobilised Growing Microbial Cells.........................................................51

2.3.1.1 Production of Amino Acids..................................................................512.3.1.2 Production of Organic Acids................................................................532.3.1.3 Production of Antibiotics......................................................................572.3.1.4 Transformation of Steroids...................................................................602.3.1.5 Production of Enzymes.........................................................................612.3.1.6 Production of Alcohols.........................................................................672.3.1.7 Production of Sugars.............................................................................67


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2.3.1.8 Applications for Food and Beverages...................................................702.3.1.9 Gel Entrapment of Microbial Cells for Use in Agriculture..................702.3.1.10 Environmental Applications.................................................................722.3.1.11 Energy Production by Gel Immobilised Cells......................................742.3.1.12 Production of Other Useful Biochemicals............................................762.3.1.13 Production of Proteins by Gel Immobilised Genetically

Engineered Micro-organisms................................................................782.3.2 Gel Immobilised Plant Cells................................................................................78

2.3.2.1 Biotransformation.................................................................................782.3.2.2 Salvage Synthesis.................................................................................782.3.2.3 De novo Synthesis.................................................................................782.3.2.4 Artificial Seeds.....................................................................................81

2.3.3 Gel Immobilised Animal Cells............................................................................823. MICRO-ENCAPSULATION..............................................................................................85

3.1 Encapsulation Materials and Methods..........................................................................863.1.1 Alginate...............................................................................................................863.1.2 Polyacrylate.........................................................................................................913.1.3 Nylon...................................................................................................................943.1.4 Polyethyleneimine...............................................................................................953.1.5 Chitosan...............................................................................................................953.1.6 Cellulose..............................................................................................................963.1.7 Agarose-acrylamide.............................................................................................973.1.8 Gelatin.................................................................................................................983.1.9 Carrageenan-chitosan..........................................................................................99

3.2 Applications...................................................................................................................993.2.1 Micro-encapsulation of Islets of Langerhans......................................................993.2.2 Production by and Growth of Micro-encapsulated Cells..................................102

3.2.2.1 Mammalian Cells................................................................................1093.2.2.2 Microbial and Plant Cells...................................................................114

4. MASS TRANSFER AND MODELLING ........................................................................1164.1 Diffusion in Immobilised Cell Systems......................................................................116

4.1.1 Definitions of Diffusion Coefficients................................................................1164.1.2 Diffusion through Support Materials................................................................1174.1.3 Methods of Measurement..................................................................................1184.1.4 Diffusion in Cell-containing Matrices...............................................................120


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4.1.5 Diffusion in Gels...............................................................................................1234.1.6 Diffusion in Micro-capsules..............................................................................139

4.2 Modelling....................................................................................................................1394.2.1 Gel Immobilised Cell Systems..........................................................................139

4.2.1.1 Introduction.........................................................................................1394.2.1.2 Intrinsic Kinetics.................................................................................1414.2.1.3 Modelling............................................................................................142

4.2.2 Micro-capsules..................................................................................................1554.2.2.1 Models without Cell Growth..............................................................1554.2.2.2 Models with Cell Growth...................................................................156

5. SUMMARY AND CONCLUSIONS.................................................................................158REFERENCES.......................................................................................................................161


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ABSTRACT

Methods, modelling and applications of gel immobilisation and micro-encapsulation of livingcells are reviewed. These two methods are most frequently used as they allow for excellentcell containment and are suitable for a wide variety of cell types. The first section provides abrief introduction to the whole area of living cell immobilisation by presenting aclassification of immobilised cell systems and giving the overall objectives of this review.The next section deals with gel immobilisation. An overview of the various materials andmethods used is presented. The physiology of gel entrapped cells (bacteria, fungi, algae plantcells as well as animal cells) is discussed and included here is a discussion on some specificexperimental techniques which have been used to obtain in situ information of gel systems.This section ends with a review of applications for entrapped microbial, plant and animalcells. The third section gives an overview of the various materials and methods used tomicro-encapsulate living cells and their applications. The micro-encapsulation of Islets ofLangerhans as a bio-artificial pancreas to treat diabetes is treated in depth. Attention is furthergiven to the production of biochemical compounds by encapsulated mammalian, microbialand plant cells. A discussion of the growth of animal cells in micro-capsules is also included.In the fourth section, engineering aspects of gel immobilised and micro-encapsulation arediscussed. The discussion of mass transfer is limited to internal mass transport by diffusion.Finally, it is shown how mathematical models can be used to quantitatively describe andanalyse the behaviour of immobilised cells and steady-state, pseudo-steady state as well asdynamic models are discussed.

1. INTRODUCTION AND SCOPE

Whole-cell immobilisation can be defined as the physical confinement or localisation ofintact cells to a certain defined region of space with the preservation of some desired activity(Karel et al., 1985). The region in which the cells are localised is called immobilised cellsystem or also immobilised cell aggregate, which can be divided into three components: thecells, the support material and the solution that fills the remainder of the space (interstitialsolution). The space around the immobilised cells is also called the "micro-environment",because the chemical properties of the interstitial solution may be quite different from thoseof the bulk solution.

Studies on immobilised biocatalysts were initiated by immobilising single enzymes forsimple reactions such as hydrolysis and isomerisation. Subsequently, multi-enzyme systems,isolated cellular organelles, and treated microbial cells have been used as biocatalysts formore complicated and conjugated reactions. Moreover, many applications have beendeveloped utilising living or growing microbial cells and cells of multicellular organisms(higher plants and animals) as well as genetically improved microbial cells.

The successful application of an immobilised cell system as a biocatalyst relies on theproper choice of the components of the system: matrix, cells, reactor type. This choice will bedirected by the type of application and possible operational conditions, like temperature, pH,


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flow pattern and rates, and substrate composition. The most desirable requirements are listedin Table 1. An ideal system that possesses all these optimal properties does not exist, and asuitable compromise has to be found. In Table 2, some advantages and disadvantages ofimmobilised living cell systems compared to immobilised enzyme systems and ordinarysuspension culture systems are listed.

TABLE 1DESIRABLE REQUIREMENTS FOR IMMOBILISED CELL SYSTEMS

(modified from Looby and Griffiths, 1990)

High cell mass loading capacityEasy access to nutrient mediaSimple "non toxic" immobilisation procedureHigh surface area-to-volume ratioOptimum diffusion distance from flowing media to centre of supportMechanical stability (compression, abrasion)Chemical stabilitySterilisableReusableLow shear experienced by cellsEasy separation of cells and carrier from mediaSuitable for conventional reactor systemsSuitable for suspension as well as anchorage-dependent cellsBiocompatibility for animal cellsEconomically feasible

1.1 Classification of Immobilised Cell Systems

Generally, immobilised cell systems can be classified into four categories based on thephysical mechanism of cell localisation and the nature of the support mechanisms. Asillustrated in Figure 1, we can distinguish "attachment to a surface", "entrapment within aporous matrix", "containment behind a barrier" and "self aggregation" (Karel et al. 1985).


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TABLE 2CELL IMMOBILISATION BY SURFACE ATTACHMENT: TYPICAL NATURAL CARRIERS

Materials Reference

Celluloses Chen and Zall, 1982; Larsson and Litwin, 1985Chitin Bianchi et al., 1995Collagen (Heparin- and laminin-coated) Denomme and Qu, 1987DEAE-dextran (Cytodex 1TM, Dorma- Hirtenstein et al., 1980; Shulz et al., 1986;

cellTM, MicrodexTM, SuperbeadsTM) Forestell et al., 1992DEAE-cellulose (DE-52TM, DE-53TM) Shahar et al., 1987; Pajunen et al., 1989Dextran (CytodexTM, SuperbeadsTM) Van Wezel, 1967; Hirtenstein and Clark, 1983;

Varani et al., 1983; Schulz et al., 1986; Karkare et al., 1992

Fibronectin-coated beads Schwarz and Juliano, 1984Gelatin (GelibeadsTM, VentragelTM) Paris et al., 1983Sugar cane De Cabrera et al., 1981; Cheung et al., 1986Wood chips Durand and Navarro, 1978; Förberg and

Häggström, 1985; Lamptey and Moo-Young, 1987; Scharer et al., 1988

1.1.1 Attachment to a Surface

Cells are immobilised by adsorption to a support material. The surface attachment can beartificially induced using linking agents (metal oxides or covalent bonding agents) or canoccur by natural adsorption on various carriers (cf. Table 2 to 5 for a list with some typicalcarriers and linking agents). This immobilisation technique is very popular because it is asimple and fast technique. A suitable adsorbent for spontaneous attachment should display ahigh affinity towards the biocatalyst and yet cause minimal denaturation. Van der Waalsforces, ionic bonds, hydrogen bridges or covalent interactions are responsible for the bindingof cells. Microbial cells exhibit a dipolar character and behave cationic or anionic dependingon the pH of the solutions. Also, the physiology of the cell has a strong influence on theadhesion. Microbial cell walls carry a charge which depend on the pH of solutions and otherenvironmental conditions. As there is no barrier between the cells and the solution, thismethod cannot be used where cell free effluent is desired. Once a monolayer is formed,further attachment occurs mainly by the same mechanisms as in self aggregation, anddepends on cell-cell interactions. Studies of immobilisation on clean surfaces are often notvery relevant to practical situations where the surfaces available for cell immobilisation arerapidly coated with a variety of solute molecules, ions and proteins or larger agglomerates, orlayers of these. This "precoat" is often a better support for cell attachment than the virginsurface. After longer periods of operation, thick biofilms may become inactive in the regionclose to the solid surface and detach. The remaining cell debris, slime and other components


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then form the new surface for immobilisation and is obviously not related to the originalsurface conditions. Given enough time, even the smoothest surfaces will become fouled. Thiscan be accelerated by circulation of a solution with the above mentioned components or bychemically pretreating the surface (e.g. glutaraldehyde). Biofilm processes are difficult tocontrol since the depth of the biofilm often varies - especially - with flow rate.

Mammalian cell culture techniques have been improved by using immobilised cell systemsbased on surface attachment. A popular system is the microcarrier where cells grow on thesurface in the form of monolayers (Reuveny, 1990).

TABLE 3CELL IMMOBILISATION BY SURFACE ATTACHMENT: TYPICAL SYNTHETIC ORGANIC

CARRIERS

Materials Reference

Nylon Linko et al., 1986b; Linko and Hujanen, 1990Polyacrylamide (BiocarrierTM) Reuveny et al., 1983; Zhaoxin and Kumakura, 1994Polyacrylates and polymetha- Daugulis et al., 1981; Krug and Daugulis, 1983; Carenza

crylates et al., 1990; Zhao Xin and Kumakura, 1993Polyester Van Den Berg and Kennedy, 1981; Kennedy and Van

Den Berg, 1982Polypropylene Blain et al., 1979; Anderson et al., 1980; Genung et al.,

1982;Polystyrene (BioplasTM, Daugulis et al., 1981; Keese and Giaever, 1982; Krug

BiosilonTM, CytospheresTM) and Daugulis, 1983; Reuveny, 1983Polystryrenedivinylbemzene Jacobson and Rayan, 1982; Burke et al., 1983;

(various coatings) Fairman and Jacobson, 1983;Polyurethane Livesy-Goldblatt et al., 1977Polyvinylchloride Livesy-Goldblatt et al., 1977; Durand and Navarro, 1978;

Hollo et al., 1979; Van Den Berg and Kennedy, 1981Polyvinyltoluene van Meel et al., 1984

1.1.2 Entrapment within Porous Matrices

We can distinguish two categories: gel entrapment and entrapment in a preformed support.In the first, the porous matrix is synthesised in situ around the cells to be immobilised;whereas in the second, the cells are allowed to move into the preformed porous matrix. Gelentrapment can be further subdivided in systems using natural polymers or syntheticpolymers.


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1.1.2.1 Gel EntrapmentIn the last decade, most of the research in the general area of immobilised cells involved

porous supports that are formed around cells. Polysaccharides, synthetic polymers andproteins can be gelled into hydrophilic matrices under mild conditions to allow cellentrapment with minimal loss of viability. Very high biomass loadings can be achieved. Thepolymer-cell mixture can be formed in different shapes and sizes. The most common formsare small beads about 1 to 5 mm in diameter.

Gel entrapment with natural polymers is a very mild technique and damage to living cellscan be minimised. Disadvantages of gel entrapment are the poor mechanical strength and thelimited longevity of the gel structure. The gel structure is easily destroyed by cell growth andcarbon dioxide production. When cell free effluent is desired, cell leakage is a seriousdisadvantage of this immobilisation technique. The gel structure can be enforced by thereaction of alginate with other molecules like polyethyleneimine, silica, genipin andpolyvinylalcohol. Mass transfer limitations can be severe because mass transfer is onlyaccomplished by molecular diffusion. Substrates usually transport sufficiently rapidly butoxygen transport limitations occur in almost all conditions, making aerobic productionsdifficult.

TABLE 4CELL IMMOBILISATION BY SURFACE ATTACHMENT: TYPICAL INORGANIC CARRIERS

Materials Reference

Activated carbon Scott and Hancer, 1976; Huysman et al., 1983; Mörsen and Rehm, 1990; Halfmeier et al., 1993

Ceramics Marcipar et al., 1979; 1983; Scharer et al., 1988; Krekeler et al., 1991Charcoal Scharer et al., 1988Clay Van Den Berg and Kennedy, 1981; Huysman et al., 1983Coal Hancher and Perona, 1982; Myerson and Kline, 1983Fluoroapatite DiSpirito et al., 1983Glass Arcuri et al., 1980; Rutter and Leech, 1980; DiSpirito et al., 1983;

Huysman et al., 1983; Varani et al., 1983; Bringi and Dale, 1985; Shuler et al., 1990

Pumice stone Cabral et al., 1984Pyrite DiSpirito et al., 1983Sand DiSpirito et al., 1983; Kosaric and Blaszczyk, 1990; Halfmeier et

al., 1993Silicates (bentinite, Kosaric and Blaszczyk, 1990; Borja and Banks, 1994

sepiolite, saponite)Stainless steel Juneja et al., 1986Titanium Paul Sr., 1990Vermiculite Bland et al., 1982Zeolite Huysman et al., 1983; Borja and Banks, 1994


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1.1.2.2 Preformed SupportIn this case, the cells are entrapped in a preformed matrix which protects them from the

flow and shear field outside the particles. As with the adsorption method, cells are notcompletely separated from the effluent in these systems. Mass transport of substrates andproducts can be achieved by molecular diffusion as well as convection, and consequentlymass transport limitations are in most cases less stringent. A high degree of cell viability isretained in most entrapment methods since the supports are inoculated directly from the bulkmedium and continuous renewal of the cell population is possible. The preformed supportparticles are more resistant to the compression and disintegration than the gel particles. Thehigh cell packing densities associated with other entrapment methods are not achieved in thiscase. Cells are retained partly by surface attachment, partly by self aggregation in the cavitiesof the matrix and partly in the zones with very low flow or dead end pockets in the material.Some typical support materials are listed in Table 6.

TABLE 5CELL IMMOBILISATION BY SURFACE ATTACHMENT: LINKING AGENTS

Materials Reference

Chelation with metal compounds:Aluminium Van Haecht et al., 1985Iron Mozes and Rouxhet, 1985Titanium oxides Kennedy, 1979; Kennedy et al., 1980; Dias et al.,

1982Zirkonium oxides Kennedy, 1979

Covalent bonding with coupling agents: Kolot, 1981; Jirku and Turkovà, 1987AminosilaneCarbodiimideDiisocyanateGlutaraldehyde Navarro and Durand, 1977; Jirku et al., 1980a, b;

Jirku et al., 1981; Kim et al., 1982

1.2.3 Containment behind a Barrier

The barrier can be preformed (e.g. hollow fibre systems, membrane reactors; typicalsynthetic membranes are listed in Table 8) or formed around the cells to be immobilised (e.g.micro-capsules).


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TABLE 6CELL IMMOBILISATION BY ENTRAPMENT WITHIN POROUS PREFORMED SUPPORTS

Materials Reference

Alumina pellets Koutinas et al., 1988Brick Messing, 1982; Monsan et al., 1987; Opera and Mann,

1988Celite Gbewonyo and Wang, 1983; Arcuri et al., 1986;

Gbewonyo et al., 1987; Wang and Wang, 1990; Chun and Agathos, 1991; 1993; Dias et al., 1994

Cellulose Reuveny, 1983; Shahar et al., 1994Ceramics Demuyakor and Ohta, 1992Collagen (VeraxTM, Agathos et al., 1990; Cahn, 1990; Looby and Griffiths,

MicrophereTM) 1990; Ray et al., 1990; Vieth, 1994Collagen-glycosamine-glycan Cahn, 1990; Looby and Griffiths, 1990

(InfomatrixTM)Cordierite Ghommidh et al., 1981; 1982; Messing, 1982Cotton cloth Joshi and Yamazaki, 1984Gelatin (CultipherTM) Nilsson et al., 1986a; Cahn, 1990; Looby and Griffiths,

1990; Ohlson et al., 1994Glass Bisping and Rehm, 1986; Looby and Griffiths, 1988;

Aivasidis and Wandrey, 1990; Reiter et al., 1992b; Halfmeier et al., 1993; Lohmeyer and Sander, 1993; Benito et al., 1994; De Backer and Baron, 1994

Kaolinite Jian et al., 1983Natural sponge Huysman et al., 1983Polyester foam Muallem et al., 1983; Black et al., 1984Polymethane + polyvinylchloride Huysman et al., 1983Polyphenyleneoxide Jirku et al., 1980Polystyrene Fletcher, 1976; Reuveny, 1983; Lee et al., 1992Polyurethane Huysman et al., 1983; Muallem et al., 1983; Mavituna

and Park, 1985; Kobayashi et al., 1990; Matsushita et al., 1990; Soetaert et al., 1990; Armentia and Webb; 1992; Borja and Banks, 1994; Pflugmacher and Gottschalk, 1994

Polyvinyl formal resin Yamaji et al., 1989; Yanagi et al., 1992Silica Navaro and Durand, 1977; Monsan et al., 1987Silicone rubber (ImmobaSilTM) Oriel, 1988a; Fuller, 1995; Knights, 1995Stainless steel particles Atkinson et al., 1980; Black et al., 1984; Webb et al.,

1986b


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This kind of cell immobilisation is ideal for several specialised systems. When cellseparation from the effluent is required, or when some high molecular weight product needsto be separated from the effluent, these systems are highly useful. In the biomedicalengineering field, micro-encapsulation leads to immunoprotection and artificial organs. Inmost cases, nutrients are supplied to, and products are removed from the cell mass bydiffusion through the barrier. Consequently, mass transfer limitations can reduce theefficiency of these systems. This is not the case with hollow fibre systems where masstransfer can be enhanced by convective mass transport (e.g. perfusion or "Starling flow") if apressure difference is imposed on the membrane. A serious problem with micro-encapsulation may be toxic effects on cells due to toxic reagents, solvents, reactive conditionsand pH levels. The barrier which immobilises cells can be as simple as the liquid /liquidphase interface between two immiscible fluids (see Table 7 for some examples) (Kaul andMattiasson, 1991). Two phase systems are useful in applications where substrates or productsare partitioned separately.

TABLE 7CELL IMMOBILISATION BY CONTAINMENT BEHIND A BARRIER: TWO PHASE ENTRAPMENT

Materials Reference

Dextran/water Andersson et al., 1984Paraffin/water Miyawaka et al., 1986Polyethyleneglycol/dextran Larsson and Mattiasson, 1992Polyethyleneglycol/water Andersson et al., 1984; 1987; Larsson and

Mattiasson, 1984Polyethyleneglycol/pottassium phosphate Chang et al., 1992Polyethyleneimine/dextran or hydroxyethyl Dissing and Mattiasson, 1993

cellulose or polyethyleneglycolTween 85/water/isopropylpalmitate Haering et al., 1987


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TABLE 8CELL IMMOBILISATION BY CONTAINMENT BEHIND A BARRIER: TYPICAL SYNTHETIC

MEMBRANES

Materials Reference

Acrylic polymers Hopkinson, 1985; Lacy et al., 1991; Lanza et al., 1994Cellulose Lee et al., 1980; Shuler, 1981; Chen and Zall, 1982;

Vasudevan et al., 1988; Jeong et al., 1991; Vieth, 1991; Schrezenmeir et al., 1994

Polyacrylonitrile Ku et al., 1981Polycarbonate Shuler et al., 1986Polyetheretherketone Zekorn et al., 1990Polyetherimide Kniebush et al., 1990Polypropylene Roy et al., 1982; Blanch et al., 1984; Inloes et al., 1985;

Nanba et al., 1985; Shuler et al., 1986; Chung et al., 1987;Chung and Chang, 1988

Polysulphone Inloes et al., 1983; 1985; Jose et al., 1983; Roy et al., 1983; Wisniewski et al., 1983; Hopkinson, 1985; Altshuler et al.,

1986; Bunch et al., 1990; Piret et al., 1991; Patkar et al., 1993; Zekorn et al.,1993Polyvinylchloride acrylic Knazek et al., 1972; Altman et al., 1986 copolymer

TABLE 9SELF AGGREGATION OF CELLS: NATURAL AGGREGATION

Materials Reference

Algal flocs Atkinson and Daoud, 1976; Calleja, 1984Animal cell aggregates Radlett, 1987; Avgerios et al., 1990; Litwin, 1990; Reiter et al.,

1992a; Moreira et al., 1995Fungal pellets Steel et al., 1955; Smith and Greenshields, 1974; Atkinson and

Daoud, 1976; Metz and Kossen, 1976; Schügerl et al., 1983; Michel Jr. et al., 1992; Lejeune and Baron, 1995; Nielsen and Carlsen, 1995

Microbial flocs Atkinson and Daoud, 1976; Arcuri et al., 1980; Prince and Barford, 1982a, b, c; Strandberg et al., 1982; Thompson and Forster, 1983; Hsiao et al., 1983; Calleja, 1984; Straver et al., 1993

Plant calli Atkinson and Daoud, 1976; Wagner and Vogelmann, 1977; Azechi et al., 1983


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1.2.4 Self Aggregation of Cells

Self aggregation systems can be artificially induced or result from natural aggregation (cf.Table 9). Natural aggregates can be formed by fungi, microbial, algal, plant and animal cells.Artificial flocculating agents or cross-linkers may be added to enhance the process ofaggregation for cells that do not naturally flocculate (cf. Table 10). Flocculation can also beinfluenced at the genetic level using genetic engineering techniques (Stratford, 1992; Watariet al., 1994) or by selection of a flocculating strain (e.g. Fein et al., 1983; Scott, 1983; Wanget al., 1994).

TABLE 10SELF AGGREGATION OF CELLS: TYPICAL LINKING AGENTS TO INDUCE ARTIFICIALLY

AGGREGATION

Materials Reference

Polyelectrolytes and positively chargedparticles: Karel et al., 1985; Attia, 1987Al3+, Ba2+, Ca2+, Fe3+, Mg2+ Kosaric and Blaszczyk, 1990Chitosan Goldberg et al., 1990Nitrosoguanidine Lee et al., 1982Poly-L-lysine Goldberg et al., 1990Proprietary polyelectrolytes (A10R, C7R) Nazly and Knowles, 1981

Covalent bonding with coupling agents:Glutaraldehyde Karel et al., 1985

Inert powders: Weeks et al, 1982; 1983Calcium carbonateIron sandIron oxideNickel powder

1.2 Scope

Over the last decade, a vast research effort has been devoted to the immobilisation of livingorganisms. Most of this research was focused on entrapment in gels and a wealth ofinformation has become available. Gel immobilisation has been applied in very differentareas, from the production of synthetic seeds in agriculture, the production of recombinant


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proteins in industrial microbiological processes to the construction of artificial organs inmedicine. The scope of this review is to bring this scattered information together in a unifiedapproach. As more and more chemical engineers are involved in the design and utilisation ofthese techniques, they should find a wealth of information in this review to assist them intheir work. Researchers involved in the various subfields of biotechnology and biochemicalengineering may also from this review learn about other methods, approaches, models orsimply "tricks of the trade" and hence not have to rediscover the wheel.

Emphasis is on materials and methods, physiological aspects and applications. The variousgel materials are described, guidelines in their selection, methods for living cell entrapmentexplained and the most important characteristics are described, such as strength, transportproperties, or biocompatibility. Since immobilised cells are surrounded by a micro-environment, their physiological behaviour can be different from their free suspensioninformation. This topic has been well investigated for gel immobilised living cells. Anoverview of this research is presented, with attention for the "new" experimental techniquesto extract in situ information. Most applications are collected and subdivided in the differentapplication fields.

Micro-encapsulation has been developed since the early 1980s as a means to construct bio-artificial organs. The micro-encapsulation membrane acts as a immunoisolation barrier whichprevents the rejection of foreign cell types. Research was focused on the micro-encapsulationof Islets of Langerhans to treat diabetes. A review of the different materials and methods usedto encapsulate living cells is presented and the bio-artificial pancreas is covered in detail.Applications using other mammalian cells as well as microbial, plant and insect cells are alsotreated.

Next, the engineering principles of these two immobilised cell systems are reviewed.Focus is on the cell aggregate itself. Compared to gel entrapment, an additional mass transferlayer is introduced in the case of micro-encapsulation. An overview of internal mass transferby diffusion in gel matrices (with and without cells), in micro-capsules and methods formeasuring diffusion is provided. An extensive database of measured diffusion coefficientshas been assembled as an aid in design. Cell immobilisation in gels and micro-capsulesresults in reaction-diffusion systems such as in heterogeneous catalysis where the reactioncan be diffusion limited. The evaluation of these systems using mathematical modelling isdiscussed and an overview of the models to describe steady-state as well as dynamicbehaviour is presented.

Since the number of papers and books produced over the last decade is so large, it was notpossible to be complete. A selection has been made but still a very large number of keyreferences is included, and constitutes an excellent start for any aspect or application. Thereare a number of books available on cell immobilisation and a list of books is presented inTable 11, with also a list of general biochemical engineering books (recent books also coversome basics of cell immobilisation).

Our major aim was to give both the novice and the expert a collection of facts, tools andrules to design and analyse gel or micro-capsule immobilised cell systems in an orderlymanner. It is our hope that chemical engineers will more easily find their way in the vast


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quantity of accumulated knowledge on the chemical and physiological aspects of thesesystems, but also to convince chemists and microbiologists to use the methods of chemicalengineering for a more rational and quantitative approach to this field.

TABLE 11REFERENCE BOOKS ON IMMOBILISATION AND BIOCHEMICAL ENGINEERING

Title Reference

ImmobilisationImmobilized cells and organelles (vol I and II) Mattiasson, 1983Immobilised cells and enzymes Woodward, 1985Immobilisierte biokatalysatoren Hartmeier, 1986Process engineering aspects of immobilised cell systems Webb et al., 1986aImmobilized cells: principles and applications Tampion and Tampion, 1987Bioreactor immobilized enzymes and cells Moo-Young, 1988Physiology of immobilised cells de Bont et al., 1990Fundamentals of animal cell encapsulation and immobilization Goosen, 1993Wastewater treatment with microbial films Iwai and Kitao, 1994Immobilized biosystems: theory and practical applications Veliky and McLean, 1994Immobilised living cell systems: modelling and experimental Willaert et al., 1995

methods

Biochemical EngineeringBiochemical reactors Atkinson, 1974Biochemical engineering Aiba et al., 1976Biotechnology (8 vols.) Rehm and Reed, 1982Biochemical engineering fundamentals Bailey and Ollis, 1986Basic biotechnology Bu'lock and Kristiansen,

1987Bioreaction engineering (vol 1 & 2) Schügerl, 1987Airlift bioreactors Christy, 1989Bioprocess computations in biotechnology Ghose, 1990Chemical engineering problems in biotechnology Winkler, 1990Biochemical engineering and biotechnology handbook Atkinson and Mavituna,

1991Bioprocess technology: modelling and transport phenomena BIOTOL, 1992Biological reaction engineering Dunn et al., 1992


20

TABLE 11 (continued)

Title Reference

Biochemical engineering Lee, 1992Bioprocess engineering Shuler and Kargi, 1992Bioprocess engineering principles Doran, 1994Advances in bioprocess engineering Galindo and Ramírez, 1994Bioreactor engineering principles Nielsen and Villadsen, 1994Bioprocess engineering Vieth, 1994Bioreactor system design Assenjo and Merchuck, 1995


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2. GEL ENTRAPMENT

Entrapment in polymeric gels has become a very popular immobilisation method. Variousmaterials has been successfully used to immobilise living cells. Fundamental and appliedresearch have been performed on gel entrapped microbial, fungal, algal, plant and animalcells. In this chapter, attention is focused on the different gel materials and methods whichhave been used to entrap living cells, the influence of the immobilisation on the cellphysiology (including the results obtained using specific experimental techniques); and anoverview of applications is provided.

2.1 Gel Materials and Methods

Various types of polymers have been used for gel immobilisation. The mechanisms for gelformation with corresponding gel materials are listed in Table 12. The gel is formed in thepresence of the cells. This requires a mild (non toxic) entrapment method. Gel materials canbe classified as natural and synthetic polymers. Natural polymers are composed ofpolysaccharides or proteins.

TABLE 12GEL FORMATION MECHANISMS FOR CELL ENTRAPMENT

Principle of gelation Material

Ionotropic gelation Alginate, chitosanThermal gelation Agar, agarose, κ-carrageenan, collagen,

gelatine, gellan gum, curdlanPrecipitation Cellulose, cellulose triacetatePolymerisation with cross-linking reagent Polyacrylamide, polymethacrylate,

polyacrylamide-hydrazidePolycondensation Polyurethane, epoxy resinRadical-mediated polymerisation by irradiation Photo-cross-linkable resin prepolymers

with near ultra violet lightCross-linking through photo-dimerisation Photo-sensitive resin prepolymers

by irradiation with visible or UV lightRadiation polymerisation Poly(2-hydroxyethyl methacrylate/

acrylate), Poly(vinyl alcohol),poly(ethylene glycol) diacrylate/ dimethacrylate

Gelation by iterative freezing and thawing or Polyvinyl alcoholcross-linking with boric acid (and Ca-alginate)


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2.1.1 Natural Polymers

2.1.1.1 Polysaccharides(a) Algal PolysaccharidesAlgal (marine) polysaccharides are the most widely used matrices for immobilisation of

biocatalysts. Alginate, carrageenan and agar are all extracted from algae.

(a.1) AlginatesIn recent years, entrapment of cells within spherical beads of calcium alginate has

become one of the most widely used methods for immobilising living cells. The success ofthis method is mainly due to very mild conditions under which immobilisation is performedand it is a fast, simple and cost-effective technique. The cell suspension is mixed with asodium alginate solution, and the mixture dripped into a solution containing multivalentcations (usually calcium). The droplets form gel spheres instantaneously, entrapping the cellsin a three-dimensional lattice of ionically cross-linked alginate. Ca2+ ions may also beintroduced in situ by adding inorganic calcium salts of low solubility (Johansen and Flink,1986a, b; Clark and Ross-Murphy, 1987). Recently, Poncelet et al. (1992) formed smalldiameter alginate beads via internal gelation of the alginate solution emulsified withinvegetable oil. Calcium ion release from an insoluble citrate complex was initiated via gentleacidification with an oil soluble acid, which partitions to the aqueous alginate phase. Bycontrolling the conditions under which the water-in-oil dispersion is produced, the beaddiameter could be controlled within the range of 200 to 1000 µm. The gelation of sodiumalginate by Ca2+ involves a diffusion and reaction process. The moving gel front has beensimulated using a mathematical model (Lin, 1991).

Alginates constitute a family of unbranched copolymers of 1,4-linked β-D-mannuronic(M) and α-L-guluronic acid (G) of widely varying composition and sequence, depending onthe organism and tissue they are isolated from. The monomers are arranged in a pattern ofblocks along the chain, with homopolymeric regions (termed M and G blocks) interspersedwith regions of alternating structure (MG blocks) (Smidsrød et al., 1972; Smidsrød, 1974).Divalent cation-induced gelling of alginates in solution reflects their specific ion bindingaccompanied by a conformational change. The divalent cations bind preferentially to the Gblocks. This process has been shown to be highly co-operative. The size of the co-operativeunit is reported to be more than 20 monomers. Guluronate sequences in alginates associateinto matched crystalline aggregates in a two-fold ribbon like form. Ions co-operatively boundduring the process are placed inside the electronegative cavities, like eggs in an egg-box (the"egg-box" model). Between theses cavities and similar sequences in other polymer chains,bonds are formed giving rise to junction zones in the gel network. The physical properties ofalginate beads depend strongly on the composition, sequential structure, and molecular sizeof the polymers. Beads with the highest mechanical strength, lowest shrinkage, best stabilitytowards monovalent cations, and the highest porosity were made from alginate with a contentof L-guluronic acid higher than 70 % and an average length of the L-guluronic acid residues(G-blocks) of about 15 (Martinsen et al., 1989).


23

Algin or alginic acid is extracted from brown seaweeds. Although it is the majorconstituent of all brown seaweeds, the principal seaweed genera used for commercialpurposes are Ascophyllum, Durvillea, Ecklonia, Fucus, Laminaria, Macrocyctis, Nereocystis,Sargassum and Turbinaria (Bucke, 1987; Landau 1992). The highest content of α-L-guluronic acid residues is usually found in alginate prepared from stipes of old Laminariahyperborea plants. Alginates from Ascophyllum nodosum and Laminaria japonica arecharacterised by a low content of G-blocks and a low gel strength. The alginate fromMacrocystis pyrifera, which is most frequently used for immobilisation, yields gels withlower strength and stability than those made from stipes of L. hyperborea. Bacterial alginate,with a more extreme composition, can be isolated from Azotobacter vinelandii which, incontrast to Pseudomonas species, produces polymers containing G-blocks (Smidsrød andSkjåk-Bræk, 1990). Mannuronic acid can be converted enzymatically to guluronic acid usingC-5 epimerase from A. vinelandii (Skjåk-Bræk et al., 1986b); this technique can be used to"tune" the alginate to the optimal guluronic acid content.

The major disadvantage of the use of calcium alginate beads is its sensitivity towardschelating compounds such as phosphate, citrate, EDTA and lactate, or anti-gelling cationssuch as Na+ or Mg2+. On the other hand, if it is desirable to free the cells from the gel (e.g.cell counting), these compounds can be used to dissolve the gel beads. Various ways toovercome this limitation is to keep the beads in a medium containing a few millimolar freecalcium ions and keep the Na+/Ca2+ ratio low (Cheetham et al., 1979; Martinsen at al., 1989).Alginate beads can also be stabilised by replacing Ca2+ with other divalent cations having ahigher affinity for alginate. The affinity series for other divalent cations used in living cellimmobilisation is: Ba2+ > Sr2+ > Ca2+ >> Mg2+. Smidsrød (1974) has shown that the rigidityof the alginate gels increased with this affinity series. Ca-alginate gels have been stabilised byadding other multivalent cations such as Al3+ (Rochefort et al., 1986) and Ti3+ (Smidsrød andSkjåk-Bræk, 1990). Polymeric polyelectrolytes such as polyethyleneimine (PEI) (Joung et al.,1987) and polypropyleneimine (Velicky and Williams, 1981) have been used to harden thegel. However, subsequent cross-linking with glutaraldehyde affects the viability of theimmobilised cells (Birnbaum et al., 1981; Bajpai and Margaritis, 1985). Fukushima and co-workers (1988) prepared a hard and flexible gel using a mixture of alginate and colloidalsilica. Gels of alginate have also been prepared by using a polycation instead of Ca2+. Gelsproduced by mixing propylene glycol ester of alginic acid with PEI were found to be strongand thermostable but brittle and were superior to beads produced by substituting gelatine forPEI (Mohamed and Salleh, 1982). The mechanical stability of Ca-alginate beads can beconsiderably increased by partially drying the gel. Kokufuta and co-workers (1987) preparedCa-alginate beads, that withstood phosphate ions in the medium, by reinforcing the networkstructure of the gel with a polyelectrolyte complex consisting of potassium poly(vinylalcohol) sulphate and trimethylammonium glycol chitosan iodide. The influence ofimmobilisation of Lactococcus lactis bacteria, Saccharomyces cerevisiae yeasts andTrichoderma viride fungal spores on the mechanical properties of alginate and agar gelshave been investigated (Nussinovitch et al., 1994). The addition of micro-organisms resultedin weakening gel strengths and deformation moduli. The addition of 105 micro-organisms/mldid not change the mechanical properties of the agar, and changed alginate strength only


24

when mold spores and yeast cells were entrapped in this range of concentration. Majorchanges in the gel properties occurred when 107-109 cells/ml gel were immobilised.Generally, agar gels are more brittle than alginate gels. After immobilisation, agar gelsbecame even more brittle while alginate gels maintained a nearly constant strain failure.

By using the dripping technique, alginate beads with a diameter of 0.5-3.5 mm areproduced. The size and sphericity of the bead depend primarily on the surface tension(viscosity) of the sodium solution and the distance between the syringe and the CaCl2solution, and not on the inner diameter of the needle (Klein et al., 1983; Matulovic et al.,1986; Smidsrød and Skjåk-Bræk, 1990). Various techniques have been developed to producesmaller beads which can be scaled up easily. An effective method appeared to be that with acontrolled stream of compressed air which blows off the alginate beads. Beads of diametersin the range 0.1-3.0 mm have been produced (Sada et al., 1981; Klein et al., 1983; Regh etal., 1986; Eikmeier and Rehm, 1987; Sirirote et al., 1988; Smidsrød and Skjåk-Bræk, 1990;Gröhn et al., 1994). A reduction in particle size can also be achieved by controlled drying(Klein et al., 1979; 1983). Another method is the resonance nozzle technique (Hulst et al.,1985b; Woodward, 1988): a jet of the cell-alginate solution is broken up in droplets by meansof a mechanical vibration. Small alginate beads have been produced by electrostatic dropletgeneration (Bugarski et al., 1994a, b; Poncelet et al., 1994b; Goosen, 1995). In this case, apolymer solution is extruded through a charged needle. The collecting solution beneath theneedle is either grounded or has a charge opposite to that of the needle. As the polymersolution passes through the needle, it accumulates charge and droplets formed at the end ofthe needle are pulled off by electrostatic attractive forces between the needle and thecollecting solution. The result is a charged stream of fine droplets. Smaller microbeadsresulted from reduced needle diameter, increased electrostatic potential, reduced needle tocollecting solution distance, reduced polymer solution concentration and flow rate. Thistechnology has been used to immobilise Spodoptera frugiperda in Ca-alginate microbeadsand no detectable effect of applied potential on the cell viability was observed. Small (< 300µm) alginate beads have also been produced by using a high voltage electrostatic pulsesystem (Hallé et al., 1994).

The scaled-up production of small (diameters between 0.25 to 1.2 mm) Ca-alginate beadsusing a spraying technique has been described (Siemann et al., 1990). A rotating ring withmore than 5000 nozzles was employed. An increased speed of the rotating nozzle-ring, asopposed to a decreased diameter of the nozzle, let to a decrease in the size of the beads. Asimilar rotating disc divice has also been constructed by Ogbonna et al. (1989) to producemicro gel beads.

Sterilisation of alginates have been performed by autoclaving for 20 min at 121°C(Kupchik et al., 1983; Edmunds et al., 1989), for 15 min at 100°C (King et al., 1989), for 20min at 80°C (Roscoe and Owsianka, 1982) and membrane filtration (Sun, 1988; Burgess andKwok, 1991). Since it was demonstrated that autoclaving has a deleterious effect on theviscosity (Coates and Richardson, 1974; Hartman et al., 1975; Leo et al., 1990), the influenceof the different sterilisation procedures on alginate dispersions have been studied bymeasuring viscosity and molecular weight changes (Vandenbossche and Remon, 1993).Autoclaving caused a 64% decrease in viscosity. Heating at low temperature over several


25

cycles was less efficient in sterilising alginates and there was a progressive breakdown of thealginate chain over succeeding cycles. Heating during ethylene oxide sterilisation alsoresulted in reduced viscosity and breakdown. Membrane filtration yielded a sterile productwith no significant reduction in viscosity or molecular weight.

(a.2) CarrageenansThere are three main types of carrageenan, in particular lambda-, kappa- and iota-

carrageenan which are extracted from red seaweeds (Landau, 1992; Thomas, 1992). Themajor sources are Chondrus crispus (κ and λ), Eucheuma cottonii (κ ), E. spinosum (ι),Gigartina stellate (κ and λ ) and G. radula (κ and λ) (Guiseley, 1989). Only κ- and ι-carrageenan are suitable for biocatalyst immobilisation where the κ-form is considered to bethe most suitable for cell immobilisation. λ-Carrageenan forms viscous solutions but isnongelling. ι-Carrageenan forms compliant, thermally-reversible gels with calcium ions. κ-Carrageenan produces firm, thermally-reversible gels with potassium ions. The carrageenanspossess a backbone of alternating 1,3-linked β-D-galactose and 1,4-linked α-D-galactose.Differences in structure arise from the number and location of ester sulphate groups on thesesugars and the extent to which the 1,4-linked residues exist as the 3,6-anhydro derivative.Gelation can be achieved by cooling or by contact with a solution containing gel-inducingreagents such as K+, NH4+, Ca2+, Cu2+, Mg2+, Fe3+, amines and water-miscible organicsolvents (Takata et al., 1977; Chibata et al., 1987). Both procedures are easy to carry out andare very mild which can lead to a high viable cell content (Wada et al., 1979; Wang andHettwer, 1982). The mechanical strength of κ-carrageenan gel can be improved by treatmentwith amines (Chao et al., 1986). Beads have also been treated with other hardening agentssuch as glutaraldehyde and hexamethylenediamine (Tosa et al., 1979). Mechanical propertiesof κ-carrageenan gels can be significantly modified by enrichment with galactomannans(Guiseley, 1989). The κ-carrageenan concentration may be reduced without reduction in gelstrength by addition of locust bean gum (Tosa et al., 1979; Miles et al., 1984; Fiszman et al.,1985; Cairns et al., 1986; Audet et al., 1988) or tara gum (Cairns et al., 1986). Casas et al.(1990) added carob bean gum and polyols (glycerol and propylene glycol) to obtain a gelwith better compression resistance, trapping capacity and storage stability, and a lesssyneresis phenomenon.

Carrageenans form hydrocolloidal gels due to double-helix formation. At temperaturesabove the melting point of the gel, thermal agitation overcomes the tendency to form helices,and the polymer exists in solution as a random coil. A three dimensional polymer networkbuilds up with double helices forming junction zones between the polymer chains (gel I) oncooling. Further cooling leads to aggregation of these junction zones (gel II) (Anderson et al.,1969; Rees et al., 1969b; Guiseley et al., 1980; Morris et al., 1980; Smidsrød, 1980; Smidsrødet al., 1980). In practice, this mechanism of interchain association seems to cease after theformation of small soluble clusters (typically consisting of about 10 chains) with exchange ofpartners at "kinking" residues (Morris, 1986). Generally, the higher the 3,6-anhydro-D-galactose content and the lower the ester sulphate level, the less readily will the carrageenanhydrate (Tye, 1989). Further cross-linking of these domains into a cohesive gel structureinvolves side-by-side association of the double helices. Cations are capable of suppressing


26

electrostatic repulsions between the participating chains by their packing within the aggregatestructure (Morris, 1986).

Although both κ- and ι-carrageenan depend on cations for gelation, their gels are (unlikethose of alginate) thermally reversible. They exhibit hysteresis in their melting and gellingtemperatures (like agar). ι-Carrageenan melts and gels at about 70°C and has a hysteresis ofonly about 2-5°C. κ-Carrageenan exhibits a range in both its gelling and melting temperaturesand in the amount of hysteresis. At low potassium ion concentrations, all three values are low.When the amount of K+ increases, the gelling and melting temperatures increase until thehysteresis reaches about 26-27°C. Since most κ-carrageenans contain some potassium ascounterions, an increase in κ -carrageenan concentration results in an increase in K+

concentration which influences the gelling and melting temperatures (Guiseley, 1989).κ-Carrageenan gel can easily be produced in different shapes (bead, cube, membrane)

according to the particular application. Bead formation can be accomplished by the drippingtechnique. The resonance nozzle technique has also been used to produce beads of a morespherical and uniform shape and to scale up the process (Buitelaar et al., 1988; 1990;Woodward, 1988). Because the gel is thermally reversible, it is not a suitable immobilisationmaterial for thermophilic micro-organisms.

(a.3) Agar and AgaroseAgar and agarose (a purified preparation of agar) are polysaccharides isolated from

marine red algae. Two kinds of agar can be distinguished by their gelling temperatures andtheir methoxyl content. Those extracted from various species of Gracilaria have gellingtemperatures in the 40s (°C) and contain methyl ether groups, whereas those obtained fromspecies of Gelidium and Pterocladia have gelling temperatures in the low to mid 30s and areessentially devoid of methyl ether groups. Agar is a complex water-soluble polysaccharideoccurring as a mixture of polysaccharides, ranging from those which are heavily sulphated orpyruvated to those carrying little or no charge. The more ionic polymers are named asagaropectin whereas the more neutral ones are called agarose. Agarose represents the basicgel-forming component of agar (Meer, 1980). Agarose is a linear polysaccharide composed ofrepeating agarobiose units consisting of alternating 1,3-linked β-D-galactopyranose (A) and1,4-linked 3,6-anhydro-α-L-galactopyranose, i.e. L-galactose anhydride (B). The otherspecies present in agar include compounds derived from the β-D-galactose by substitution ofanhydride in position 6, e.g. the 6-methyl ether and 6-sulphate, and the compounds obtainedby substitution of anhydride in position 2 (the 2-sulphate derivative). Consequently, chains ofagarose residues may deviate from the ideal repeating (AB)n structure. They are believed toplay an important role in the gelation mechanism of agarose by preventing perfect orderingand precipitation of agarose chains during sol-gel transformation (Clark and Ross-Murphy,1987). The mechanism of gelation of agarose involves a shift from a random coil in solutionto a double helix in the initial stages of gelation and then to bundles of helices in the finalstage, like gelation of carrageenan (Rees, 1969a; Arnott et al., 1974). For the gelation of thehydroxyl derivatives, it has been suggested that there is a looser packing of the double helicesat the unchanged fibre bundle diameter (Serwer et al., 1983). The net result is more fibres offewer helices each and, smaller pores among the fibres and a less rigid texture of the gel.


27

Pentagonal pores are the essential feature of agarose supports. The pores are large enoughto be readily penetrated by a protein with a molecular mass of several millions. The stabilityof the pores is dependent on the hydrogen bond formation between the strands of the triplehelix of agarose chains (Scouten, 1981). Disruption of these bonds results in dissolving thesoluble monomeric agarose. The hydrogen bonds may be disrupted by urea, guanidinehydrochloride, chaotropic agents and certain detergents. The strength of the bonds and,therefore, the porosity and size of the beads are altered by a change in ionic strength(Scouten, 1981).

The polymer (2-5% w/w) is dissolved by heating in a buffer or a medium that iscompatible with the cells. The polymer solution is cooled to a temperature 5-10°C above thegel-forming temperature and subsequently mixed with the cells. The physical form of thefinal immobilised cell preparation can be chosen to suit the particular application. It can becast as a sheet, bead or cylinder. The gels have been processed in two different ways, i.e. bymoulding beads in a mould or by producing a "gel-block" with mechanical disintegration intosmaller particles (Brodelius, 1983; Kierstan and Coughlan, 1985). Spherical beads can also beproduced by adding the molten preparation dropwise to ice-cold buffer (Brodelius andNilsson, 1980). A simpler method has been developed by Wikström and co-workers (1982):the preparation is poured into a stirred, heated bath of vegetable oil to produce an aqueousemulsion in oil which is then cooled to 5°C. Recently, the resonance nozzle immobilisationtechnique has also been used (Buitelaar et al., 1988; 1990).

(b) Chitin and ChitosanThe term chitin is used for the designation of fibrous 1,4-linked 2-deoxy-β-D-glucan

where the acetylation is usually incomplete to various extents. Chitin, partially deacetylatedchitin and chitosan contain amino groups which have been used as reactive groups in anattempt to develop inexpensive matrices for biocatalyst immobilisation (Gemeiner et al.,1994). Chitin exists in three polymorphic forms with various degrees of cristallinity. The termchitosan is preferred when the nitrogen content is higher than 7% w/w, and is generally usedfor artificially deacetylated chitins (Muzzarelli, 1977; Gemeiner et al., 1994). Chitin isinsoluble in water, but many of its derivatives, i.e. chitosan, are soluble in water and producesolutions which are sometimes capable of gelation (Moore and Roberts, 1980). Chitosan gelscan be formed by an ionotropic mechanism similar to that described above for alginate: gelformation will occur using a chitosan solution with a pH value smaller than 6 (the NH2groups are then protonated) and multivalent anion counterions. Cross-linking of chitosan withhigh molecular weight counterions results in capsules while cross-linking with low molecularweight counterions results in globules in which the cells are entrapped in a real network. Bothlow molecular weight ions such as ferricyanide, ferrocyanide and polyphosphates; and highmolecular weight ions such as poly(aldehydocarbonic acid), poly(1-hydroxy-1-sulfonate-2-propene) and alginate are used (Vorlop and Klein, 1981; 1987; Klein and Kressdorf, 1989).With more hydrophobic counterions (like octyl sulphate, lauryl sulphate, hexadecyl sulphateand cetylstearyl sulphate), it is also possible to produce hydrophobic gels (Vorlop and Klein,1987). Bead formation in a cross-linking solution can also occur at a pH above 7.5, but in this


28

case it is merely a precipitation of chitosan. At pH values greater than 7.5, chitosan is totallydeprotonated and becomes water insoluble (Vorlop and Klein, 1987).

Chitosan ionotropic gel beads are, unlike calcium alginate and potassium carrageenan,stable in phosphate buffered media and their mechanical stability is comparable to that of Ca-alginate beads (Vorlop and Klein, 1987; Klein and Kressdorf, 1989).

(c) Pectins, Pectates and PectinatesPectins are acidic polysaccharides which are present in the cell wall of plant cells where

they fulfil a structural role. Although pectins are branched in their native form, whenextracted they are predominantly linear polymers, based on a 1,4-linked α-D-galacturonatebackbone, which are randomly interrupted by 1,2 linked L-rhamnose (Clark and Ross-Murphy, 1987). Pectin is – like alginate – based on a diaxially linked backbone and formsgels with calcium ions. Gel formation studies indicate a very strong binding of thesecounterions (even in dilute solutions) (Thibault and Rinaudo, 1985). Due to the variability ofthe chemical structure of the commercialy available pectins, gels can be formed in severalways. Polygalacturonic acid as the principal constituent, is partly esterified with methoxylgroups. The free acid groups may be partly or fully neutralized with monovalent ions (i.e.Na+, K+, NH4+). The degree of methoxylation (DM) has an essential influence on theproperties of pectin, especially on its solubility and its requirements for gelation which isdirectly derived from the solubility (Pedersen, 1980). Pectins with a DM lower than 5% arecalled pectic acids and those with a higher DM pectinic acids. Pectinic acids with a DMgreater than 50% are termed high methoxyl (HM) pectins and those with a lower DM lowmethoxyl (LM) pectins. The LM and HM pectins are gelled by different mechanisms. Gelformation of HM pectins requires a minimum amount of soluble solids (glucose) and a pHwithin a moderate range around 3. The gelation of LM pectins requires the presence of acontrolled amount of calcium ions and need neither sugar nor acids. LM pectins must becombined with the soluble calcium source at a temperature above the gelling temperature ofthe system, because the gelation process with Ca2+ ions is temperature dependent (Pedersen,1980). The gelation of LM pectin in the presence of Ca2+ ions and at higher pH, resemblesthe behaviour of alginate gelation but in this case the mechanism is different. Three-foldhelices are formed and the inter-chain association occurs in a different way because pectincontains only one type of uronic acid (Morris et al., 1982; Clark and Ross-Murphy, 1987).The 1,2-linked L-rhamnose residues in the polymer backbone introduce "kinks" whichterminate the inter-chain association. These rhamnosyl linkages are comparatively labile.Accordingly, the chain is cleft preferentially at these positions on mild hydrolysis. Calciumpectate gel network incorporates two solubilising features: rhamnosyl "kinks" which sharplydelimit potential junction zones, and esterification which provides a more subtle mechanismfor control of physical properties (Morris, 1986; Clark and Ross-Murphy, 1987).

The ionotropic gelation of pectate is a simple, mild and inexpensive method. Beads can beeasily produced by dropping the polymer-cell solution into a cross-linking solution. Calcium(Navarro et al, 1983; 1984; Berger and Rühlemann, 1988; Gemeiner et al., 1989c; Tóth et al.,1989) as well as aluminium (Berger and Rühlemann, 1989) have been used as counterions for


29

the gellation of pectinate (Navarro et al, 1983; 1984; Berger and Rühlemann, 1988) andpectate (Berger and Rühlemann, 1988; Gemeiner et al., 1989; Tóth et al., 1989).

Gelation of pectate by diffusion of calcium ions results in a gel concentrationinhomogeneity similar to what has been observed with alginate gelation, i.e. the polymerconcentration is higher at the surface than in the centre of the gel (Skjåk-Bræk et al., 1989).Calcium pectate as well as aluminium pectate beads are much less sensitive to small mono-and multivalent anions, i.e. citrate, phosphate, lactate, gluconate and chloride, and Ca2+

(Al3+) complexing agents which diminish the stability of the beads (Berger and Rühlemann,1988). Stabilisation and hardening of pectate beads can be performed using a treatment withpolyethyleneimine followed by glutaraldehyde (Kurillová et al., 1992).

(d) Cellulose and Cellulose AcetatesCellulose is a polydisperse, linear syndiotactic polymer of plant origin and it is composed

of 1,4-linked β-D-glucose residues. Cellulose is insoluble in water, but it can be dissolved incertain organic liquids. Although cellulose carriers have been widely used for living celladsorption, entrapment in the gel form is less effective due to loss of viability upon contact ofthe cells with organic solvents. Cells may be entrapped by a precipitation mechanism bychanging the solvent: the polymer-cell suspension is transfered from an organic phase to anaqueous phase. Whole cells have been immobilised with the intention to retain only thecatalytic activity of certain enzymes. Linko and co-workers (1977; 1980; 1987) haveentrapped numerous bacteria and yeast cells inside cellulose beads using a solvent systemconsisted of N-ethylpyridinium chloride, dimethylformamide and sulphoxide. Similartechniques have been developed to entrap cells in cellulose di- and triacetate (Linko et al,1980; Linko, 1982). Aceton (Joshi and Yamazaki, 1986; Giovenco et al., 1987; Marconi etal., 1987), n-hexane (Sakimae and Onishi, 1981) and methylene chloride (Dinelli, 1972;Ghose and Kannan, 1979) have also been used for the immobilisation of bacteria and yeastsin cellulose acetates.

(e) Gellan GumGellan gum is a gel-forming polysaccharide secreted by the bacterium Pseudomonas

elodea and is produced by aerobic fermentation (Kang et al., 1980; 1981; 1982; 1983; Kangand Veeder, 1982; 1983; Gibson, 1994). Gellan gum is a linear, anionic heteropolysaccharidewith tetrasaccharide repeating units consisting of two β-D-glucose, one β-D-guluronic acidand one α-L-rhamnose residue (O'Neil et al., 1983). The polymer is secreted by P. elodea,and contains approximately 1.5 acyl substituents per tetrasaccharide repeating unit. Thesesubstituents have been identified as an L-glyceric ester on C-2 of the 3-linked D-glucose andan acetic ester on C-6 of the same glucose residue (Kuo et al., 1986). The presence of thesesubstituents, in particular the bulky glycerate groups, hinders chain association and accountsfor the change in gel texture brought about by de-esterification. The de-esterified product is apolymer with a well-defined, unsubstituted tetrasaccharide repeating unit. In the solid statethis molecule forms a parallel, half-staggered intertwined double helix in which eachpolysaccharide chain is a left-handed, threefold helix (Chandrasekaran et al., 1988a, b). Thesubstituted form produces soft, elastic and cohesive gels whereas hard, firm and brittle gels


30

are obtained from the unsubstituted form (Rinaudo, 1988; Gibson, 1994). The rheologicalproperties of unsubstituted gels are superior to those of other common polysaccharides suchas agar, κ-carrageenan, and alginate at similar concentrations (Sanderson et al., 1989).

Gelation initially occurs by the formation of double helices followed by ion-inducedassociation of these helices. Heating and cooling in the absence of gel-promoting cationsfavours the formation of fibrils by double helix formation between the ends of neighbouringmolecules (Gunning and Morris, 1990). Gel formation occurs when the fibrils associate in thepresence of gel-promoting cations. Gelation depends upon the gum concentration, ionicstrength and the type of stabilising cation (divalent are more effective than monovalentcations) (Moorhouse et al., 1981; Kang et al., 1982; Norton and Lacroix, 1990). A dispersionof gellan gum with a minimum amount of sequestrant (needed to hydrate the gum fully at anelevated temperature of 80-90°C) and divalent cations (for optimal gel strength), will form acoherent, demouldable gel on cooling to ambient temperature. The gelation temperaturedepends on the cation concentration and increases from 35 to 55°C with increasing cationconcentration. Gels can also be prepared at ambient temperature upon reaction of gellan gumsolutions with mono- and divalent cations, but these gels are unstable and exhibit syneresis(Gibson, 1994).

Gel beads with entrapped cells can be produced by extrusion or emulsification of the warmgellan gum-cell solution followed by cooling (Grasdalen and Smidsrød, 1987; Norton andLacroix, 1990; Camelin et al., 1993). Gellan gum is suitable for the immobilisation ofthermophilic bacteria (up to 60°C) (Norton and Lacroix, 1990). The high setting temperature(over 50°C) of gellan gum at high concentrations, can be decreased by using sequestrants(such as citrate, metaphosphate and EDTA) so that also more temperature-sensitivemesophilic bacteria can be entrapped (Camelin et al., 1993).

2.1.1.2 Proteins(a) CollagenThe collagens are a family of highly characteristic fibrous proteins found in all

multicellular animals. The central feature of all collagen molecules is their stiff, triple-stranded helical structure. Collagen chains are extremely rich in glycine and proline, both ofwhich are important in the formation of the stable triple helix (Bornstein and Sage, 1980;Eyre, 1980). Collagen is hydrophilic and swells in the presence of water. At low pH values, itcan be dissolved and recovered from the solution at higher pH values.

The mechanism of cell immobilisation in collagen involves the formation of multiple ionicinteractions, hydrogen bonds and van der Waals forces between the cells and collagen (Viethand Venkatsubramanian, 1979; Cheetham, 1980; Kolot, 1981). Preparation of the collagensolution and mixing with cells has to be performed at low temperatures (4°C). Gelification isaccomplished by raising the pH and ionic strength of the collagen solution and exposure to37°C. Collagen has been wideley used to immobilise enzymes in a membrane form, whichrequires cross-linking with glutaraldehyde to stabilize the structure. Cell immobilisationmethods have also used the stabilisation reaction with gluteraldehyde. However, excessiveexposure to glutaraldehyde will damage the cells. Recently, behaviour studies of mammalian


31

cells have been performed by using collagen matrices to mimic the original three-dimensionalmicro-environment found in tissues (examples can be found in Table 35).

(b) GelatineGelatine is a hydrolytic derivative of collagen. Gelification is accomplished by cooling the

gelatine solution below a temperature of 30 to 35°C. The sol-gel transformation is reversible(De Alteriis et al., 1988). The gel structure can be stabilised by adding organic or inorganiccompounds (Sungur and Akbulut, 1994). Gel particles have been stabilised withglutaraldehyde (e.g. Sungur et al., 1992b) or formaldehyde (Dhulster et al., 1983), which canhave, however, a negative impact on the cell viability. Sungur et al. (1992a) used chromiumsalts (acetate or sulphate) as inorganic stabilisers. The three dimensional structure of gelatineis formed by secondary interactions between the polypeptide chains. These interactions arebroken upon heating. The stabilisation by aldehydes is based on covalent bond formationsbetween the gelatine strands.

In a typical procedure, the cell suspension is mixed with an aqueous gelatine solution at40°C, the suspension is cooled and the gel lyophilised. Subsequently, the dry preparation isdisintegrated into small particles. The glutaraldehyde treatment can be performed before thecooling of the suspension or after the lyophylisation process (Brodelius and Vandamme,1987). Uniform beads can be formed by dripping the hot suspension in a hydrophobic liquidlike butyl acetate.

2.1.2 Synthetic Polymers

The application of synthetic polymers for the immobilisation of living cells has someinteresting benefits compared to the use of natural polymers, since synthetic polymers ofadequate properties can be easily and artificially designed. The porosity of the gel as well asthe ionic and hydrophobic or hydrophilic properties can be easily adjusted. Additionally, themechanical strength and longevity of the gels formed from synthetic polymers are generallysuperior to those from natural polymers.

(a) Polyacrylamide, Polyacrylamide-hydrazide and Copoly(NIAAm/AAm)(a.1) PolyacrylamideThe first synthetic gel used to entrap living microbial cells was polyacrylamide.

Entrapment is performed by the polymerisation of an aqueous solution of acrylamidemonomers in which the cells are suspended. It is an easy immobilisation technique, but thepolymerisation of the acrylamide monomers in the presence of viable cells usually results in areduction of the viability of the entrapped cells due to the toxicity of the monomers (e.g.acrylamide and bisacrylamide) and the heat evolved during polymerisation. Analysis of thefactors affecting the cells during immobilisation has shown that acrylamide monomer, whoseaction increased with increasing concentration and temperature, is the most toxic (Lusta et al.,1990). Introduction of acrylamide monomer into the growth medium of any bacteria blockedtheir division and, under certain conditions, it also caused a strong elongation of living gram-negative bacteria. Electron microscopy of elongated Escherichia coli grown in the presence


32

of acrylamide monomer revealed on the outer surface numerous vesicles, meyelin-like bodiesand clusters of membrane-like formations, due to the destruction of the outer bacterialmembrane. Treatment of bacterial cells with high acrylamide concentrations (over 20%) at40˚C completely disturbed the plasmalemma semi-permeability and resulted in cell death.The level of the "immobilisation shock" of polyacrylamide entrapped cells depended to alarge extent on the initial physiological state of the population (Starostina et al., 1983; 1985;Lusta et al., 1990). Bacterial clones of E. coli and Pseudomonas putida with increasedresistance to polyacrylamide immobilisation and acrylamide monomer action have beenisolated (Starostina et al., 1986; Lusta et al., 1990). By carefully controlling thepolymerisation time and temperature, 100% viability of the Arthrobacter globiformis cellscould be retained (Koshcheenko et al., 1983; Skryabin and Koshcheenko, 1987). Usually,small biocatalyst particles are obtained by casting blocks of immobilised cells which are cutor passed through meshes to produce smaller particles (Mattiasson, 1983; Kierstan andCoughlan, 1985; Skryabin and Koshcheenko, 1987). Spherical beads can be prepared bysuspending the cell-monomer solution in an inert hydrophobic phase (i.e. oils such as soy ofparaffin oil) before polymerisation (Mosbach, 1984; Nilsson et al., 1987).

Polymerisation of polyacrylamide is a free radical process in which linear chains ofpolyacrylamide are formed. The polymers are cross-linked by inclusion of a bifunctionalreagent (e.g. N,N'-methylene-bisacrylamide) which has two unsaturated double bonds. Thecross-linking degree is a function of the relative amounts of acrylamide and bifunctionalreagent and determines the porosity and fragility of the gel. Initiation of the free radicalpolymerisation can be performed by a chemical or a photochemical reaction. Persulphate andβ-dimethylaminopropionitrile or N,N,N',N'-tetramethylenediamine (TEMED) are chemicalcatalysts, whereas sodium hydrosulphite, riboflavin and TEMED act as photochemicalpolymerisation initiators.

(a.2) Polyacrylamide-hydrazideTo eliminate the unfavourable influences caused by the acrylamide monomer,

techniques have been developed which use pre-polymerised linear polyacrylamides, partiallysubstituted with acylhydrazide groups, for the entrapment of living cell with good retention ofviability (Freeman and Aharonowitz, 1981; Freeman, 1987). The prepolymerised material iscross-linked in the presence of viable cells by the addition of controlled amounts ofdialdehydes (glyoxal, glutaraldehyde, periodate-oxidised polyvinylalcohol). The porosity ofthese gels is affected by the cross-linking agent (Pines and Freeman, 1982; Freeman, 1984).However, the best results were obtained using glyoxal. The concentration of polymericbackbone also affects gel porosity. The mechanical stability of this gel is superior to gelsmade with similar concentrations of polymeric backbone from acrylamide-bisacrylamidecopolymerisation. This polyacrylamide-hydrazide (PAAH) gel is less brittle, chemicallystable and does not undergo deformation as a result of changes in salinity or pH.

PAAH was used to study the effect of gel entrapment on yeast tolerance to a series ofwater miscible solvents (Dror et al., 1988): PAAH entrapment increased the tolerance toethanol and ethyleneglycol, but not to other solvents such as methanol anddimethylformamide.


33

(a.3) Copoly(NIAAm/AAm)Copoly(N-isopropylacrylamide/acrylamide) (NIAAm/AAm) is a "lower critical

solution temperature" (LCST) hydrogel (Taylor and Cerankowski, 1975; Hoffman et al.,1986; Bae et al., 1987; Galaev and Mattiasson, 1993). This gel gradually shrinks astemperature is raised and then collapses when it is warmed through the LCST region. Itexpands and reswells as it is cooled below LCST. Cell immobilised gel beads have beenprepared by inverse suspension polymerisation (Park and Hoffman, 1990). Paraffin oil wasused as the continuous phase and Pluronic L-81 as the surfactant. The monomers (NIAAmand AAm) and methylene-bis-acrylamide cross-linker were dissolved in Tris-HCl buffer, andmixed with the cell suspension. This solution was then poured into the paraffin oil containingthe surfactant. After stable droplets were formed, TEMED was injected into the continuousparaffin phase to initiate the polymerisation. After polymerisation (30 min), the mixture wastransferred to a separatory funnel and excess buffer was added to separate the system in twophases. It has been demonstrated that microbial cells can be successfully immobilised. Theconversion and activity of the immobilised cells may be enhanced by thermal cycling of thegel below its LCST, due to the reduced mass transfer resistance as the gel bead "squeezesout" and "draws in" substrate when it shrinks and swells during the thermal cycling (Park andHoffman, 1990). Pore sizes and their interconnections will change significantly as the gel isshrunk or swelled and this can significantly affect the diffusion rates of substrate in andproduct out of the gel (Engasser and Horvath, 1974; Zhong et al., 1988).

(b) MethacrylatesThe preparation of methacrylate gels is analogous to that of polyacrylamide gels.

Methacrylate monomers such as methylacrylamide, hydroxyethylmethacrylate ormethylmethacrylate are polymerised in the presence of a cross-linking agent (e.g.tetraethyleneglycol dimethacrylate) to form a porous gel (Brodelius and Vandamme, 1987).Methacrylate gels have been used to a rather limited extent for the immobilisation of cells(Kumakura et al., 1978; 1979; Klein and Schara 1981; Cantarella et al., 1983) because theviability of the entrapped cells is reduced due to the immobilisation method. Living cells canalso be immobilised in methacrylate by γ-ray irradiation at low temperatures (Kumakura etal., 1984a). Low temperature (-78°C) radiation polymerisation has proven to be fruitful toprepare porous hydrogels for entrapment of yeast cells (Carenza and Veronese, 1994).Aqueous solutions of a wide variety of monomers (Carenza et al., 1990; 1993) or co-monomers (Fujimura et al., 1988; Carenza et al., 1989; Xin et al., 1992) have been used. Thepolymeric matrices were swollen in water and then aerobically incubated with yeast cells.High ethanol productivities could be obtained by batch (Fujimura et al., 1988; Xin et al.,1992; Carenza et al., 1993) as well as by continuous fermentation (Carenza et al., 1989; 1990)

Poly(ethylene glycol) dimethacrylate was also used as a cross-linking agent for theentrapment of biocatalysts by radical polymerisation of acrylic acid and N,N-dimethylaminoethyl methacrylate (Sakata et al., 1981).


34

(c) Photo-cross-linkable Resin PrepolymersPrepolymer methods using synthetic resin prepolymers as gel-forming starting materials

have some specific features and advantages (Fukui and Tanaka, 1984): (1) entrapmentprocedures are very simple under very mild conditions; (2) prepolymers do not containmonomers which have adverse effects on the biocatalysts to be entrapped; (3) the networkstructure of gels can be controlled by using prepolymers of optional chain length; (4) optionalphysicochemical properties of gels, such as hydrophobicity-hydrophilicity balance and ionicnature, can be changed by selecting suitable prepolymers which had been synthesised in theadvance in absence of biocatalysts.

Illumination with near-ultraviolet light of photo-cross-linkable resin prepolymers initiatesradical polymerisation of the prepolymers and completes gel formation within only 3 to 5minutes. Various types of prepolymers possessing photo-sensitive functional groups havebeen developed. Poly(ethylene glycol) dimethacrylate (PEGM) was synthesised frompoly(ethylene glycol) (PEG) and methacrylate (Fukui et al., 1976). ENT and ENTP wereprepared from hydroxyethylacrylate, isophorone diisocyanate and poly(ethylene glycol) orpoly(propylene glycol), respectively. Each prepolymer has a linear skeleton of optionallength, at both terminals of which are attached the photo-sensitive functional groups such asacryloyl or methacroyl. PEGM (Fukui et al., 1976; Tanaka et al., 1977a) and ENT (Tanaka etal., 1977b; 1978) containing PEG as the main skeleton are water soluble and give hydrophilicgels, while ENTP (Omata et al., 1979; Sonomoto et al., 1979) with poly(propylene glycol) asthe main skeleton is water insoluble and forms hydrophobic gels. Using PEG orpoly(propylene glycol) of different molecular weight, prepolymers of different chain lengthcan be prepared: from PEGM-1000 to PEGM-4000 (MW of main chain from ~1000 to 4000,respectively), ENT-1000 to ENT-6000, and ENTP-1000 to ENTP-4000. The chain length ofthe prepolymers correlates to the size of the network of gels formed from these prepolymers.Anionic and cationic prepolymers can also be prepared by introducing anionic and cationicfunctional group(s) to the main skeleton of the prepolymers.

The entrapment of cells can be obtained by illumination of a mixture consisting of aprepolymer, a photo-sensitizer such as benzoin ethyl ether or benzoin isobutyl ether, and thecell suspension. A suitable buffer is used for the hydrophilic prepolymer and an adequateorganic solvent for the hydrophobic prepolymer. Suitable mixtures of these two types ofprepolymers can also be utilised (Sonomoto et al., 1979). In some cases, a detergent isemployed to mix a hydrophobic prepolymer with the suspension of biocatalysts (Omata et al.,1979).

An emulsion-type photo-cross-linkable resin (ENTE) is useful when swelling of the gels isto be avoided (Itoh et al., 1979). ENTE-1 was prepared as follows: hydrophobic poly(vinylacetate) was coated with hydrophilic poly(vinyl alcohol) to be readily dispersed in an aqueoussolution and photosensitivity was introduced in the particles by reacting with N-hydroxymethylacrylamide in the presence of an acid catalyst; after neutralisation, theresulting prepolymer was used as an emulsion-type photo-cross-linkable resin prepolymer.

Recently, laser-induced photo-polymerisation of poly(ethylene glycol) diacrylates andmultiacrylates have been used to entrap living mammalian cell (Pathak et al., 1992). Thesemolecules of various molecular weights were synthesised by reaction of PEG with acryloyl


35

chloride, using triethylamine as a proton acceptor. Tetrahydroxy PEG was used for themultiacrylate synthesis. A solution of the PEG acrylate was mixed with the cell suspensionalong with ethyl eosin and triethanolamine as the photo-sensitizer/electron donor initiatingsystem and N-vinylpyrrolidinone. A coextrusion apparatus was used to form micropheres(0.5-0.8 mm in diameter) by extruding the liquid macromer into droplets and subsequentlygelling them by exposure to laser light. The viability (measured by a trypan blue exclusionassay) of encapsulated human fibroblasts (HFF), Chinese hamster ovary cells (CHO-K1) andβ cell insuloma cells (RiNm5F) was unchanged upon immobilisation. Rat islets ofLangerhans have been entrapped by this method and the ultrastructure (examined bytransmission electron microscopy) was different from the control only at the outermost singlecell layer, where the β cells showed fewer secretory granules than untreated control islets.The advantages of this entrapment method are: the laser light is not absorbed by the cells inthe absence of an exogenous, cell-binding chromophore; there is no significant heat ofpolymerisation due to the nature, size and dilution of the macromers used; the polymerisationcan proceed extremely rapidly in oxygen-containing aqueous environments at physiologicalpH; gels with the proper formulation are capable of being immunoprotective and can be usedfor cell therapy purposes.

(d) PolyurethaneUrethane prepolymers have isocyanate groups at both terminals of the linear chain and are

synthesised by heating for 1-2 hours at 80°C from toluene diisocyanate and polyether diolscomposed of poly(ethylene glycol) and poly(propylene glycol) or poly(ethylene glycol)alone. Prepolymers with a different hydrophilic or hydrophobic character can be obtained bychanging the ratio of poly(ethylene glycol) and poly(propylene glycol) in the polyether diolmoiety of the prepolymers. For example, PU-3 with a high content of poly(propylene glycol)gives hydrophobic gels, while PU-6 and PU-9 with a high content of poly(ethylene glycol)give hydrophilic gels, although all the prepolymers are water miscible (Sonomoto et al.,1980). The chain length and the content of isocyanate group can also be changed.

Cells can be entrapped by the "self-cross-linking" gel. The prepolymers are water-miscibleand when a liquid prepolymer is mixed with an aqueous cell suspension, the isocyanatefunctional groups at both terminals of the molecule react with each other only in the presenceof water, forming urea linkages with liberation of carbon dioxide (Fukushima et al., 1978).

Cells have also been entrapped in conventional polyurethanes which were obtained bypolycondensation of polyisocyanates (Klein et al., 1979; Klein and Kluge, 1981). Thepolyurethane can be made in a foam or in a gel structure depending on the type andconcentration of the polycyanate used .

(e) Photo-sensitive Resin PrepolymersPhoto-sensitive resin prepolymers are derivatives of poly(vinyl alcohol) (PVA) introduced

by styrylpyridinium (SbQ) groups as photo-sensitive sites and are polymerised by photo-dimerisation with irradiation of visible or ultra-violet light (PVA-SbQ gel). Hydrophilicity ofthe prepolymers can be controlled by changing the saponification degree of poly(vinylalcohol) (Ichimura and Watanabe, 1982; Ichimura, 1984).


36

(f) Radiation PolymersLiving cells can be entrapped by γ-irradiation of a wide variety of functional monomers or

prepolymers at low temperature. Utilisation of this method is limited because radiationequipment is required. Kumakura and co-workers (1984a) entrapped living Trichodermareesei cells using 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate by irradiation (1Mrad) at -78°C. Irradiation of cells at low temperatures was necessary to avoid the radiationdamage (Kumakura et al., 1979). It was found that the production of cellulase from the cellsat the initial stage of the culture was retarded by irradiation and freeze treatment, but wasrecovered immediately. The rigidity of the polymer matrix can be increased and the porositydecreased, by increasing the monomer concentration.

Several prepolymers, such as poly(vinyl alcohol) (Maeda et al., 1973), poly(ethyleneglycol) diacrylate and poly(ethylene glycol) dimethacrylate (Yoshii et al., 1981) are useful gelmaterials for entrapment by irradiation. A mixture of poly(ethylene glycol) dimethacrylateand poly(vinyl alcohol) has also been used. When 10% of poly(vinyl alcohol) was used, 5-7Mrad of irradiation was sufficient to obtain rigid gels (Maeda et al., 1973). A technique forthe immobilisation of biocatalysts by means of radiation polymerisation of glass-formingprepolymers (i.e. poly(ethylene glycol) diacrylate and poly(ethylene glycol) dimethacrylate)at low temperature, has been developed by Yoshii and co-workers (1981): entrapment wascarried out at -24°C with 10% of prepolymer and an irradiation of 1 Mrad.

(g) Epoxy ResinsEpoxy resins are formed by the reaction of bifunctional epoxy oligomers with

polyfunctional amino compounds. Direct immobilisation by entrapment of cells in an epoxyresin is generally possible, but due to low activity and limited porosity such systems cannotbe recommended. Introduction of porosity is possible by combining the epoxy resinformation with ionotropic gelation (Na-alginate), where the ionotropic gel is used as a processto stabilise the bead shape and size, and to form pores for mass transport (Klein andKressdorf, 1987). Klein and co-workers developed two methods for cell immobilisation. Thefirst method is without whereas the second is with precondensation of the curing agent andepoxy compound. The first method is useful for the immobilisation of dead and resting cellsfor one-enzyme reactions or insensitive cells (Klein and Eng, 1979; Klein et al., 1981). Thesecond method has to be used to attain a high viability of entrapped cells (Klein andKressdorf, 1982). The residual activity of the immobilised cells depends strongly on theprecondensation time.

Epoxy biocatalysts show high mechanical strength even with high cell loading. Thestability also depends on the alginate concentration, which is optimised with regard toporosity and stability.

(h) Polyvinyl AlcoholPoly(vinyl alcohol) (PVA) is a raw material of vinylon and is a low-cost material. PVA is

non toxic to micro-organisms and, consequently, can be used to entrap living cells. A PVAsolution becomes gelatinous by freezing and the gel strength increases during iterations of


37

freezing and thawing (Nambu, 1983). Using this technique, a rubber-like, elastic hydrogel canbe obtained without using any chemical reagent. The gel stength increases with the iterationnumber of freezing-thawing until seven iterations (Ariga et al., 1987). Activated sludge andEscherichia coli cells have been immobilised in PVA. A decrease of activity due to thefreezing and thawing could be prevented by adding cryoprotectants such as glycerol and skimmilk to the PVA-cell solution (Ariga et al., 1987). PVA cryogels can be used up to atemperature of 65°C and have also been used to immobilise thermophilic micro-organismslike Clostridium thermocellum, C. thermosaccharolyticum and C. thermoautotrophicum toperform fermentations at 60°C (Varfolomeyev et al., 1990). PVA immobilised Candidarugosa cells have been used for the industrial production of L-malic acid since 1990 (Yang,1991; Yang et al., 1992).

Elastic PVA gels with a high strength and durability can also be formed by cross-linkingPVA with a boric acid solution: a monodiol-type PVA-boric acid gel lattice is produced(Ochiai et al., 1981). Activated sludge was successfully immobilised using this technique,with no apparent loss of biological activity (Hashimoto and Furukawa, 1987). However, thistechnique has two potential problems. First, the saturated boric acid solution used to cross-link the PVA is highly acidic (pH of approximately 4) and could cause difficulty inmaintaining cell viability. Second, PVA is an extremely sticky material. As a result, PVAbeads have a tendency to agglomerate which can cause problems in fluidised bed reactors.This latter problem can be solved by using a combination of PVA-boric acid and a smallamount of calcium alginate (0.02%), by hardening a mixture of PVA and sodium alginatewith a mixed solution of boric acid and calcium chloride (Wu and Wisecarver, 1992). Thistechnique has been used to entrap Pseudomonas cells and used in a fluidised bed reactor todegrade phenol. Recently, Chen and Lin (1994) developed a new method based on the usageof phosphorylated PVA. PVA was first cross-linked with boric acid for a short time to form aspherical structure, which was followed by solidification of the gel beads by esterification ofPVA with phosphate. The short contact time with boric acid prevented severe damage to theentrapped micro-organisms. This method was successfully employed to entrap denitrifyingsludge to treat waste water.

2.2 Physiology of Gel Immobilised Cells

The widespread development and application of gel immobilised cell technology hassignificantly increased interest in the physiology of immobilised cells. A number of reportshave appeared suggesting that the immobilisation has a profound effect on the metabolicbehaviour of immobilised cells compared with free cells. On the other hand, results of otherreports suggest that the observed changes are caused by a change in local concentrations dueto mass transfer limitations.

2.2.1 Physiology and Experimental Techniques

The study of immobilised cells requires specific experimental techniques. Detailed in situinformation is needed to get a clear picture of immobilised cell systems. Many biophysical


38

techniques have already been used to study immobilised cells in situ. Only a few of thesetechniques are non invasive. Some involve the removal of the cells from the immobilisationmatrix and/or destruction of the sample. Interest in non invasive studies has increased rapidlyas improved instrumentation with expanded capabilities became available. The physicalmethods for direct examination of immobilised cells are listed in Table 13.

The specific growth rate of S. cerevisiae cells attached to gelatine and immobilised in Ca-alginate was decreased by roughly a factor two compared with free cells. The specific ethanolproduction and glucose consumption was approximately 50 % greater for immobilised cellsthan for suspended cells (Doran and Bailey, 1986; Bailey et al., 1987; Galazzo and Bailey,1990a). Bailey and co-workers have used a variety of techniques to characterise themetabolic state of immobilised cells and proposed an explanation for their peculiarbehaviour. Measurements of the polysaccharide content were made and larger quantities ofreserve carbohydrates and structural polysaccharides have been found in cells attached togelatine than in suspended cells (Doran and Bailey, 1986), indicating that cellularcomposition was also affected by immobilisation. Flow cytometry has been used to obtainDNA, RNA and protein frequency functions. The results showed that the cells attached togelatine had higher ploidy and a lower RNA content than cells in suspension. The quantity ofprotein per unit size for the two cell types was roughly equal. Based on these observations, itwas proposed that the immobilisation may have altered the normal cell cycle by preventingbud development in some cells undergoing DNA replication and nuclear division.

TABLE 13EXPERIMENTAL TECHNIQUES FOR EXAMINING GEL IMMOBILISED CELLS

Method Information Comments Referenceavailable

Enzyme assays Enzyme activities Sample removal and Hilge-Rotmann and Rehm,destruction 1990; Galazzo and Bailey,

1990a; Höötmann et al., 1991; Senac and Hahn-

Hägerdal, 1991Flow cytometry DNA, RNA, protein Capable of multipara- Doran and Bailey,

content, cell size meter analysis of 1986single cells; detach-ment and staining ofcells required

NMR spectroscopy Intra- and extracel- Relative insensitive Galazzo et al., 1987;Galazzo

lular concentrations and Bailey, 1989; Karel etand pH al., 1987; Neeman et al.,

1987; McGovern et al., 1993; Fernandez, 1995


39


Method Information Comments Referenceavailable

Fluorescence NADH On-line and non- Doran and Bailey, 1987;measurement invasive Müller et al., 1988; Anders

et al., 1990; Scheper et al., 1990

Microfluorimetry Spatial distribution Requires staining of Monbouquette et al., 1990;of biomass, RNA cells and sectioning Kuhn et al., 1991; 1993;and DNA of the matrix Ollis, 1995

Radioisotope Rate of cell mass Sample removal and Karel et al., 1987; Stewartlabelling and synthesis, location destruction and Robertson, 1988; KarelAutoradiography and size of cell and Robertson, 1989a, b

growth regionMicroelectrodes pH, O2, NH4+, Limited by the de Beer and Sweerts, 1989;

N2O, NO3-, fabrication of the de Beer et al., 1990; Geeseyglucose appropriate electrode and White, 1990; Hooijmansconcentrations et al., 1990a, b, c; Revsbech

et al., 1989; Cronenberg et al., 1991, 1993, 1994; Masson et al., 1994; Müller et al., 1994; Schrezenmeir et al., 1994; Beuling et al.,

1995; Ottengraf and Van den Heuvel, 1995

Mass spectrometry Simultaneous On-line and in situ Willaert et al., 1990;measurement of measurement Willaert, 1993;1995various compounds

Microscopy Morphology, Off-line analysis; e.g. Eikmeier et al., 1984;surface coverage necessary for micro- Bailliez et al., 1985; Godia

etfluorimetry and al., 1987; Marin-Iniesta et autoradiography al., 1988; Barbotin et al.,

1990bMicroscope reactor Morphology, On-line and non- Willaert and Baron, 1993

growth rate, spatial invasivebiomass distribution

Membrane reactor Measurements of On-line and in situ Hannoun andsubstrate and measurement Stephanopoulos, 1990; product Willaert et al., 1990; De

Backer et al., 1992


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Fluorescent measurements of intracellular NADH of S. cerevisiae cells grown on agelatine surface and free cells have been performed (Doran and Bailey, 1987): dynamicresponses of NADH for immobilised cells were different from those of yeast cells grown insuspension, indicating that immobilisation affects cellular regulation and control functionswithin the glycolytic pathway. The influence of the immobilisation of S. cerevisiae in Ca-alginate has also been studied by using on-line measurements of NADH fluorescence (Mülleret al., 1988; Anders et al., 1990; Scheper et al., 1990). A comparison of the immobilised andsuspended cell system showed that the rate of ethanol consumption during an aerobicfermentation was significantly slower for the immobilised cells. This reduced rate ofoxidative decomposition was attributed to mass transfer limitations of oxygen.

NMR spectroscopy is a powerful tool for monitoring metabolic processes within wholecells and intact tissue (Fernandez, 1995). A unique advantage of NMR spectroscopy is that itpermits the simultaneous assay of a wide range of naturally occurring, or externally supplied,metabolites in a non destructive and non invasive manner. Uses of in vivo NMR techniquesrange from relatively simple measurements of intracellular and transmembrane pH to detailedanalyses of metabolic pathways and cellular regulation mechanisms. Freely suspended aswell as immobilised cells are amenable to study by NMR (Vogel et al., 1987). NMRmeasurements have shown that the internal pH of S. cerevisiae cells immobilised in Ca-alginate was lower than the internal pH of suspended cells, and in vitro studies revealed that alower pH increased the rate of phosphofructokinase (PFK) and hexokinase. Theseobservations could explain the increased rates of fermentation pathways in immobilised cells(Galazzo et al., 1987). By using NMR analysis, Galazzo and Bailey (1989) found substantialdifferences in glucose-6-phosphate, fructose-6-phosphate and 3-phosphoglycerate levels insuspended and immobilised cells. Adenosine tri- and diphosphate, and fructose-diphosphateconcentrations were very similar in both systems. The uridine-diphospho-glucose (UDPG)was also higher in immobilised cells, which is consistent with the higher polysaccharideproduction in immobilised cells since UDPG is a key intermediate in polysaccharidebiosynthesis. The NMR measurements of intracellular metabolite concentrations have beenutilised in a detailed kinetic model of the glucose catabolic pathways in S. cerevisiae(Galazzo and Bailey, 1990a, b), which showed that cells grown in alginate exhibit fluxcontrol centred almost entirely in the glucose uptake step, while both fructosephosphorylation by PFK and ATP utilisation by all cellular ATPase functions drop fromsubstantial control in suspension grown cells to negligible influence in alginate grown cells.The estimated change in PFK maximum velocity was also consistent with in vitro assays ofPFK activity in extracts of suspension and alginate grown yeast. 31P NMR studies withagarose immobilised Candida tropicalis have shown that C. tropicalis metabolising xylosewas not capable of building up polyphosphate (Lohmeier-Vogel et al., 1990a). Moreover, thisstrain did not grow inside agarose beads when xylose was supplied. However, cell growthand polyphosphate metabolism resumed immediately when the perfusate was changed to aglucose containing medium. Although glucose metabolising C. tropicalis more closelyresembled freely suspended cells in their metabolic response, they displayed a slight up fieldshift of the cytoplasmic inorganic phosphate (Pi) peak indicating an acidification of this


41

compartment by 0.3 pH units. A pH drop close to one pH unit was observed for xylosemetabolising C. tropicalis. In addition, sodium-23 and potassium-39 NMR data showed thatimmobilised C. tropicalis cells had higher intracellular levels of Na+ and K+ than suspendedcells. Moreover, measurements of the relaxation times of the intracellular cations suggestedthat the intracellular environment is more viscous in immobilised cells. Santos et al. (1990)used NMR techniques to examine the phosphorus and carbon metabolism of free and κ -carrageenan immobilised Propionibacterium acidi-propionici. It was shown thatpolyphosphate was present both in the intracellular space and on the outside of the externalmembrane; and polyphosphate is used to phosphorylate glucose. Immobilised cells led tomuch higher yields of propionic acid compared to suspended cells. Taipa et al. (1990) used31P NMR to probe the anaerobic metabolism of glucose by suspended and κ-carrageenanentrapped S. bayanus cells. It was shown that the intracellular pH was slightly higher in thecase of immobilised cells (7.15 compared to 7.05) and is kept constant during the course offermentation, in contrast with the suspended cells for which a steady state decrease in pH wasobserved. This difference was attributed to the differences in the extracellular pH whichdecreased much faster in the free cell fermentation medium compared to the gel matrix.

The intracellular metabolism of gel entrapped plant cells has been studied by 31P NMR(Brodelius, 1984; Brodelius and Vogel, 1984; Brodelius, 1988; Lohmeier-Vogel et al.,1990a). The spectra of freely suspended Catharanthus roseus cells, and agarose and alginateentrapped cells have been recorded under oxygenation conditions (incubation time of 48, 72and 72 hours respectively). The same resonances have been observed in all three spectra andthe chemical shifts of the cytoplasmic sugar phosphate and inorganic phosphate resonanceswere almost identical. Also, the ADP/ATP ratio was the same, within experimental error,indicating a similar energy status for the three cases. Although it is well known that changesin the intracellular pH exert regulatory effects on the metabolism of plant cells, it was clearfrom these NMR studies that the cytoplasmic pH as well as the vacuolar pH are not altered bythe entrapment of the cells in agarose or alginate gel.

NMR spectroscopy has also been used as a probe of the physiological state of Escherichiacoli contained by microporous hollow fibre membranes (Karel et al., 1987). Two pH valueswere recorded during the anaerobic growth corresponding to the nutrient medium (pH 7.3)and the glucose deprived region of the cell layer (pH 6.5). The internal pH of non starving E.coli could not be measured because only a small fraction of the reactor was populated bygrowing cells. The energy state of the cells was determined by measuring the concentrationof nucleotide triphosphates.

Rehm and co-workers have used enzyme assays to measure enzyme activities of S.cerevisiae cells entrapped in Ca-alginate beads (Hilge-Rotmann and Rehm, 1990) and Pichiafarinosa cells immobilised on sintered glass Raschig rings (Höötmann et al., 1991). Cellsgrown in the form of micro-colonies in the alginate beads showed faster glucose uptake andethanol productivity with simultaneously decreased product and cell yields. In these cellsincreased specific hexokinase and phosphofructokinase activities could be determined. Thesealterations in physiology were not found in immobilised single cells. In the case of adsorbedP. farinosa, activities of some of the key enzymes involved in polyol and glycerol formation


42

were determined. The course of the activities of glucose-6-phosphate dehydrogenase andglycerol-3-phosphate dehydrogenase showed differences between free and immobilised cells.

Concentrations of intracellular intermediary metabolites fructose 1,6-diphosphate,pyruvate, citrate and malate in free and Ca-alginate immobilised S. cerevisiae fermenting D-glucose anaerobically have been determined with NAD+/NADH linked enzymatic assays(Skoog and Hahn-Hägerdal, 1989; Senac and Hahn-Hägerdal, 1990) when the sugar uptakerate and ethanol production rate were constant (Senac and Hahn-Hägerdal, 1991). No cellgrowth was observed and the fermentation yields and rates were the same in both types ofcells. The concentrations of intermediary intracellular metabolites were also identical forimmobilised and free cells.

Another non invasive technique which can be used to probe cell metabolism in theimmobilised state is radioisotope labelling/autoradiography. Radioisotope precursors for thesynthesis of all major biological polymers are readily available. This technique has beenapplied to look at overall distributions of isotopes incorporated in cell mass in dense cellsystems. By appropriate choice of a pulse-chase labelling protocol, and in combination withmass balance measurements on activities in the inlet and outlet streams of a reactor, a varietyof parameters of immobilised cell growth have been obtained (Karel et al., 1987; Stewart andRobertson, 1988; Karel and Robertson, 1989a, b). These include: (i) the net rate of cell masssynthesis at a given time, (ii) the yield coefficients for cell mass synthesis, (iii) thedegradation rate of cell mass in the immobilised cell system as a function of time after itssynthesis, (iv) the size of the region in which cell growth takes place, (v) the specific rates ofsubstrate consumption and cell growth within the growth region, (vi) the rate and pattern ofcell movement within the cell layer. For the case of E. coli in membrane reactors and gelbeads, these measurements have led to the conclusion that the intrinsic reactions are verysimilar to those for free cells.

Scanning microfluorimetry has been used for in situ investigations of spatialheterogeneities in biomass density, cell RNA content and DNA concentration (Monbouquetteand Ollis, 1988a, b; Ollis et al., 1990; Ollis, 1995). RNA content was related to the growthrate and used to infer the growth rate history of Ca-alginate immobilised Zymomonas mobiliscells (Monbouquette et al., 1990). A more accurate gauge of the cell growth rate is the rate ofDNA synthesis. Immunofluorescent measurements of DNA synthesis rate of Sr-alginateentrapped E. coli cells were accomplished by bromodeoxyuridine (BrdU) pulse-labellingcoupled with the use of anti-BrdU monoclonal antibodies (Kuhn et al., 1991, 1993). By usingthis technique in combination with mathematical modelling, they showed that gelimmobilised E. coli cells exhibit behaviour similar to free cells.

Al-Rubeai and Spier (1989) used a cytometabolism diffusion test in conjuction with imageanalysis for assessing the viability and metabolism of mammalian hybridoma cellsimmobilised in agarose gel. The test is based on the capacity of mitochondrial enzymes ofviable cells to transform the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl (MTT) tetrazolium salt(yellow) into MTT formazan (blue). It was observed that the activity of the cells decline withtime in a manner consistent with oxygen diffusion limitation.

To investigate the effect of diffusional limitations and heterogeneous yeast cell distributionin a gel immobilised cell system, a Ca-alginate gel membrane reactor has been constructed


43

(Willaert et al., 1990; De Backer et al., 1992). The membrane reactor consists essentially of agel membrane with immobilised S. cerevisiae cells, flanked by two well-mixed chambers.Substrate is pumped continuously through the first chamber, and this chamber can beconsidered as the equivalent of the space around a gel bead. The second closed measuringchamber contains a small quantity of liquid. Analysis of the liquid in this chamber gave directinformation on substrate (glucose) concentrations at the gel surface, and is an indication ofthe situation in the centre of a gel bead. The results were used in a dynamic mathematicalmodel of reaction and diffusion to determine the kinetic parameters of the immobilised cellsand revealed that they were comparable with those of free cells. Due to the diffusionallimitations, yeast cells grew preferentially closer to the feed edge of the membrane. Hannounand Stephanopoulos (1990) have also used a membrane reactor to determine the intrinsicreaction rates of alginate-entrapped S. cerevisiae by using diffusion-reaction analysis. Underanaerobic conditions, the specific growth rate of the immobilised cells decreased by 20%compared to the growth rate for suspended cells. The specific glucose uptake rate andspecific ethanol production rate increased by a factor 4 compared to those of suspended cells.The ethanol yield remained the same, and the biomass yield decreased to one-fifth of theyield for suspended cells. Further experiments were conducted under aerobic conditions toinvestigate the effects of dissolved oxygen: oxygen appeared to affect immobilised cells in away similar to suspended cells.

Microelectrodes can be inserted into immobilised cell systems to measure concentrationprofiles. This technique is necessarily invasive, and limited by the fabrication of appropriateelectrodes. Microelectrodes have been frequently used for biofilm research and especiallyoxygen microelectrodes are very popular (e.g. Lee and Tsao, 1979; Revsbech et al., 1989; deBeer and Sweerts, 1989; de Beer et al., 1990; Geesey and White, 1990; Lewandowski et al.,1991; Lens et al., 1993; Ottengraf and Van den Heuvel, 1995). An oxygen microsensor incombination with mathematical modelling has been used to determine the behaviour ofThiosphaera pantotropha immobilised in agarose (Hooijmans et al., 1990b) and of E. coliimmobilised in Ca-alginate (Hooijmans et al., 1990a; Huang et al., 1990). Schrezenmeir et al.(1994) used a oxygen microelectrode to measure O2 concentration profiles in Ba-alginatebeads with entrapped Brockmann bodies (islet organs) of Osphronemus gorami. Müller andco-workers (1994) investigated the oxygen monitoring technique with microelectrodes in Ca-alginate with and without entrapped S. cerevisiae . To obtain real Po2-profiles it wasimportant to be exactly informed about the physical, chemical and biological properties of thematerial to be investigated. It was recommended to apply a special stepwise puncturetechnique with distinct step-in/step-out movements of the electrode. Improper use of theelectrode can result in serious errors (pseudo-Po2-gradients) which could be explained byformation of artefacts and diffusion barriers in front of the electrode tip or oxygen"availability" at the tip and consumption of oxygen by the electrode itself.

A membrane mass spectrometric inlet reactor has been constructed which allowed themeasurement of oxygen and carbon dioxide "in" an immobilisation matrix (Willaert et al.,1990; Willaert 1993; 1995). This reactor has a thin but very low permeability barriermembrane (PET) on which a layer of immobilised cells is deposited, so that the samplewithdrawal is very low and the micro-environment of the immobilised cells is practically not


44

disturbed. The reactor has been used to monitor the behaviour of immobilised S. cerevisiaeby measuring oxygen and carbon dioxide. The results in combination with mathematicalmodelling were used to assess the influence of the immobilisation on the kinetics and showedthat the immobilised and free cell kinetics were comparable.

It has already been proven that light microscopy as well as electron microscopy (e.g. LarretaGarde et al., 1981; Eikmeier et al., 1984; Bailliez et al., 1985; Godia et al., 1987; Marin-Iniestaet al., 1988; Barbotin et al., 1990b) can serve as a valuable tool in studying the morphology ofimmobilised cells, the surface coverage of carriers and the dynamic behaviour of immobilisedcells (cf. scanning microfluorimetry and autoradiography). Recently, κ-carrageenan gel beadswith immobilised Nitrosomonas europaea and Nitrobacter agilis cells, were sliced in thincross sections after fixation and embedding; and the spatial biomass distribution wasdetermined by a specific fluorescent-antibody labelling technique (Hunik et al., 1993). Thedisadvantage of most of these techniques is that they are unsuitable for on-line analysis. Byconstructing a continuous "microscope reactor", microscopy could be used to investigate on-line and in situ the growth kinetics of S. cerevisiae in 2 % Ca-alginate (Willaert and Baron,1993). The specific growth rate of single immobilised cells and free cells were measured. Thegrowth of a microcolony in Ca-alginate was followed on-line and the specific growth rate ofthe yeast cells in the microcolony determined. Using a simple growth model for themicrocolony, the cell volume fraction of the cells in a colony was estimated: the packing ofyeast cells in a microcolony was comparable to the hexagonal close packing of spheres.Immobilised growth rates were found to be identical to those of free cells when internal andexternal mass transfer limitations are negligible.

2.2.2 Additional Physiological Aspects

2.2.2.1 BacteriaRecombinant plasmid stability in host cells can be increased upon gel immobilisation.

Plasmid pTG201 (ampicillin resistant, tetracycline-sensitive derivative of pBR322 containingthe Pseudomonas putida Xyl E gene that codes for catechol 2,3-dioxygenase) was unstable infree Escherichia coli cells, but stable when immobilised in κ-carrageenan gel (De Taxis duPoët et al., 1986; Nasri et al., 1987a, b; Marin-Iniesta et al., 1988; Briasco et al., 1990). Themaintenance of the plasmid of immobilised cells was always accompanied by the stabilisationof the plasmid copy number (Sayadi et al., 1988; 1989; Ollagnon et al., 1993). Enhancedplasmid stability has also been encountered for other plasmids in various immobilisationmatrices (De Taxis du Poët et al., 1987; Joshi and Yamazaki, 1987; Oriel, 1988a, b; Walls andGainer, 1988, 1991) and micro-organisms such as Myxococcus xanthus (Jaoua et al., 1986),yeast cells (Sode et al., 1988a, b) and Lactococcus lactis (D'Angio et al., 1994). The increasedplasmid stability may have resulted from the mechanical properties of the gel bead system thatallows only a limited number of cell divisions to occur in each microcolony before the cellsescape from the gel bead (Nasri et al., 1987b; Barbotin et al., 1990a; Barbotin, 1994).

Immobilisation can confer protection to cells exposed to toxic or inhibitory substrates orenvironments. It has been demonstrated that better phenol degradation rates are obtained withPseudomonas putida cells immobilised in Ca-alginate or polyacrylamide-hydrazide gel than


45

free cells, and that the immobilised bacteria could be exposed to higher phenol concentrationswithout loss of cell viability (Bettmann and Rehm, 1984). The phenol tolerance for bacterialacking the potential for phenol degradation (Escherichia coli and Staphylococcus aureus) wasalso enhanced when they were grown in Ca-alginate (Keweloh et al., 1989). It was found thatthe strength of the effect was correlated with the formation of colonies in the gel matrix(Keweloh et al., 1989; 1990a). The membranes of E. coli cells grown entrapped in Ca-alginate,showed low lipid-to-protein ratios which were also encountered in the membranes of free-grown cells in the presence of phenol (Keweloh et al., 1990b). Immobilisation of E. coli cellsalso markedly changed the protein pattern of the outer membrane. Heipieper et al. (1991)observed that cells immobilised and grown in alginate suffered a small loss of cations whenexposed to phenols. The re-establishment of gradients was observed at a higher phenolconcentration with immobilised cells compared to free cells and less membrane damage wasobserved.

It has been demonstrated that Ca-alginate immobilised cultures of Streptococcus lactis andS. cremoris were protected from attack by lytic bacteriophages due to the exclusion of phageparticles from the gel matrix (Steenson et al., 1987). These cultures were also functionallyproteinase-deficient when immobilised and grown in milk. Acid was produced by immobilisedcultures at a lower rate than cells freely suspended in milk due in part to the inability of theimmobilised cells to hydrolyse milk proteins, and to diffusional limitations of substrate into thebeads.

The effect of pH on the morphology of Lactobacillus helveticus in free-cell batch and κ-carrageenan-locust bean gum immobilised-cell continuous fermentation has been studied(Norton et al., 1993b). In free-cell batch fermentation, chain elongation of L. helveticusoccurred progressively when the pH set point was in the 5.1-6.3 range. Morphology was muchless pH-dependent during the immobilised-cell continuous fermentations. This phenomenonwas attributed to the long response time of entrapped cells to daily random changes in pH setpoints.

Lactococcus lactis ssp. lactis bv. diacetylactis has been entrapped in Ca-alginate to studythe bioconversion of citrate into flavour compounds such as diacetyl and acetoin (Cachon andDivies, 1993). Citric acid was totally utilised in a radius of 100 µm from the periphery of thegel bead. As a result, the activities of NADH oxidase, alcohol dehydrogenase, diacetylreductase and acetoin reductase, which are repressed in the presence of citrate, were higher inthe deeper zones than at the surface of the bead.

A sodium dodecyl sulphate (SDS) degrading strain of Pseudomonas fluorescens has beenimmobilised in polyacrylamide (White and Thomas, 1990). Immobilised cells lost their SDS-metabolising ability, possibly due to limited substrate diffusion caused by the polyacrylamide.

It has been demonstrated that enzyme production can be enhanced upon gel immobilisation.Ca-alginate entrapped Leuconostoc mesenteroides produced more dextran and dextransucrasethan free cells (El-Sayed et al., 1990a, b). Improved α-amylase production by gel immobilisedBacillus spp. has also been reported (Shinmyo et al., 1982; Chevalier and de la Noue, 1987;1988). Bacillus subtilis has been entrapped in polyacrylamide to produce α-amylase (Kokubeet al., 1978). The amount of α-amylase produced by the immobilised cells was approximatelythree times higher than that produced by free cells under optimised conditions and immobilised


46

cells were longer active than free cells. In contrast, α -amylase production by Bacillusamyloliquefaciens was reduced upon Ca-alginate immobilisation (Argirakos et al., 1992). Mostof the time higher protease production was found when the cells were immobilised(Vuillemard et al., 1988; Aleksieva et al., 1991; Kanasawud et al., 1992). Protease productionby free Myxococcus xanthus cells was inhibited by peptone and gelatine (Fortin andVuillemard, 1990). This repressive effect was eliminated when the cells were immobilised inalginate, since gel immobilisation could reduce the diffusion of the repressors to the cells.Substantially different metabolic activities have been observed when Escherichia coli cellswere immobilised in alginate to produce β -galactosidase: cells released from the gelsynthesised 1.6 (aerobic growth) and 4.9 (anaerobic growth) times as much β-galactosidase percell as suspended cells (Zhang et al., 1989).

The increased production of alginate by Pseudomonas aeruginosa and of gellan gum byPseudomonas elodea which were immobilised in κ-carrageenan, compared to free cells, wasattributed to a favourable media supplement (probably metal ions such as Ca2+, Mg2+ andZn2+) with the aqueous soluble components of the matrix (Brito et al., 1990b).

The physiology of Mycobacterium sp. entrapped in Ca-alginate gel was compared to that offree cells, using the biotransformation of propene to 1,2-epoxypropane (Smith et al., 1990). Inthe absence of mass transfer limitations there were no significant differences between free andimmobilised cells with respect to NADH levels and energy status.

It has been demonstrated that the germination time of Bacillus subtilis cells wassignificantly longer than for free cells; and that after a time lag due to encapsulation, thegrowth of the cells was uninhibited and no difference between entrapped and free cells werefound (Pepeljnjak et al., 1994).

The properties of immobilised bacteria may differ fundamentally from those of free cells,since the morphology of certain species is modified by confinement or by attachment to asurface. The luminous marine bacterium Vibrio fischeri can express one phenotype for growthin liquid medium ("swimming" phenotype) and another for growth on surfaces ("swarming"phenotype) (Krieg and Holt, 1984). Confinement of Vibrio cells has also an influence on themorphology, because it has been observed that Vibrio alginolyticus cells in the interior of aswarming colony (Ulitzur and Kessel, 1973) or Vibrio fischeri cells in micro-colonies in Ca-alginate beads (Willaert, 1993) do not have the swarmer phenotype. The regulation of thelateral flagella gene (laf) transcription in Vibrio parahaemolyticus has been studied by Belasand co-workers (1986). They discovered that laf gene transcription was induced when cellswere propagated on agar media (swarming phenotype) but not when cells were grown in liquidmedia (swimming phenotype), and induction could also be accomplished by increasing theviscosity of the liquid medium.

Usually, gel immobilised micro-organisms give rise to micro-colonies when they are grownin gels. It has been demonstrated that the morphology of cells in a colony can be markedlydifferent compared to free-grown cells. Sharply delineated zones of bacterial populationsdifferentiated from each other with respect to cell sizes, cell shapes and patterns ofmulticellular alignment were found in colonies of E. coli , and it was reported that extracellularmaterial is deposited (Shapiro, 1987). Furthermore, micro-colonies of Pseudomonas putida


47

(Shapiro, 1985) and E. coli (Keweloh et al., 1989) were surrounded by a "membrane" ofextracellular material.

2.2.2.2 FungiCa-alginate immobilised cells of Aspergillus niger needed a lower initial sucrose

concentration than free cells in order to obtain maximal yields of citric acid production(Honecker et al., 1989). High sucrose concentrations led to reduced yields and increased polyolformation (i.e. glycerol, erythritol, arabitol). Continuous fermentation with media containinglow sugar concentrations prevented the formation of polyols. The change from nitrogen-limited to phosphate-limited precultivation of immobilised spores significantly increased theproductivity of the mycelium. The ratio of citric acid to residual sugar in the effluent distinctlylay in the direction of citric acid. Inside the alginate beads mainly large bulbous cells wereobserved.

Physiological studies related to the immobilisation of Penicillium chrysogenum in κ -carrageenan and penicillin production have been performed by Mussenden et al. (1993).Reducing the immobilised viable spore loading from 4 104 to 2 103 spores/ml gel and initialbead diameter from 3.5-4.0 to 1.5-2.0 mm, gave rise to an increase in the penicillin titer from0.2 to 1.2 g/l. Using these conditions in immobilised cell culture, the growth phase wasprolonged and the duration of expression of isopenicillin N synthase gene (pcbC) wassignificantly extended when compared with free cell culture (150 h as opposed to 100 h).During the period of maximum penicillin production, different penicillin biosyntheticintermediates accumulated in the broth of free and immobilised cell culture, reflecting afundamental difference in cell physiology. Although the maximum specific productivity ofpenicillin production was reduced by immobilisation, the average specific productivityincreased when compared to free cell fermentation.

The zygomycete Mortierella isabellina has been entrapped in alginate beads to transformdehydroabietic acid into non toxic metabolites (Kutney et al., 1985). It was shown thatimmobilisation resulted in greater long term stability of enzyme activity compared with freemycelia and the breakdown products differed which was attributed to a greater level ofsecondary metabolism.

The growth and alkaloid production by Claviceps purpurea depends on the inorganicphosphate concentration. The phosphate optimum of production was shifted towards lowerconcentrations when the cells are entrapped in Ca-alginate, being only about a quarter of theone determined for free cells (Lohmeyer et al., 1990b). The physiological activity of Ca-alginate entrapped Claviceps fusiformis have been studied during 550-day semi-continuouscultivation (Kren et al., 1987). No extracellular glucans were produced by the immobilisedcells. Alginate concentrations in the range of 2 to 4% did not influence the yield and spectrumof produced alkaloids. After 3 days, the cytoplasm of the immobilised cells became condensed,polysaccharides disappeared and centres of lipid synthesis were formed in the cytoplasm. After60 days, the cells harboured a great number of lipid particles, mitochondria were diminishingand their cristae disappeared, indicating a decreased respiration capacity. After 350 - 500 days,the volume of most cells was increased many times and the cells were filled with large ovalbodies of electron-dense materials.


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It has been shown that the kind and concentration of the immobilisation support (alginate,carrageenan or polyurethane) for Fusarium moniliforme affected the production of secondarymetabolites (i.e. gibberellins, carotens and bikaverins) for a given type of culture (NavaSaucedo et al., 1989b; 1990). Changes in the growth development, morphological appearance,mycelial viability under starvation conditions and induction of resting forms in post-immobilisation cultures using alginate as support under different conditions, have beenobserved (Nava Saucedo et al., 1989b). Significant ultrastructural differences between free andCa-alginate immobilised mycelia have been found: the presence of large glyoxisome-likebodies and active vesicle-generating systems, the formation of endocells resulting in hyphaewith up to three cell walls and the concomitant accumulation of secondary metabolites (mainlypigments) in peripheral cell compartments, the progressive development of autophagicvacuoles involved in the turnover of cell constituents (Nava Saucedo et al., 1989c).

It has been shown that alginate entrapped Candida pseudotropicalis is more effective for thetreatment of dairy effluents than free cells, probably due to the protective sheath of the alginatematrix (Marwaha et al., 1990).

The ethanol yield was increased when sand was co-entrapped in alginate withSaccharomyces formosensis (Fang et al., 1983). This was explained as a stimulation of thefermentation process by Si4+ by permeabilising the cell wall and/or membrane. Theimprovement of the ethanol fermentation of high concentrations of glucose by κ-carrageenanimmobilised S. bayanus, compared to free cell fermentation, was attributed to a favourablemedia supplement (probably metal ions such as Ca2+, Mg2+ and Zn2+) with the aqueoussoluble components of the matrix (Vieira et al., 1989; Brito et al., 1990b).

Protoplasts of S. cerevisiae may regenerate the cell wall and revert to cells if entrapped in2%-5% Ca-alginate gel and cultured in an osmotically stabilised medium (Svoboda andOurednicek, 1990). The reversion yield was dependent on the actual gel concentration, gelshape (beads versus sheets) and the medium molarity. The morphology of the cell wallregeneration and morphology of reversion to the cell forms corresponded to protoplastdevelopment in gelatine or agar gels (Necas and Svoboda, 1985). The fatty acid composition ofS. cerevisiae immobilised in Ca-alginate beads has been determined and compared to that offreely suspended cells under different fermentation conditions (Hilge-Rotmann and Rehm,1991). The fermentation product ethanol was found to cause a shift towards saturation in thefatty acid composition under anaerobic conditions. Immobilised cells contained significantlyhigher percentages of saturated fatty acyl residues, especially of palmitic acid (16:0), and adecreased amount of oleic acid (18:1) compared to free cells. The percentage saturation of totalfatty acid composition correlated positively with improved fermentation rates obtained withthe immobilised cells. The researchers suggested that this enhanced saturation in immobilisedcells may be due to altered osmotic conditions in the micro-environment of the cells.

Co-immobilising cells (e.g. S. cerevisiae) with vegetable oils (castor, soy bean, olive, peanutoil) can be used to protect the cells against inhibitory substances (e.g. p-hydroxybenzoic acidester or Preventol GD) in the broth (Tanaka et al., 1994; Ohta et al., 1994). In such a system,the cells grow only in the water phase of the gel beads where most components of thefermentation medium are retained, whereas the p-hydroxybenzoate is retained mainly in the oilphase. Consequently, the p-hydroxybenzoate concentration in the water phase remains too low


49

to inhibit the metabolic activities of the immobilised cells. The effectiveness of a vegetable oilin protecting the immobilised cells against an inhibitory substance depended on the partitioncoefficient of the substance between the oil and water, the concentration of the oil and theinitial cell concentration. Longer process stability was achieved when p-hydroxybenzoate wasreplaced with Preventol GD which has a higher partition coefficient between castor oil andwater (Ohta et al., 1994).

The destiny and behaviour of S. cerevisiae cells entrapped for a long-term period in Ca-alginate beads have been studied. During long-term continuous anaerobic process (1100 hcultivation), performed in a packed bed reactor on complete medium at 30°C, immobilisedcells retained high metabolic activity. It was further confirmed that beads may be stored for along time (even longer than 1 year) before application or between cultivations without loosingglycolytic activity and viability (Melzoch et al., 1994). Simon et al. (1990) found that thecarbohydrate and protein content of Ca-alginate entrapped S. cerevisiae could be considerablydifferent from those of free cells after long term culture.

Recently, it has been shown using mathematical modelling that the intrinsic ethanolproductivities of Ca-alginate entrapped S. cerevisiae was similar to that of free yeast and thereductions in productivity were caused by lower substrate concentrations towards the centre ofthe bead due to mass transport limitations (Gilson and Thomas, 1995).

The ethanol inhibition effect in the fermentation of xylose by free and immobilised Pichiastipitis in κ-carrageenan has been determined (Chamy et al., 1994): the tolerance of theimmobilised yeasts to ethanol was lower than that of free cells. It was explained that this effectmay be due to diffusion and counter diffusion problems of ethanol in the gel bead. However,Norton et al. (1993a) used a method based on yeast survival following an ethanol shock toevaluate the effect of κ-carrageenan entrapment on the ethanol resistance of brewer's yeast. Avery significant protection effect of cell entrapment was observed for ethanol shock. Releasedcells did not exhibit a significantly higher ethanol tolerance as compared to free cells whichwas an indication that no durable alteration of cell physiology or composition occurred inrelation to stress resistance.

Osmotic inhibition of the anaerobic growth and ethanol productivity of free andimmobilised Kluyveromyces marxianus in whey permeate concentrate has been studied byDale and co-workers (1994). The cell growth of the immobilised cells was more stronglyinhibited than the free cells at higher osmolalities, whereas the productivity of the immobilisedcells was less inhibited by high osmolality.

2.2.2.3 AlgaeImmobilisation of Botryococcus braunii in Ca-alginate resulted in a decreased growth rate

and to a marked improvement in hydrocarbon production compared to free cells (Bailliez etal., 1985). It was also demonstrated that these alginate immobilised cells exhibited anincreased chlorophyll content and chlorophyll photosynthetic activity, relative to free cells, atany stage of standard batch cultures (Bailliez et al., 1986). Immobilisation exerted aprotective influence on ageing cultures both under standard and air lift conditions. Decreasesin chlorophyll content and photosynthetic activity were delayed and slowed down and theorganisation of protein-chlorophyll complexes was stabilised. These positive effects were


50

related to protection of entrapped cells against photo-inhibition owing to gel screening andself-shadowing. The higher photosynthetic activity was explained by an increased ionicconcentration in the micro-environment of the cells.

Entrapped Chlamydomonas reinhardtii cells in alginate matrix exhibited a full viabilityand improvement of their nitrite uptake rate when used as biocatalyst for ammoniumphotoproduction (Santos-Rosa et al., 1989a), reaching a yield higher than free-living cells(Santos-Rosa et al., 1989b). These alginate immobilised cells showed also higher totalphotosynthetic and photosystem I activities than free cells, but no significant changes on theactivity of photosystem II as well as on the respiratory rate of the cells were observed afterimmobilisation (Galvan et al., 1990).

Chlorella emersonii Shihira and Kraus var. emersonii cells entrapped in Ca-alginate matrixhad reduced respiratory and growth rates, and a higher chlorophyll content than free cells(Robinson et al., 1985). Respiratory rate per cell was reduced as cell stocking densityincreased. The green alga Chlorella fusca and the cyanobacterium Synechoccus leoppoliensishave been immobilised in Ca-alginate, Ca-alginate + polyanhydrid, chitosan, chitosan +Eudragit, agar and carrageenan for the asymmetrically reduction of ketones (Fooladi et al.,1990). Resting cells of both micro-organisms showed the highest activity in Ca-alginate;immobilised cells showed the same reaction kinetics as free cells and immobilisationincreased the stability of the biocatalysts.

2.2.2.4 Plant CellsAlginate entrapped plant cells have shown in several instances an enhanced product

formation as compared to freely suspended cells under the same conditions: ajmalicine(Brodelius and Nilsson, 1980; Asada and Shuler, 1989) and serpentine (Brodelius et al.,1981) production by Catharanthus roseus, anthraquinones by Morinda citrifolia (Brodelius etal., 1980a), codeine by Papaver somniferum (Furuya et al., 1984), methylxanthine by Coffeaarabica (Brodelius, 1988), capsaicin by Capsicum frutescens (Lindsey and Yeoman, 1984;Sudhakar Johnson et al., 1991), and solavetivone production by Hyoscyamus muticus(Ramakrishna et al., 1993). Various studies have shown that diffusion limitations are not sopronounced in slow growing immobilised cell preparations: e.g. immobilised cells areappropriately oxygenated (Vogel and Brodelius, 1984; Hulst et al., 1985a) and the phosphatemetabolism is essentially unaffected by immobilisation (Vogel and Brodelius, 1984).Reversible interaction between alginate and pectic acid (a component of the cell wall) couldbe established by Ca2+-ions. In this way, alginate could function as a glue between cells andthereby mediate a cell to cell interaction mimicking that of a differentiated plant tissue(Brodelius, 1988). Of the various polymers used for entrapment of plant cells, only alginatecan function in this manner and only in alginate entrapped cells an increased secondaryproduct formation has been observed. An alternative or additional explanation to theincreased production may be found in the suggestion that alginate can function as an elicitorin plant cell systems (Wolters and Eilert, 1983; Brodelius, 1988). Asada and Shuler (1989)have shown that ajmalicine production and excretion from C. roseus could be stimulated byadsorption in situ (using the neutral resin Amberlite XAD-7), elicitors (i.e. autoclaved


51

cultures of the mold Phytophthora cactorum) or Ca-alginate immobilisation. It was furtherdemonstrated that these 3 factors had a synergistic effect when applied together.

Ca2+ ions or immobilisation in Ca-alginate gel increased the alkaloid production by Daturainnoxia (Thomasset et al., 1990). Sudhakar Johnson et al. (1991) demonstrated an elicitationof capsaicin production by alginate immobilised C. frutescens cells by curdlan and xanthanwith a synergistic influence when both elicitors were present in the medium. Also,immobilised placental tissues from C. frutescens exhibited greater potentiality for capsaicinsynthesis than immobilised cells (Sudhakar Johnson et al., 1990).

Glandular epithelial cells of resin ducts of the desert tree Comniphora wightii have beenentrapped in Ca-alginate to study the production of gugulipids (Jacob John et al., 1990). Theresin ducts retained their differentiated state in the immobilised state, whereas nonimmobilised tissue gave rise to callus formation. The neutral lipid production by theimmobilised system was much higher than by the conventional (free) cell culture systems.

The influence of entrapment material (i.e. Ca-alginate, κ-carrageenan and agarose), cellloading and, in case of alginate, bead diameter on the rate of respiration of immobilisedDaucus carota cells has been investigated (Hulst et al., 1985a). No significant differenceswere observed between the three immobilisation materials and no loss of respiration activityoccurred as a result of immobilisation per se. Furthermore, above a critical combination ofcell loading and bead diameter limitations of the rate of respiration by diffusion of oxygenincreased with increasing loading and diameter.

The growth of carrot plantlets (D. carota) was promoted by encapsulation of somaticembryos compared with the plantlets from naked, unencapsulated embryos due to theremoval of the water and nutrient stress to which embryos are readily exposed at early stagedevelopment (Tsuji et al., 1993).

2.2.2.5 Animal CellsCells within tissues encounter a complex chemical and physical micro-environment: each

cell is surrounded by a matrix of hydrated glycoproteins, each cell can contact other cells ofthe same or different type and each cell is bathed in an aqueous medium containing solublefactors that provide essential signals for cell growth and differentiation. The in vitro culture ofthese cells require a three-dimensional culture system which mimic the environment of tissues.Gel encapsulation has been used for this purpose.

A wide range of liver-specific functions were compared between adult rat hepatocytessandwiched by two layers of collagen gel (sandwich system) and those on a single layer ofcollagen matrix (Dunn et al., 1989; 1991; 1992; Yarmush et al., 1992). The sandwich systemprovided an environment that more closely mimics the normal histological organisation of theliver. The relatively simple addition of the top collagen overlay in the sandwich systemproduced significant effects both morphologically and functionally. The effect of the geloverlay correlated with (i) a change in the cytoskeletal organisation, (ii) the elaboration of acomplex extracellular matrix, and (iii) the expression of surface receptors in culturedhepatocytes. Also, the growth and function of chicken hepatocytes encapsulated in collagengels and attached to tissue culture polystyrene (TCPS) have been compared (Saltzman et al.,1992). It was demonstrated that hepatocytes immobilised in collagen gel reached a


52

significantly higher cell density, DNA and protein content after 96 h of culture. The albuminsecretion rate was significantly greater for high-cell-density gel cultures when compared toTCPS cultures. Rat hepatocytes have been immobilised in Ca-alginate beads and detoxificationfunction was assessed quantitatively by measuring the kinetics of several specificdetoxification systems: the cytochrome P450 system, the urea cycle and two conjugationsystems. Reaction rates for all enzyme systems were similar in immobilised and nonimmobilised cells (Tompkins et al., 1988).

Chondrocytes are cartilage-specific cells which in the tissue do not ensure direct cell-cellinteractions. They secrete a series of cartilage-specific molecules (collagen types II, IX, XI, Xand the proteoglycan aggrecan). When cultured in monolayer, human articular chondrocytesrapidly lose their differentiated phenotype, becoming dedifferentiated cells which synthesisemolecules normally expressed by mesenchymal cells (collagen types I, III, V and versican).Entrapment of isolated articular chondrocytes within agarose gel has become a popular culturesystem (Benya et al., 1978; Aydelotte et al., 1986; Bruckner et al., 1989). A matrix thatappears to share many of the physicochemical properties of normal cartilage matrix, is formedaround the entrapped cells (Buschmann et al., 1992). Several studies have now established thatthe entrapment of isolated chondrocytes in agarose gel helps to promote the synthesis ofcollagen type II and keratan sulphate-containing aggrecan, which are markers of thechondrocytic phenotype (Benya and Shaffer, 1982; Aydelotte et al., 1991; 1992). Recently,chondrocytes have also been entrapped in alginate (Kupchik et al., 1983; Guo et al., 1989;Häuselmann et al., 1992; Tamponnet et al., 1992; Chan et al. 1993). It has been demonstratedthat rabbit articular chondrocytes cultured for 38 days (Guyot et al., 1990) and bovine adultarticular chondrocytes cultured for 8 months (Häuselmann et al., 1994) in Ca-alginate beadshave retained their phenotype and are synthesising collagens and proteoglycans that are typicalof those found in the cartilage matrix from which the cells are isolated. In the latter case, thecells were present as two populations that differ in shape depending upon their location (nearthe surface of the bead or further inside the bead) within the alginate bead (Häuselmann et al.,1994). It has been shown that dedifferentiated human articular chondrocytes were able torevert to a chondrocytic phenotype in the alginate system, even when cells were expanded onplastic flasks by subculture (Bonaventure et al., 1994).

Mouse neuroblastoma cells have been entrapped in Ca-alginate (Simonneau et al., 1990).Growth of these cells was demonstrated and the membrane properties of the cells were notmodified as far as voltage-dependent ionic channels are concerned. Furthermore, storage of 2to 3 days at 4°C appeared not to modify cell growth and surface membrane proteins.

Shirai and co-workers (1988) measured the specific oxygen uptake rate of hybridoma cellsimmobilised in Ca-alginate gel beads. The observed data were compared with those of non-immobilised cells. The uptake rate of the immobilised cells coincided with that of the free cellsjust after immobilisation, but increased with cell growth. On the other hand, the cellularglucose consumption rate decreased slightly during the experiments. The increased oxygenuptake rate by immobilised cells was closely related to the formation of cell colonies in the gelparticles. It has been demonstrated that the stability of antibody productivity is improved whenhybridoma cells are entrapped in Ca-alginate beads, due to the limited cell growth in entrappedcell culture (Lee and Palsson, 1993).


53

2.3 Applications

2.3.1 Gel Immobilised Growing Microbial Cells

2.3.1.1 Production of Amino AcidsAmino acids are widely used in the food, feed, pharmaceutical, cosmetic and chemical

industries. The L-isomers of the amino acids are used for food and nutritional applications(since the L-isomers are compatible with human metabolism) whereas the D-isomers areuseful for the synthesis of antibiotics. The use of amino acids as food additives constitute amultibillion dollar market (Hamilton et al., 1985). With about 370,000 tons per year, L-glutamic acid is the amino acid with the largest production volume by fermentation. L-Glutamic acid and L-alanine are mainly used as taste enhancers whereas L-aspartic acid andL-phenylalanine are used for the synthesis of the diet sweetener Aspartame. Essential aminoacids, such as L-phenylalanine, L-tryptophan and L-lysine, can be used to supplementdeficient diets.

Gel immobilised technology have been applied on growing cells for the production ofvarious amino acids as listed in Table 14. An interesting feature of biotechnological aminoacid production is that it can be conducted in several steps since some amino acids are used asprecursors for others (Norton and Vuillemard, 1994).

TABLE 14PRODUCTION OF AMINO ACIDS BY GEL ENTRAPPED GROWING MICROBIAL CELLS

Amino Acid Micro-organism Entrapment ReferenceMethod

L-Alanine Corynebacterium Polyacrylamide, Sarker and Mayaudon, 1983dismutans κ-carrageenan,

DEAE-celluloseEscherichia coli κ−Carrageenan Sato et al., 1982; Takamatsu et

al., 1982Pseudomonas dacunhae κ−Carrageenan Yamamoto et al., 1980; Sato et

al., 1982; Takamatsu et al., 1982

L-Arginine Serratia marcescens κ−Carrageenan Fujimara et al., 1984Bacillus subtilis κ−Carrageenan Kolot, 1981

L-Aspartic acid Escherichia coli κ−Carrageenan Sato et al., 1979; Tosa et al.1979; Nishida et al., 1979; Sekoet al., 1990

Polyacrylamide Chibata et al., 1974; Takamatsuet al., 1984a, b

N-Carbamoyl- Bacillus sp. Polyacrylamide Yamada et al., 1980 D-amino acids


54


Amino Acid Micro-organism Entrapment ReferenceMethod

L-Glutamic acid Corynebacterium Polyacrylamide Slowinski and Charm, 1973glutamicum κ-Carrageenan Kim and Ryu, 1982

Euchema gel + Li et al., 1990cellulose acetate

Brevibacterium Ca-alginate Lu and Chen, 1988ammoniagenesBrevibacterium flavum Collagen Constantinides et al., 1981Corynebacterium lilium Collagen Vieth and Venkatasubramanian,

1979L-Glutamic acid Corynebacterium Sr-alginate Ogbonna et al., 1991a, b + glutamine glutamicumGlycolate Chlamydomonas Ba- and Ca-algi- Galvan et al., 1990

reinhardtii nate5-Hydroxytryp- Escherichia coli Polyacrylamide Chibata et al., 1974 tophanL-Isoleucine Serratia marcescens κ-Carrageenan Wada et al., 1979; 1980bL-Lysine Bacillus subtilis Ca-alginate Israilides et al., 1989

Corynebacterium PVA Velizarov et al., 1992glutamicumMicrobacterium Polyacrylamide Kanemitsu, 1975ammoniaphilum

L-Phenylalanine Paracoccus κ-Carrageenan Nakamichi et al., 1989denitrificans

L-Serine Corynebacterium Ca-alginate Tanaka et al., 1989glycinophilumProtomonas Ca-alginate Sirirote et al., 1988extorquens

L-Tryptophan Escherichia coli Polyacrylamide Marechal et al., 1979; Bang et al., 1983

κ-Carrageenan da Fonseca et al., 1995Escherichia coli + Alginate Ishiwata et al., 1990Pseudomonas putida

2.3.1.2 Production of Organic AcidsOrganic acids are extensively used in the food and pharmaceutical industries and some of

them are products of microbial processes. Organic acids can be produced by gel immobilisedmicrobial cells via bioconversion (cf. Table 15) or de novo (cf. Table 16). Acetic acid, lacticacid and propionic acid are growth-related primary metabolites. In this case, the advantages


55

of cell immobilisation are mainly linked to the increase in productivity due to high biomassconcentration and the improved stability in continuous operation. Citric acid and kojic acidare secondary metabolites. Another advantage is the possibility of continuous operation of areactor containing non growing cells starved of nitrogen or other limiting substrates (Nortonand Vuillemard, 1994). Production of lactic acid occurs by simple fermentation since onlylactic acid is produced when homofermentative strains are employed. Therefore, thisfermentation is frequently used as a model system for immobilisation. Lactic acid can be usedas polylactide for the production of biodegradable packaging films, food acidulant andpreservative in a wide range of foods and beverages. Approximately 50% of the lactic acidproduction is manufactured synthetically by hydrolysis of lactonitrile. Citric acid is the mostwidely produced organic acid with a world market of 350,000 tons per year (Milsom, 1987).Approximately 75% of this acid is used as food acidulant. The conversion of fumaric acidinto malic acid was the first successful industrial process for the production of organic acidsby immobilised cells (Yamamoto et al., 1976). Itaconic acid is used as a starting material forplastics. Itaconic, citric and lactic acid have been produced by immobilised fungal cells(Horitsu et al., 1988).

TABLE 15PRODUCTION OF ORGANIC ACIDS VIA BIOCONVERSION BY GEL ENTRAPPED LIVING

MICROBIAL CELLS

Organic Acid Substrate Micro-organism Entrapment ReferencesMethod

Acetic acid Ethanol Acetobacter aceti κ-Carrageenan Osuga et al., 1984; Mori, 1985

α,ω-Alkanedi- n-Alkanediol Candida tropicalis κ-Carrageenan Yi and Rehm, 1982 oic acidα-Keto-iso- L-Leucine Providencia sp.+ Agarose Wikström et al., 1982 capric acid Chlorella vulgarisα-Keto-γ-methi- D-Methionine Providencia sp. Ca-alginate Swajcer et al., 1982 ol-butyric acid L-Methionine Trigonopsis Ca-alginate Brodelius et al., 1980b

variabilisL-Malic acid Fumaric acid Brevibacterium Polyacrylamide Yamamoto et al., 1976

ammoniagenesBrevibacterium κ-Carrageenan Takata et al., 1979;flavum 1980; 1983a, bCandida rugosa Polyacrylamide Yang and Zhong, 1980

Poly(vinyl Yang et al., 1992alcohol)

Saccharomyces Agarose Neufeld et al., 1991cerevisiae (gen-etically engineered)


56


Organic Acid Substrate Micro-organism Entrapment ReferencesMethod

Saccharomyce Polyacrylamide Oliveira et al., 1994cerevisiae

Vanillic acid Vanillin Pseudomonas Ca/Ba-alginate Baré et al., 1994fluorescens

TABLE 16PRODUCTION OF ORGANIC ACIDS BY GEL ENTRAPPED LIVING MICROBIAL CELLS

Organic Acid Micro-organism Entrapment ReferenceMethod

Acetic acid Acetobacter sp. Collagen Vieth and Venkatasu-bramanian, 1979

Acetobacter aceti Ca-alginate Sun and Furusaki, 1991a, b

Acetogenium kivui Poly(vinyl alcohol) Rainina et al., 1994Acetic acid Propionibacterium sp. Polyacrylamide Vorobeva et al., 1977Acrylic acid Clostridium propionicum Ca-alginate O'Brien et al., 1990ε-Aminocarpoic acid Achromobacter guttatus Polyacrylamide Kinoshita et al., 1975Chorismic acid Enterobacter aerogenes Polyacrylamide Keller and Lingens,

1984Citric acid Aspergillus niger Agarose Khare et al., 1994

Ca-alginate Vaija et al., 1982; Eikmeier et al., 1984; Horitsu et al., 1984; Honecker et al., 1989

κ-Carrageenan Eikmeier et al., 1984Collagen Vieth and Venkatasu-

bramanian, 1979Polyacrylamide Horitsu et al., 1985

Candida lipolytica Polyacrylamide Stottmeister, 1980; Berger and

Langhammer, 1980Yarrowia lipolytica Polyurethane, Ca- Kautola et al., 1991

alginate, κ-carra-geenanCa-alginate Rymowicz, 1993


57



Gibberellic acid Fusarium moniliforme Alginate Nava Saucedo et al., 1989a

Carrageenan Nava Saucedo et al., 1989b

Gluconic acid Aspergillus niger Ca-alginate Rao and Panda, 1994;Moresi et al., 1995

Polyacrylamide Kolot, 1981Gluconobacter oxydans Alginate Tramper et al., 1983Saccharomyces cerevisiae Polyacrylamide Nadkarni and D'Souza,

1981Itaconic acid Aspergillus terreus Agar, Ca-alginate Kautola et al., 1985

Polyacrylamide Horitsu et al., 19832-Keto-L-gulonic Gluconobacter oxydans + Agar, alginate, Yuan et al., 1992 acid Bacillus cereus κ-carrageenan,

chitosan, CM-cel-lulose, polyacryl-amide

Kojic acid Aspergillus orizae Ca-alginate Kwak and Rhee, 1992a, b

Lactic acid Aspergillus awamori + Alginate Kurosawa et al., 1988Streptococcus lactisLactobacillus sp. Pectate Richter et al., 1991Lactobacillus bulgaricus Ca-alginate Stenroos et al., 1982;

Linko et al., 1984Lactobacillus casei + κ-Carrageenan Hoshino et al., 1991amylaseLactobacillus casei Agar Tuli et al., 1985

Alginate Guoqiang et al., 1991κ-Carrageenan Arnaud et al., 1992bPolyacrylamide Divies and Siess, 1976

Lactobacillus casei, κ-Carrageenan, Roukas andLactabacillus lactis agar, Ca-alginate, Kotzekidou, 1991

polyacrylamideLactobacillus delbrueckii Ca-alginate Stenroos et al., 1982

κ-Carrageenan Yabannavar and Wang,1991

Lactobacillus delbrueckii, κ-Carrageenan/ Audet et al., 1990;


58



Streptococcus salivarius locust bean gum Lacroix et al., 1990Lactobacillus helveticus Ca-alginate Linko et al., 1984; Roy

et al., 1987; Boyaval and Goulet, 1988

κ-Carrageenan Norton et al., 1993bκ-Carrageenan/ Norton et al., 1994a, blocust bean gum

Lactobacillus pentosus Ca-alginate Linko et al., 1984Lactobacillus Ca-alginate Tipayang and Kozaki,vaccinostercus 1982Leuconostoc Agar/Agarose, Totsuka and Hara, 1981mesenteroides alginate

Lactic acid Leuconostoc oenos Alginate Spettoli et al., 1982Methylobacillus Polyacrylamide, Dinarieva andflagellatum agarose, Netrusov, 1991

carrageenanRhizopus oryzae Ca-alginate Horitsu et al., 1984;

Hang et al., 1989Schizosaccharomyces Agar/agarose, Totsuka and Hara, 1981pombe alginateStreptococcus lactis Ca-alginate Ohta et al., 1994Streptococcus salivarius κ-Carrageenan/ Audet et al., 1991

locust bean gum12-Ketochenodeoxy- Brevibacterium flavum κ-Carrageenan Sawada et al., 1981 cholic acid2-Ketogluconic acid Gluconobacter melanoge- Polyacrylamide Martin and Perlman,

nus + Pseudomonas 1976ayringaeSerratia marcescens Collagen Venkatasubramanian et

al., 1978Methylperhydro- Nocardia sp. Polyacrylamide Glomon et al., 1982 indanone propionic acidPropionic acid Propionibacterium sp. Polyacrylamide Vorobeva et al., 1977

Propionibacterium Alginate, κ-carra- Bégin et al., 1992shermani geenan

Tartaric acid Achromobacter sp. Polyacrylamide Kawabata and Ichikura,1977


59

2.3.1.3 Production of AntibioticsAbout 150 antibiotics are presently commercially available, and most of them are

produced by microbial processes. Other production processes make use of enzymaticreactions, chemical synthesis or a combination of these methods. Batch processes dominate,because antibiotics are non growth associated secondary metabolites which are synthesised denovo after the exponential growth phase, and because the production activities are oftenunstable. Furthermore, mycelium-forming microbial cells are used for antibiotic productionwhich makes the continuous production difficult. These problems can be overcome to someextent by using immobilised cells. The major problem in antibiotic production usingimmobilised cells is to attain a continuous stable production of secondary metabolites withlimited growth (Vandamme, 1988; Tanaka and Nakajima, 1990; Furusaki and Seki, 1992).This implies a proper regulation of the concentration of growth limiting substrates such ascarbon source (Ogaki et al., 1986), oxygen (Trueck et al., 1990a), nitrogen (Morikawa et al.,1980b; Deo and Gaucher, 1985; Trueck et al., 1990b), phosphate (Trueck et al., 1990b;Constantinides and Mehta, 1991) and amino acids (Chun and Agathos, 1991). The variousantibiotics produced by gel entrapped microbial cells and entrapment materials are listed inTable 17.

TABLE 17PRODUCTION OF ANTIBIOTICS BY GEL ENTRAPPED LIVING MICROBIAL CELLS

Antibiotic Micro-organism Entrapment ReferenceMethod

Actinomycin D Streptomyces parvullus Ca-alginate Dalili and Chau, 1988Ampicillin Kluyvera citrophila Polyacrylamide Morikawa et al.,

1980aBacitracin Bacillus sp. Polyacrylamide Morikawa et al., 1979;

1980bCandicidin Streptomyces griseus κ-Carrageenan Constantinides and

Mehta, 1991Collagen Vieth and Venkatasu-

bramanian, 1979Cephalexin Xanthomonas citri κ-Carrageenan, Kim et al., 1983a

polyacrylamideCephalosporins Streptomyces clavuligeris Polyacrylamide- Freeman and

hydrazide Aharonowitz, 1981Cyclosporin A Tolypocladium inflatum κ-Carrageenan Foster et al., 1983Cyclosporin C Cephalosporium Ca-alginate Khang et al., 1988a;

acremonium Kundu et al., 1992Ca-, Ba- and Sr- Park and Khang, 1995alginate + polyethy-eneimine


60


Antibiotic Micro-organism Entrapment ReferenceMethod

Daunorubicin Streptomyces peucetius Ca-alginate, Takashima et al., 1987photo-sensitive resin

β-Lactam antibiotics Cephalosporium Ca-alginate Khang et al., 1988aacremoniumC. acremonium + Ca-alginate Khang et al., 1988bChlorella pyrenoidosa

Nikkomycin Streptomyces sp. Ca-alginate, agar, Veelken and Pape,carrageenan 1982

Oxytetracycline Streptomyces rimosus Ca-alginate Farid et al., 1994Polyurethane Tanaka et al., 1984a;prepolymer Ogaki et al., 1986

Patulin Penicillium urticae κ-Carrageenan Berk et al., 1984a, b;Deo and Gaucher, 1983; 1985

Penicillin G Penicillium chrysogenum Alginate Kurzatkowski et al., 1982; 1984; El-Sayed and Rehm, 1987

κ-Carrageenan Deo and Gaucher, 1984; Mussenden et al., 1993

Polyacrylamide Morikawa et al., 1979;Deo and Gaucher, 1984

Tylosin Streptomyces sp. Ca-alginate, agar, Veelken and Pape,carrageenan 1982

TABLE 18TRANSFORMATION OF STEROIDS BY GEL ENTRAPPED LIVING MICROBIAL CELLS

Substrate Reaction Micro-organism Entrapment ReferenceMethod

4-Androstene- 9α-Hydroxylation Coryne- Photo-cross- Sonomoto et al., 3,17-dione bacterium sp. linked resin 1983bCortexolone 11β-Hydroxylation Curvularia lunata Ca-alginate, Ohlson et al.,

polyacrylamide 1980Ca-alginate Sukhodolskaya

et al., 1990


61


Substrate Reaction Micro-organism Entrapment ReferenceMethod

Photo-cross- Sonomoto et al.,linked resin 1981; 1983a

Tieghemella Polyacrylamide Skryabin and orchidis Koshchenko,

1987Cortisol ∆1-Dehydrogenation Arthrobacter Polyacrylamide, Vlahov et al.,

simplex Ca-alginate, 1990carrageenanCopoly(NIPA- Park andAm/AAm) Hoffman, 1990

Cortisol ∆1-Dehydrogenation Arthrobacter Ca-alginate Hocknull and simplex Lilly, 1990

Cortisol, 4AD, ∆4-Reduction Clostridium Polyacryl- Abramov et al., ADD, proges- paraputricum amide- 1990 terone, 16-dehydroprogesterone hydrazideDehydroepi- 16α-Hydroxylation Streptomyces Photo-cross- Chun et al., androsterone roseochromogenes linked resin 1981Esteron 16α-Hydroxylation Sepedonium Photo-cross- Kim et al.,

ampullosporum linked resin 1983b; Tanaka et al., 1984a

Hydrocortisone ∆1-Dehydrogenation Arthrobacter Ca-alginate Larsson et al.,simplex 1976; Ohlson et

al., 1978; 1979Arthrobacter Polyacrylamide Skryabin andglobiformis Koshchenko, 1987

IAA Hydroxylation Aspergillus niger Polyacrylamide Baklashova et al., 1984

6-α-Methyl- ∆1-Dehydrogenation Arthrobacter κ-Carrageenan, Pinheiro and hydrocortiso- simplex agar, polyure- Cabral., 1992 ne-21-acetate thaneProgesterone 11α-Hydroxylation Aspergillus Ca-alginate + Houng et al.,

ochraceus polyurea coat 1994Fe-alginate Chen et al.,hardened with 1994polyacrylamide

Rhizopus nigricans Agar Maddox et al., 1981Rhizopus stolonifer Photo-cross- Sonomoto et al.,

linked resin 1982


62

2.3.1.4 Transformation of SteroidsConsiderable research has been devoted to the biotransformation of various steroids for the

production of pharmaceutical steroid hormones. Because steroid transformation involvescomplex reactions including activation of molecular oxygen and the continuous supply ofreductive power, immobilisation of living cells can be advantageous. Moreover,immobilisation can stabilise the steroid transforming enzymes such as hydroxylase anddehydrogenase which normally have poor stability. The first example of steroid conversionby immobilised living cells was reported on ∆1-dehydrogenation of hydrocortisone to yieldprednisolone (Larsson et al., 1976). Since then, immobilised micro-organisms have been usedfor hydroxylation, dehydrogenation and reduction reactions such as 9α-hydroxilation, 11α-hydroxylation, 16α-hydroxylation, 11β-hydroxylation, ∆1-dehydrogenation and ∆4-reduction(cf. Table 18).

2.3.1.5 Production of EnzymesGel immobilised cell technology is well suited to produce industrially important

extracellular microbial enzymes. Research has been mainly focused on production ofcarbohydrate hydrolysing and proteolytic enzymes (cf. Table 19). Microbial extracellularglucoamylase, α-amylase, β -amylase and pullulanase are able to convert starchpolysaccharides to glucose and maltose. They are used in the brewing industry and for theproduction of glucose, maltose and fructose syrups, and dextrins (Norton and Vuillemard,1994). Cellulose as a potential source of glucose has a higher availability than starch.However, cellulose is not a significant source of glucose for technical reasons. Cellulases areused in the brewing industry. Proteases are used for cheese making, meat tenderizing,improving dough fermentation, improving the functional properties of food proteins,recovering proteins from parts of animals, decoloration of blood, also used in the brewingindustry, in detergents and in the leather and wool industries. Lipases are useful foraccelerating cheese ripening and enzyme modification of cheese. Enzyme-modified cheeseshave a strong flavour and are used in various soups, dips, dressings and snacks. β -Galactosidase is used in the production of ice cream and sweetened flavored condensedmilks, lactose-reduced products, flour and bread. Catalase is used to degrade hydrogenperoxide to water and dioxygen. Recently, enzymes have also been produced usinggenetically engineered micro-organisms (cf. Table 29).

TABLE 19PRODUCTION OF ENZYMES BY GEL ENTRAPPED LIVING MICROBIAL CELLS

Enzyme Micro-organism Entrapment ReferenceMethod

α-Amylase Bacillus sp. Ca-alginate J a m u n a a n dRamakrishna, 1992

Bacillus cereus Ca-alginate Ramakrishna et al., 1993Bacillus subtilis Polyacrylamide Kokubu et al., 1978; 1982


63



κ-Carrageenan Chevalier and de la Noüe,1987

α-Amylase Bacillus amylo- Ca-alginate Argirakos et al., 1992liquefaciens κ-Carrageenan Shinmyo et al., 1982Bacillus subtilis+ κ-Carrageenan Chevalier and de la Noüe,Scenedesmus obliquus 1988Bacillus cereus Ca-alginate Ramakrishna, 1995Bacillus licheniformis Agar Tonkova et al., 1994Clostridium sp., Thermo- Ca-alginate Klingeberg et al., 1990anaerobacter finnii

Catalase Saccharomyces cerevisiae Polyacrylamide Seip and Di Cosimo, 1992Cellulase Clostridium thermocellum Poly(vinyl alcohol) Varfolomeyev et al.,1990

Streptomyces sp. Ca-alginate Chatel et al., 1990Trichoderma reesei κ-Carrageenan Frein et al., 1982

Poly(2-hydroxy- Kumakura et al., 1984a, bethyl methacrylate/acrylate)Polymerised 2,6- Jirku et al., 1984dimethylphenol

Talaromyces emersonii Ca-alginate McHale, 1988Dextransucrase Leuconostoc Ca-alginate El-Sayed et al., 1990a, b

mesenteroidesΕndoglucanase Bacillus thermo- Polyacrylamide Krishna and Varma, 1990

alkalophilusSaccharomyces cerevisiae Ca-alginate Cahill et al., 1990

β-Galactosidase Escherichia coli Alginate Zhang et al., 1989Glucoamylase Aureobasidium pullulans Ca-alginate Gallo Federici et al.,

1990; Federici et al., 1990Aspergillus phoenicus Ca-alginate Kuek, 1991

Glucoamylase, Aspergillus niger Ca-alginate Li et al., 1984; Linko et α-amylase al., 1988β-D-Glucosidase Penicillium funiculosum Polyurethane gel Linko et al., 1988 + β-D-glucanasesHydrolases Myxococcus xanthus κ-Carrageenan Younes et al., 1984Leulysin Saccharomyces cerevisiae Photo-crosslinked Okada et al., 1987

resinLigninase Phanerochaete Agarose Linko, 1986a

chrysosporium


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Lipase Sporotrichum Ca-alginate Johri et al., 1990thermophile

Lisosomal Tetrahymena thermophila Ca-alginate Kily and Tiedtke, 1991 enzymesPeptidase Saccharomyces cerevisiae ENT-2000 Sonomoto et al., 1990bProtease Flavobacterium sp. Agar Zakran and Zayed, 1992

Humicola lutea Polyacrylamide Aleksieva et al., 1988Polyhydroxyethyl- Aleksieva et al., 1991methacrylate

Penicillium chrysogenum Ca-alginate El-Aassar et al., 1990Streptomyces fradiae Polyacrylamide Kokubu et al., 1981Serratia marcescens Ca-alginate Vuillemard et al., 1988;

Vuillemard and Amiot, 1988

Myxococcus xanthus Ca-alginate Fortin and Vuillemard, 1990

Penicillin acylase Kluyvera citrophila Polyacrylamide Morikawa et al., 1980a, bProteolytic Myxococcus xanthus κ-Carrageenan Younes et al., 1984 enzymePullulanase Clostridium sp., Thermo- Ca-alginate Klingeberg et al., 1990

anaerobacter finniiβ-Xylanase Streptomyces sp. Ca-alginate Chatel et al., 1990

TABLE 20ETHANOL PRODUCTION BY GEL ENTRAPPED LIVING CELLS

Substrate Micro-organism Entrapment ReferenceMethod

Cane + Saccharomyces cerevisiae Al-alginate Fukushima and fruit juices Hatakeyama, 1983Carob pod extract S. cerevisiae Ca-alginate Roukas, 1994Cassava starch S. cerevisiae Ca-alginate Ramakrishna et al., 1988 hydrolysateCellulose + Kluyveromyces Ca-alginate Barron et al., 1995 cellulase marxianus


65



Corn starch S. cerevisiae, Ca-alginate Converti, 1994 hydrolysate S. oviformisInulin Kluyveromyces marxianus Ca-alginate Margaritis and Bajpai,1983Glucose Clostridium beijerinckii Ca-alginate Krouwel et al., 1983a, b

Kluyveromyces marxianus Alginate Nolan et al., 1994Saccharomyces sp. Photo-crosslinked Oda et al., 1983

resinSaccharomyces bayanus κ-Carrageenan Amin and Verachtert,

1982;Barros et al., 1987;

S. carlsbergensis κ-Carrageenan Wada et al., 1980aS. cerevisiae Ca-alginate, agar, Holcberg and Margalith,

κ-carrageenan 1981κ-Carrageenan Wada et al., 1981; Gòdia et

al., 1987Ca-alginate, Shiotani and Yamané, 1981polyacrylamideCa-alginate Cho et al., 1981; Siess and

Divies, 1981; Williams andMunnecke, 1981; Hahn-Hägerdal and Mattiasson, 1982; McGhee et al., 1982a; Lee et al., 1983; Gamarra et al., 1986; Qureshi et al., 1987; Wöhrer, 1989a, b, c; 1989c;Ramakrishna et al., 1991; Vives et al., 1993; Arasaratnam, 1994; Ohta etal., 1994; Gilson and Thomas, 1995

Ca-alginate + Chotani and silica Constantinides, 1984Ca-alginate/κ-car- Constantinides andrageenan + silica Chotani, 1984Chitosan Freeman and Dror, 1994PAAH-glyoxal Pines and Freeman, 1982

S. formosensis Polyacrylamide Furusaki et al., 1983


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Glucose Zygosaccharomyces Ca-alginate Hamada et al., 1990arouxiiZymomonas mobilis κ-Carrageenan Amin and Verachtert,

1982;Luong, 1985; Keay et al., 1990b

κ-Carrageenan + McGhee et al., 1982blocust bean gumCa-alginate Grote et al., 1980;

Margaritis et al., 1981; Klein and Kressdorf, 1986; Seki at al., 1990

Poly(vinyl Varfolomeyer et al., 1990alcohol)

Glucose + xylose S. cerevisiae Alginate Grootjen et al., 1990+ Pichia stipitis

Jerusalem Kluyveromyces marxianus Ca-alginate Margaritis and Bajpai,1983 artichokeLactose Kluyveromyces fragilis Ca-alginate Droulez et al. 1990Molasses S. cerevisiae Ca-alginate Nagashima et al., 1984;

Tyagi et al., 1992Pectin Navarro et al., 1984

S. uvarum Agarose Shankar et al., 1985Gelatine Raman et al., 1982

Starch Aspergillus awamori + Ca-alginate Kurosawa et al., 1989bS. cerevisiaeS. cerevisiae Polethyleneimeine- Joung and Royer, 1990+ amylase alginateZymomonas mobilis + Ca-alginate Tanaka et al., 1986;Aspergillus awamori John et al., 1995

Agar Hellendoorn et al., 1995Sucrose S. cerevisiae Ca-alginate Agrawal and Jain, 1986;

Mandenius et al., 1987; Bravo and Gonzalez, 1991; Melzoch et al., 1994

κ-Carrageenan Agrawal and Jain, 1986Whey Kluyveromyces fragilis κ-Carrageenan, King and Zall, 1983

polyacrylamide


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Xylose Fusarium lini Alginate Chiang et al., 1982Mucor sp. Alginate Chiang et al., 1982Pachysolen tannophilus Alginate Maleszka et al., 1981;

Schneider et al., 1981; Slininger et al., 1982

Pichia stipitis Ca-alginate Kuzmanova et al., 1994κ-Carrageenan Chamy et al., 1994;

Sanromán et al., 1994S. cerevisiae Alginate Chiang et al., 1982

TABLE 21PRODUCTION OF ALCOHOLS BY GEL ENTRAPPED LIVING CELLS

Product Micro-organism Entrapment ReferenceMethod

Arabitol Aspergillus niger Ca-alginate Bisping et al., 19902,3-Butanediol Enterobacter sp. Ca-alginate, gelatineKautola et al., 1984

κ-Carrageenan Chua et al., 1980Butanol Clostridium Alginate Häggström and Molin, 1980;

acetobutylicum Reardon and Bailey, 1989; Keayet al., 1990b

Clostridium Alginate Krouwel et al., 1983a, b; Groot et beyerinckii al., 1991

Chiral alcohols Chlorella fusca Ca-alginate, algi- Fooladi et al., 1990nate+polyanhydridagar, chitosan,chitosan+Eudragit,carrageenan

Synechococcus Ca-alginate, algi- Fooladi et al., 1990leoppoliensis nate+polyanhydrid

agar, chitosan,chitosan+Eudragit,carrageenan

Erythritol Aspergillus niger Ca-alginate Bisping et al., 1990Glycerol Aspergillus niger Ca-alginate Bisping et al., 1990


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Glycerol Chlamydomonas Ca-, Ba-alginate, León et al., 1995reinhardtii agar, carrageenanSaccharomyces κ-Carrageenan Bisping and Rehm, 1982cerevisiae

Isopropyl alcohol Clostridium Alginate Krouwel et al., 1983a, bbeijerinckii

L-Phenylacetyl- Saccharomyces Ca-alginate Mahmoud et al., 1990a, b carbinol cerevisiae

2.3.1.6 Production of AlcoholsSince the world energy crisis, special attention has been focused on the production of

ethanol from renewable energy by immobilised cell technology. Large-scale industrialethanol production is already beyond the stage of pilot plant operation, but its economicimportance still depends on the oil market. A considerable amount of research has also beencarried out on ethanol production using gel immobilised micro-organisms as a model systemfor entrapped living cells. Ethanol production and production of other alcohols is summarisedin Table 20 and 21 respectively. Glycerol is used as humectant and plasticizer in food,whereas mannitol and sorbitol are used as food sweeteners.

2.3.1.7 Production of SugarsImmobilised cell technology has been applied for the production of sugars as summarised

in Table 22.

TABLE 22PRODUCTION OF SUGARS USING GEL IMMOBILISED GROWING MICROBIAL CELLS


Dextran Leuconostoc mesenteroides Alginate El-Sayed et al., 1990cFructo-oligo- Aureobasidium pullulans Ca-alginate Yun et al., 1990 saccharidesIsomaltuose Erwinia rhapontici Ca-alginate Cheetham et al., 1982Invert sugar Saccharomyces cerevisiae Polyacrylamide Ghose, 1990L-Sorbose Acetobacter suboxydans κ-Carrageenan Wada et al., 1979

Gluconobacter melanogenes Polyacrylamide Martin and Perlman, 1976b


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TABLE 23APPLICATIONS FOR FOOD AND BEVERAGES USING GEL ENTRAPPED CELLS

Application Micro-organism Entrapment ReferenceMethod

Alginate production Pseudomonas aeruginosa κ-Carrageenan Brito et al., 1990bApple juice fermen- Saccharomyces cerevisiae Alginate Dallmann et al., 1987 tation (ciders) Leuconostoc oenos, Alginate Simon et al., 1995

Saccharomyces bayanusBacteriocin production Lactococcus lactis Ca-alginate Zezza et al., 1993Beer brewing Brewer's yeast Ca-alginate Hsu and Bernstein, 1985;

Nakanishi et al., 1985; Curin et al., 1987

κ-Carrageenan Mensour et al., 1995Saccharomyces uvarum Ca-alginate Onaka et al., 1985

Bioflavour production Saccharomyces cerevisiae Bilayered algi- Kogan and Freeman,1994

Saccharomyces ginatecarlsbergensisTyromyces sambuceus

Champagne Saccharomyces bayanus Ca-alginate Durand et al., 1987Saccharomyces cerevisiae Ca-alginate Godia et al., 1991

(+ Ca-alginatecoat)

Cheese manufacture Lactobacillus helveticus Ca-alginate Gobetti and Rossi, 1990; Rossi et al., 1990

Lactococcus diacetylactis, Ca-alginate Prevost and Divies, 1987Lactococcus lactis andLactococcus cremorisLactococcus lactis and Ca-alginate Champagne et al., 1992mixed mesophilic culture

Cream fermentations Lactococcus sp. Ca-alginate Prevost and Divies, 1992Dairy fermentations Lactococcus lactis Ca-alginate Schmitt et al., 1988;

Morin et al., 1992a, b; Cachon and Divies, 1993

Lactococcus cremoris, κ-Carragee- Lacroix, 1995Lactococcus diacetylactis nan/locustLeuconostoc bean gummesenteroidesStreptococcus lactis, Ca-alginate Steenson et al., 1987Streptococcus cremoris


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Application Micro-organism Entrapment ReferenceMethod

Decaffeination Pseudomonas putida Agar/Agarose Middelhoven and Bakker,1982

Diacetyl Lactococcus lactis Ca-alginate Takahashi et al., 19904-Ethylguaiacol Candida versatilis Alginate Hamada et al., 1990a, bFrozen desserts Lactobacillus bulgaricus Ca-alginate Sheu et al., 1993Gellan gum production Pseudomonas elodea κ-Carrageenan Brito et al., 1990bLimonin degradation Rhodococcus fascians κ-Carrageenan Martinez-Madrid et al.,

1989; Manjon et al.,1991; Iborra et al., 1994Mead production Saccharomyces cerevisiea Alginate Qureshi and Tamhane,

1985Meat fermentations Lactobacillus plantarum, Ca-alginate Kearney et al., 1990

Pediococcus pentosaceusNisin Streptococcus lactis Polyacryl- Kozlova et al.,

amide 1980Single cell protein Kluyveromyces fragilis Ca-alginate Kierstan et al., 1984Soy sauce fermen- Pediococcus halophilus, Alginate Osaki et al., 1985 tation Saccharomyces rouxii,

Torulopsis versalitisZygosaccharomyces Ca-alginate Hamada et al., 1989rouxii

Vitamin B12 Propionibacterium sp. Photo-cross- Yongsmith et al., 1982linked resin,κ-carrageenan,alginate, agar/agarose

Whey fermentations Bifidobacterium longum Gellan gum Camelin et al., 1993Wine fermentation Leuconostoc oenos Ca-alginate Spettoli et al., 1984

Schizosaccharomyces sp. Ca-alginate Taillandier and Strehaiano, 1994

Schizosaccharomyces Ca-alginate Yokotsuka et al., 1993pombe, Saccharomycescerevisiae

Yoghurt Streptococcus thermo- Ca-alginate Champagne et al., 1993;philus and Lactobacillus Prevost and Divies, bulgaricus 1988a, b; Prevost et al.,

1985


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2.3.1.8 Applications for Food and BeveragesThe use of entrapped microbial cells for food and beverage applications is listed in Table

23. Recently, immobilised cell technology for the dairy industry (Champagne et al., 1994),for food bioconversions and metabolite production (Norton and Vuillemard, 1994), andphysiological effects of yeast cell immobilisation for brewing (Norton and D'Amore, 1994)have been reviewed.

2.3.1.9 Gel Entrapment of Microbial Cells for Use in AgricultureEncapsulation of microbial cells may enhance survival of both genetically engineered and

non engineered strains useful in agriculture, forestry and biodegradation of polluants. Theslow release of microbial cells from gel beads allows for efficient root colonisation.Encapsulation reduces water-induced movement of cells in soil. Moreover, inoculantconsistency and effectiveness may be enhanced due to the defined nature of some carriers.Bead inoculants can be used with conventional seed-sowing machinery and this may reducedispersal to undesirable locations (Trevors et al., 1992).

TABLE 24GEL ENCAPSULATION OF MICROBIAL CELLS FOR USE IN SOIL

Micro-organism Encapsulation Method Reference

Azospirillum brasilense Alginate(+skim milk) Bashan, 1986; Bashan et al., 1987; Bashan and Levanony, 1990

Azospirillum lipoferum Alginate+skim milk Fages, 1990Bradyrhizobium sp. Alginate Galiana et al., 1994Pseudomonas sp. Alginate(+skim milk) Bashan, 1986Pseudomonas cepacia Alginate+clay Fravel et al., 1985Pseudomonas fluorescens Ca-alginate Trevors, 1991; van Elsas et al., 1992(genetically engineered) Ca-alginate+skim milk Trevors et al., 1992

Ca-alginate+skim milk+bentonite

Rhizobium sp. Polyacrylamide Dommergues et al., 1979; Jung et al., 1982

Rhizobium phaseoli Polyacrylamide Sparrow and Ham, 1983a, b

Alginate is the most promising carrier for fungi and bacteria due to its non toxic nature andbiodegradability (Kitamikado et al., 1990), and long shelf life and appropriate survival in soilas well as sufficient cell density and activity are attained (Trevors, 1992; van Elsas et al.,1992). Immobilisation methods for microbial cells to be used in soil are reviewed in Table 24.

Alginate entrapment has been used to produce inocula of fungi which were employed asmycoherbicides, mycorrhizae and biocontrol agents (cf. Table 25) (McLoughlin, 1994).Encapsulation of fungi is also useful for the mass production of pycnidium-forming fungi,and for the production for host-plant resistance studies (Walker and Connick, 1983). Artificial


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inoculation with efficient and resistant strains of ectomycorrhizal fungi increases the vitalityand resistance of forest tree seedlings. Fungal mycelium, which was produced in fermentors,was immobilised in gel beads and provided inoculum with high viability due to the protectionby the gel after incorporation into the soil (Le Tacon et al., 1985; Mauperin et al., 1987;Kropácek et al., 1989). Biological control strategies are dependent on the establishment andmaintenance of a threshold level of microbial cells in the environment. Introduced inoculawill require an efficient formulation and delivery system which can offer protection to themicrobial culture both during storage and when exposed to field conditions (McLoughlin,1994). The application of alginate immobilisation techniques to biocontrol agents has beeninvestigated by a number of workers and effective disease control could be attained.

TABLE 25THE USE OF IMMOBILISED FUNGI IN AGRICULTURAL APPLICATIONS

Fungi Encapsulation Method Reference

MycoherbicidesAlternaria cassiae, Ca-alginate Walker and Connick, 1983Alternaria macrospora,Fusarium malvarum,Phyllosticta sp.

MycorrhizaeHebeloma crustuliniforme Ca-alginate+peat/ Mauperin et al., 1987

bentoniteHebelona cylindrosporum Ca-alginate+silica/ Le Tacon et al., 1985

epioliteLaccaria laccata, Ca-alginate+perlite Kropácek et al., 1989Suillus luteus

Biocontrol AgentsGliocladium virens Ca-alginate+Pyrax Fravel et al., 1985

Ca-alginate+vermiculite+ Lumsden and Locke, 1989wheat bran

Laetisaria arvalis Ca-alginate Lewis and Papavizas, 1988Penicillium oxalicum Ca-alginate+Pyrax Fravel et al., 1985Talaromyces flavus Ca-alginate+Pyrax Fravel et al., 1985

Ca-alginate+Pyrax/ Papavizas et al., 1987wheat bran

Trichoderma harzianum Ca-alginate(+wheat bran) Knudsen and Bin, 1990;Knudsen and Eschen, 1991

Trichoderma viride Ca-alginate+Pyrax Fravel et al., 1985


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2.3.1.10 Environmental ApplicationsDegradation of specific compounds can be accomplished by micro-organisms.

Consequently, a lot of research has been focused on the use of gel immobilised celltechnology for waste treatment as summarised in Table 26.

TABLE 26ENVIRONMENTAL APPLICATIONS OF GEL ENTRAPPED MICROORGANISMS


Ammonia Nitrosomonas europaea Ca-alginate van Ginkel et al., 1983κ-Carrageenan Wijffels and Tramper,

1989Nitrosomonas europaea + Polyelectrolyte Kokufuta et al., 1987Paracoccus denitrificans complex-stabili-

sed Ca-alginateThiosphaera pantotropha Agarose Hooijmans et al., 1990bScenedesmus obliquus κ-Carrageenan Chevalier and de la

Noüe, 1985aScenedesmus quadricauda κ-Carrageenan Chevalier and de la

Noüe, 1985bBenzene Pseudomonas putida Polyacrylamide Somerville et al., 19773-Chloroaniline Pseudomonas acidovorans Ca-alginate Ferschl et al., 1991 4-Chloro-2-phenol Enterobacter cloacae, Ca-alginate Beunink and Rehm,

Alcaligenes sp. 1990Cholesterol Pseudomonas pictorum Agar Garofalo and Chang,

1991Dairy effluents Candida pseudotropicalis Alginate Marwaha et al., 1990Decolorised water Coriolus decolorize Alginate Livernoche et al., 1983Dehydroabietic acid Mortierella isabellina Alginate Kutney et al., 1985Dihexylsulfosuccinates Comamonas terrigena Ca-alginate, Húska et al., 1995 surfactants Ca-pectate

(+PEI+ gluta-raldehyde)

Mercury Chlorella emersonii Agarose, Robinson and Ca-alginate Wilkinson, 1994

Pseudomonas putida PVA Becker et al., 1995(genetically engineered)

Methanol Methanosarcina barkeri Ca-alginate Scherer et al., 1981


74



Morpholine Mycobacterium aurum κ-Carrageenan Poupin et al., 1995Nitrate Anacystis nidulans Collagen Vieth and Venkatasu-

bramanian, 1979Denitrifying sludge PVA Chen and Lin, 1994Pseudomonas Ca-alginate Nilsson et al., 1980;denitrificans Nilsson and Ohlson,

1982Thiobacillus denitrificans Agar/Agarose Salleh and Aminuddin,

1981Nitrite Chlamydomonas Ca-alginate Vilchez and Vega, 1994;

reinhardtii 1995Nitrobacter agilis Ca-alginate Tramper and de Man,

1986Carrageenan Tramper and Grootjen,

1986Nitrobacter sp. PVA Willke and Vorlop,

1995Nitrogen Anabaena CH3 Ca-alginate Lee et al., 1995

Nitrifiers Polyethylene Tanaka et al., 1995glycol resin

Pentachlorophenol Arthrobacter sp. Alginate Lin and Wang, 1991Phenol Candida tropicalis Alginate Hackel et al., 1975;

Klein et al., 1979Pseudomonas sp. Ca-alginate, Bettmann and Rehm,

polyacryl- 1984amide hydrazidePVA-boric Wu and Wisecarver,acid-Ca-alginate 1992

Phosphate Scenedesmus obliquus κ-Carrageenan Chevalier and de la Noüe, 1985a

Scenedesmus κ-Carrageenan Chevalier and de la quadricauda Noüe, 1985b

Plutonium Pseudomonas aeruginosa Collagen Vieth and Venkatasu-bramanian, 1979

Pyridine Pimelobacter sp. Ca-alginate Lee et al., 1994Sodium dodecyl Pseudomonas fluorescens Polyacrylamide White and Thomas, sulphate 1990


75



1,1,1-Trichloro-2,2- Alcaligenes sp. and Ca-alginate Beunink and Rehm, bis (4-chlorophenyl) Enterobacter cloaccae 1988 ethane (DDT), 4,4'-dichloro-dophe- nylmethane (DDM)Trimethyl lead Arthrobacter sp., Polyacrylamide Macaskie and Dean,

Phaeolus schweinitzii 1990Uranium Streptomyces uiridochro- Polyacrylamide Nakajima et al., 1982

mogenes, Chlorella regularisStreptomyces Alginate, Nakajima et al., 1982viridochromogenes polyacrylamide

Waste water Activated sludge Poly(vinyl Ariga et al., 1987;alcohol) Hashimoto and

Furukawa, 1987

2.3.1.11 Energy Production by Gel Immobilised CellsThe biochemical production of energy is attractive as a new energy source. Besides

ethanol production (cf. Table 20), studies have been carried out on the production ofhydrogen and methane as summarised in Table 27. Hydrogen is evolved photosyntheticallyby various algae and photosynthetic bacteria. The purple non-sulphur bacteria Rhodospirillumrubrum and R. capsulata produce hydrogen via the nitrogenase enzyme which acts as ahydrogenase on substrates such as lactate, glutamate and acetate, as well as other organicacids. The bacteria utilize only a single photosystem and thus do not evolve oxygen whichwould otherwise inactivate the nitrogenase (Birnbaum, 1994). Hydrogen is known as one ofthe cleanest fuel sources and exhibits excellent reactivity in electroactive materials (Delducaet al., 1963). The generation of methane from organic wastes by micro-organisms consists oftwo phases. The organic substrate is initially converted anaerobically (by free cells) toorganic acids such as formic, acetic, propionic and butyric acid. In the second stage, theorganic acids are further converted anaerobically by immobilised methanogens to methaneand carbon dioxide. Methane can also be produced from synthesis gas (syngas) withimmobilised micro-organisms.


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TABLE 27PRODUCTION OF ENERGY BY GEL ENTRAPPED MICRO-ORGANISMS


Hydrogen Clostridium butyricum Polyacrylamide Karube et al., 1976Collagen Suzuki et al., 1980Agar/agarose Matsunaga et al., 1980a, b;

Karube et al., 1982; Suzuki et al., 1983

Clostridium thermo- Poly(vinyl alcohol) Nikitina et al., 1993saccharolyticumRodopseudomonas Alginate Paul and Vignais, 1980;capsulata Francou and Vignais, 1984Rhodospirillium Agar/agarose Vincenzini et al., 1981;rubrum Bennett and Weetall, 1976

Ca-alginate Karube et al., 1984;Karube and Suzuki, 1988

Agar, agarose, κ-car- von Felten et al., 1985rageenan, pectine,Ca- and Ba-alginate

Methane Methanogenic bacteria Agar, collagen, Karube et al., 1980polyacrylamideUrethane pre- Anasawa and Beppu, 1982polymer

Methanosarcina Ca-alginate Scherer et al., 1981barkeri

TABLE 28PRODUCTION OF VARIOUS BIOCHEMICALS BY GEL ENTRAPPED LIVING MICROBIAL CELLS


Aceton Clostridium Alginate Häggström and Molin, 1980;acetobutylicum Reardon and Bailey, 1989

Alkaloids Claviceps sp. Ca-alginate, Kren, 1990Ca-pectate,κ-carrageenan

Claviceps purpurea Alginate Kopp and Rehm, 1983; 1984;Lohmeyer et al., 1990b


77



ATP Saccharomyces cerevisiae Photo-crosslinked Asada et al., 1979resin

CDP-choline Hansenula jadinii Photo-crosslinked Kimura et al., 1978; 1981resin

Coenzyme A Brevibacterium Polyacrylamide Samejima et al., 1978;ammoniagenes Shimizu et al., 1979; Fukui

and Tanaka, 1982Photo-crosslinked Asada et al., 1982resin

Dihydroxy- Acetobacter suboxydans Polyacrylamide Schnarr et al., 1977 acetone Acetobeacter xylinum Carrageenan Nabe et al., 1979

Gluconobacter oxydans Polyacrylamide Makhotkina et al., 1981Ca-alginate Holst et al., 1982; Adlercreutz

et al., 198513-Dyhydro- Streptomyces Gelatine Marek et al., 1981 xydauno- aureofaciens mycinone1,2-Epoxides Mycobacterium sp. Ca-alginate, Habets-Crützen et al., 1984;

κ-carrageenan Brink and Tramper, 1986a, b;Smith et al., 1990

Nocardia corallina Ca-alginate, Habets-Crützen et al., 1984;κ-Carrageenan Kawakami et al., 1990

Glutathione Escherichia coli Polyacrylamide Murata et al., 1980Escherichia coli κ-Carrageenan Kimura, 1984(genetically engineered)

Hydrocarbons Botryococcus braunii Ca-alginate Bailliez et al., 1985Nucleoside and Propionibacterium Polyacrylamide Ikonnikov et al., 1982 nucleotides shermaniiPorphyrin Rhodobacter sphaeroides Agar, carrageenan Ishii et al., 1990Propylene Nocardia corallina Polyacrylamide Furuhashi et al., 1982 oxide

2.3.1.12 Production of Other Useful BiochemicalsA list of various other useful biochemicals produced by gel immobilised cells is presented

in Table 28.


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TABLE 29PRODUCTION OF PROTEINS BY GEL IMMOBILISED GENETICALLY ENGINEERED

MICRO-ORGANISMS

Cloned Gene Micro-organism Plasmid Entrapment ReferenceProduct Method

Acid phospha- Myxococcus κ-Carrageenan Younes et al., 1987 tase xanthusα-Amylase Escherichia coli pHI301 κ-Carrageenan Ariga et al., 1991

Saccharomyces p520 Gelatine + cross- Walls and Gainer, 1991cerevisiae linking with

glutaraldehydeCatechol-2,3- Escherichia coli pTG201 κ-Carrageenan Dhulster et al., 1984; De dioxygenase Taxis du Poët et al.,

1986; Sayadi et al., 1989; Huang et al.,

1989; Briasco et al., 1990pTG205 κ-Carrageenan Berry et al., 1990

Chrorionic S. cerevisiae κ-Carrageenan Karkare et al., 1986 gonadotropinβ-Galactosidase Escherichia coli pMLB1108 Ca-alginate Vieth, 1989β-Glucanase S. cerevisiae pJG317 Ca-alginate Cahill et al., 1990Hydrogenase E. coli, Citro- pCBH4 Polyacrylamide- Kanayama et al., 1988

bacter freundii hydrazideα-2-Interferon S. cerevisiae pYCB115 Ca-alginate Perez et al., 1990β-Lactamase Escherichia coli pKK223-200 Ca-alginate Georgiou et al., 1985

pBR322, κ-Carrageenan Sayadi et al., 1988pBR325,pBR328pKBF367-11 κ-Carrageenan Ollagnon et al., 1993

Luciferase Escherichia coli pCSS108 Ca-alginate Akerman et al., 1990Polygalacturo- Myxococcus κ-Carrageenan Younes et al., 1987 nate lyase xanthuα-Peptide S. cerevisiae pBR322 Ca-alginate Sode et al., 1988bProinsulin Bacillus subtillis pPCB6 Agarose Mosbach et al., 1983

Escherichia coli pBR322 Agarose Birnbaum et al., 1988Somatomedin C Yeast BJ 1991 p336/1 Polyacrylamide Sode et al., 1988a

hydrazide+glyo-xal+Ca-alginate

Xylanase Escherichia coli pKK223-200 κ-Carrageenan De Taxis du Poët et al., 1987


79

2.3.1.13 Production of Proteins by Gel Immobilised Genetically Engineered Micro-organismsRecombinant DNA technology has been used to "improve" micro-organisms for the large-

scale production of proteins. Gel immobilised cell technology can be used to increase theplasmid stability (cf. 2.2.2.1). Applications are listed in Table 29.

2.3.2 Gel Immobilised Plant Cells

Plant cells are important sources of industrially important compounds: pharmaceutical,fragrances, flavours and coloring principles. Examples of these substances are: alkaloids,glycosteroids, terpenoids, phenolics, enzymes, insecticides, pigments and vitamins. Theproduction of these compounds has been performed using cultured cells. Immobilisationtechnology can be beneficial for exploiting plant cells. The productivity of secondarymetabolites and product release from the cells sometimes increase by immobilisation, sincefavorable stress is provided to cultured plant cells by immobilisation. The materials for cellentrapment can serve as elicitors in some cases for inducing the production of secondarymetabolites (Wolters and Eilert, 1983; Brodelius, 1988; Tanaka and Nakajima, 1990).Moreover, the forced aggregation of plant cells by immobilisation will in some cases beeffective for stabilising cell viability and metabolism. The first report on the immobilisationof plant cells appeared in 1979 (Brodelius et al., 1979) and since then considerable researchhas been performed on this topic. The use of immobilised plant cells can be classified intothree categories: biotransformation, salvage synthesis and de novo synthesis.

2.3.2.1 BiotransformationVarious bioconversions, which include stereo- and regio-specific hydroxylations,

glycosylations, acetylations and methylations, have been accomplished by gel entrapped plantcells as sumarised in Table 30.

2.3.2.2 Salvage SynthesisThe yield of a particular compound in plant cell cultures may be increased considerably by

feeding appropiate precursors. This production formula is called salvage synthesis orsynthesis from added precursors. Although this method to synthesise complex organiccompounds appears to be attractive, application has been limited to the production of indolealkaloids (cf. Table 31), since the information on the biosynthetic pathways of usefulsecondary metabolites is insufficient to apply this method and proper precursors are notavailable in most cases.

2.3.2.3 De novo SynthesisA wide variety of compounds have been isolated from plant tissue cultures (Staba, 1980;

Neumann et al., 1985). Consequently, immobilised cell technology has been used for theproduction of such compounds (cf. Table 32).


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TABLE 30USE OF GEL ENTRAPPED PLANT CELLS FOR BIOCONVERSIONS

Product Substrate Plant Cell Entrapment ReferenceMethod

Ajmalicine Cathenamine Catharanthus Agarose Felix et al., 1981; Felix roseus and Mosbach, 1982

Codeine Codeinone Papaver Ca-alginate Furuya et al., 1984;somniferum Furusaki et al., 1988

Digoxin Digitoxin Digitalis lanata Alginate Brodelius et al., 1979; 1981

DOPA Tyrosine Mucuna Alginate Wichers, 1983;pruriens Wichers et al., 1983;

19855-Hydroxygi- Gitoxigenin Daucus carota Alginate Veliky and Jones, 1981 toxigeninMethyldigoxin Methyldigitoxin Digitalis lanata Alginate Alfermann et al., 1980;

1983(+)Neomenthol (-)Menthone Mentha sp. Polyacrylamide Galun et al., 1983Nerol Geraniol Vitis vinifera Ca-alginate Guardiola et al., 1995Periplogenin Digitoxigenin Daucus carota Alginate Jones and Velicky, 1981Purpureagly- Digitoxin Digitalis lanata Ca-alginate Alfermann et al., 1980 coside A

TABLE 31USE OF GEL ENTRAPPED PLANT CELLS FOR THE SYNTHESIS OF BIOCHEMICALS FROM

PRECURSORS

Product Substrate Plant Cell Entrapment ReferenceMethod

Ajmalicine Tryptamine+ Catharanthus Alginate Brodelius et al., 1979secologanin roseus Alginate, agar, Brodelius and Nilsson, 1980

agarose, carrageenanAlginate, agarose Brodelius and Nilsson, 1983


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TABLE 32USE OF GEL ENTRAPPED PLANT CELLS FOR THE NOVO SYNTHESIS

Product Plant Cell Entrapment ReferenceMethod

Ajmalicine Catharanthus roseus Ca-alginate Asada and Shuler, 1989Ajmalicine+ Catharanthus roseus Ca-alginate, Brodelius and Nilsson, 1980; 1983 serpentine agarose, agar

κ-carrageenanAlkaloid Coffea arabica Ca-alginate, Thomasset et al., 1990

Datura innoxia κ− and ι-carra-geenan

Anthraquinones Morinda citrifolia Ca-alginate Brodelius et al., 1979; 1980Berberine Thalictrum minus Ca-alginate Kobayashi et al., 1987; 1988Blue Pigments Lavandula vera Ca-alginate, Nakajima et al., 1985; 1986; 1989;

photosensitive 1990; Sonomoto et al., 1990aresinPolyurethane Tanaka et al., 1984a, b

Capsaicinoids Capsicum frutescens Ca-alginate Sudhakar Johnson et al., 1990; (+curdlan/ 1991a, b; Suvarnalatha et al., 1993xanthan)

Catechols Mucuna pruriens Alginate Pras et al., 1990Cryptotanshi- Salvia miltiorrhiza Ca-alginate Myasaka et al., 1986 one+ferruginolEchinatin Glycyrrhiza echinata Ca-alginate Ayabe et al., 1986Guggulipid Commiphora wightii Ca-alginate Jacob John et al., 1990Methylxan- Coffea arabica Ca-alginate Haldimann and Brodelius, 1987 thinesMilk Cyanara Alginate Esquivel et al., 1988 coagulation cardunculusPhenolics Nicotiana tabacum Ca-alginate Haigh and Linden, 1989Serpentine Catharanthus roseus Ca-alginate Brodelius et al., 1981

Ca-alginate+ Lambe and Rosevear, 1983polyacrylamide

Shikonin Lithospermum Ca-alginate Kim and Chang, 1990erythrorhizon

Solasodine Solanum surattense Ca-alginate Barnabas and David, 1988Solavetivone Hyoscyamus muticus Ca-alginate Ramakrishna et al., 1993Verbenone Solanum aviculare Pectate Vanek et al., 1989


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TABLE 33GEL IMMOBILISATION OF SOMATIC EMBRYOS (ARTIFICIAL SEEDS)

Crop Immobilisation ReferenceMaterial

Apium Graveolens (celery) Ca-alginate Redenbaugh et al., 1986Brassica napus (oilseed rape) Ca-alginate Uragami et al., 1993

Alginate-chitosan Tay et al., 1993Brassica oleracea (cauliflower) Ca-alginate Redenbaugh et al., 1986Coffea canephora (coffee) Ca-alginate Hatanaka et al., 1994Daucus carota (carrot) Ca-alginate; Ca-algi- Bazinet et al., 1993;

nate + gellan gum/ Barbotin et al., 1995silica/kaolinCa-alginate Dereuddre et al., 1991; Tsuji et al.,

1993Medicago sativa (alfalfa, Agar, alginate, algi- Redenbaugh et al., 1986; 1987 lucerne) nate/gelatine, carra-

geenan/locust beangum, Gelrite (KelcoCo.), pectateCa-alginate Redenbaugh, 1986; 1988; Fujii et al.,

1987Pelargonium x hortorum Ca-alginate Gill et al., 1994 Bailey cv. Scarlet (geranium)

2.3.2.4 Artificial SeedsArtificial or synthetic seeds ("Synseeds") consist of tissue-culture produced somatic

embryos in a protective coating. Hydrated and desiccated artificial seeds have been developed(Fujii et al., 1987). Hydrated synthetic seeds are somatic embryos entrapped in a hydrated gel,usually Ca-alginate which has a low toxicity to the embryo. The gel can also potentially serveas a reservoir for nutrients ("artificial endosperm") that may aid the survival and speed up thegrowth of the embryo (Fujii et al., 1987). For albuminous seed crops, such as celery, anartificial endosperm may be necessary and it is therefore necessary that coating gels retaincompounds such as sucrose (Bornman, 1993). Desiccated artificial seeds are produced byeither coating somatic embryos with a water-soluble resin, polyoxyethylene glycol, andallowing them to dry, or desiccation without coating. Advantages of artificial seed technologyare: maintenance of genetic uniformity, direct delivery of propagules to field, rapidmultiplication rate, and high volume and medium-scale propagation method; and the highcost per plantlet compared to true seed is the main disadvantage (Fujii et al., 1987). Potentialapplication of synthetic seed will vary by crop, but will most probably be determined by the


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need for improvement in the production of the crop, as well as in the ability to producesomatic embryos of that crop. Crops with a potential application include conifer, cotton,grape, lucerne, maize, orchard grass, soybean, sugarbeet, sugarcane, tomato and watermelon(seedless) (Redenbaugh et al., 1987; Gray, 1990; Redenbaugh, 1990; Bornman, 1993). AfterCa-alginate encapsulation, embryos could be successfully cryoperserved (Dereuddre et al.,1991; Uragami et al., 1993; Hatanaka et al., 1994). Therefore, the embryos are encapsulated,precultured with a highly concentrated sucrose medium, dehydrated in air and immersed inliquid nitrogen. By alginate coating, the size and shape of the material can be made uniformand a uniform water loss can be achieved during the course of drying. Alginate coating canalso work as a buffering agent by preventing the direct effect of environmental changes onthe encapsulated material. The coating also prevents rapid dehydration and absorption ofwater. Table 33 summarises gel encapsulation of various somatic embryos.

2.3.3 Gel Immobilised Animal Cells

Animal cells are used to produce many natural and recombinant protein therapeutic drugs,vaccines and monoclonal antibody products. Some of these products can also be made inbacterial or yeast expression systems. Many of the more complex recombinant proteins,however, require an animal (insect or mammalian) cell expression system, since correctglycosylation and other structural modifications can not be carried out in microbial cells.Intensification of the production of high-value biologicals can be achieved by usingimmobilised cell technology. The use of gel entrapped mammalian cells for the production ofbiochemicals is listed in Table 34. Gel immobilisation has also been used to study the growthof certain cell types in vitro, since gel entrapment can mimic the three-dimensionalenvironment of tissues (Table 35). Collagen is the major protein of connective tissue andentrapment in this gel matrix has been frequently used to study cell behaviour in a tissuelikeenvironment. Immunoisolation of mammalian cells has also been achieved by gel entrapment,e.g. gel entrapped Islets of Langerhans as a bioartificial pancreas (Table 34), although mostresearch has been focused on micro-encapsulated cells (cf. Chapter 3).

TABLE 34PRODUCTION OF BIOCHEMICALS BY GEL ENTRAPPED MAMMALIAN CELLS

Product Cell Type Entrapment Method Reference

Albumine Chicken hepatocytes Collagen Saltzman et al., 1992Cytokine Cytokine-transfected Cells on Cytodex in Savelkoul et al., 1994

monkey fibroblasts CV1 Ca-alginate beadErythropoietin Baby hamster kidney Ca- or Sr-alginate Shirai et al., 1988

Bowes melanoma cells Ca-alginate Keay et al., 1990aInterleukin 2 Gibbon lymphoblastoid Agarose Nilsson et al., 1983Insulin Brockmann bodies of Ba-alginate Schrezenmeir et al.,

Osphronemus gorami 1994


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Product Cell Type Entrapment Method Reference

Insulin Islets of Langerhans Ba-alginate Zekorn et al., 1992a, b; 1993; Siebers et al.,

1993; 1994; Horcher et al., 1994; Ca-alginateWiegand et al., 1993

Agarose Dupuy et al., 1987; Aomatsu et al., 1994;

Iwata et al., 1992; 1994; Yang et al.,1994

Poly(ethylene glycol) Pathak et al., 1992multiacrylates

Monoclonal anti- Hybridoma 63D3 Ca-alginate Wohlpart et al., 1991 body Hybridoma HP-6 Chitosan stabilised Overgaard et al., 1991

Ca-alginateHybrydoma S3H5/g2bA2 Alginate Lee and Palsson, 1990Mouse hybridoma Collagen Ray et al., 1990a, b

Monoclonal IgA Hybridoma 4H11 Ca-alginate Shirai et al., 1987; Hashimoto and Shirai, 1990

Monoclonal Hybridoma 16-3F Ca-alginate + Iijima et al., 1988; anti-α-amylase polyurethane Kamihira et al., 1990Monoclonal IgG Mouse/mouse hybridoma Agarose Nilsson et al., 1983

Human/mouse hybridoma Agarose Nilsson et al., 1986bHybridoma 4C10B6 Ca-alginate Shirai et al., 1988Hybridoma AcV1 Ca-alginate Bugarski et al., 1989Mouse/mouse hybridoma Ca-alginate Al-Rubeai et al., 1990TB/C3Murein hybridoma Ca-alginate Hsu and Chu, 1992Rat hybridoma Ca-alginate Savelkoul et al., 1994

Monoclonal IgM Mouse/mouse hybridoma Agarose Scheirer et al., 1984Proinsulin Mouse fibroblast Ltk- Agarose Taniguchi et al., 1992 (human) (genetically engineered)


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TABLE 35GROWTH OF MAMMALIAN CELLS IN GELS

Cell Type Entrapment ReferenceMethod

Chicken hepatocytes Collagen Saltzman et al., 1992Chondrocyte Agarose Benya et al., 1978; Aydelotte et al., 1986;

Bruckner et al., 1989Ca-alginate Kupchik et al., 1983; Guo et al., 1989;

Bonaventure et al., 1994; Häuselmann et al., 1992, 1994

Hamster melanoma Collagen Schorr et al., 1981Human foreskin fibroblast Collagen Bell et al., 1979; Schorr et al., 1981Human neutrophil Collagen Brown, 1982; Grinnell, 1982; Parkhurst and

Saltzman, 1992; Saltzman et al., 1992Mammary tumor epithelial Collagen Yang et al., 1979; Richards et al., 1983 cellsMouse neuroblastoma Ca-alginate Simonneau et al., 1990; Tamponnet et al., 1990PC12 rat adrenal pheochro- Collagen Saltzman et al., 1992 mocytomaPig Islet of Langerhans cells Collagen Ohgawara et al., 1990Rat exocrine pancreatic cells Collagen Chen et al., 1985Rat hepatocytes Collagen Mooney et al., 1992; Yarmush et al., 1992Rat Islet of Langerhans cells Collagen Montesano et al., 1983


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3. MICRO-ENCAPSULATION OF LIVING CELLS

Recently, the immobilisation of living cells in micro-capsules has become a valuableimmobilisation technique. A typical micro-encapsulation process involves the formation of aspherical gel mould containing cells, on which is deposited a polymeric membrane. Theinternal gel matrix can also be liquefied and allowed to diffuse out of the capsule, leavingbehind the membrane and the contained cells. The type and porosity of the membrane, andsize of the micro-capsules can be varied to accommodate many reactant-product systems.Capsule diameters from 20 µm to 2 mm are possible. The porosity of the membrane can bevaried over a range of several orders of magnitude such that on the low end, molecules assmall as glucose (180 daltons) can be constrained to remain intracapsular while on the highend molecules as large as IgG (155,000 dal) can be made to be freely permeable. Thepolymer membrane should offer minimal resistance to the mass transfer of essentialmolecules as well as the toxic end products of cell metabolism in order for the encapsulatedmass to maintain normal physiological activity.

Using this technology, enzymes as well as living cells have been encapsulated. Ofparticular interest, is the immobilisation of animal cells. A dramatic process intensificationover conventional cell suspension culture with low density and low productivity, can beachieved. Cell encapsulation and long-term continuous culture lead to significantly highercell densities which results in higher productivities (Looby and Griffiths, 1990). The highculture densities provide a high degree of cell-cell contact and interaction, resulting inpossible more favourable micro-environmental conditions. In addition, this technique canprovide protection from shear for the sensitive animal cells and other sudden changes in theculture medium. It also permits direct aeration by air bubbles without risk of damaging thecells. The produced toxic metabolites, such as lactic acid and ammonium, will diffuse out ofthe capsule due to the concentration gradient, resulting in higher growth and productformation rates. Micro-encapsulation can provide simultaneous product separation and cellcultivation, resulting in concentration of high molecular weight metabolic products (e.g.monoclonal antibodies) within the capsule. The preconcentration of products within thecapsule facilitates further purification steps. The immobilised cells are totally separated fromthe culture medium which results in an easier and cost effective downstream processing. Thisis not true for conventional gel immobilisation growing cell systems where cell release fromthe matrix is observed.

Micro-encapsulation of human cells or tissues provides a new technology to overcomebiomedical problems, because the membrane may create an immunological barrier betweenthe host and the transplanted cells. The immunoisolation of the encapsulated cells or tissuefrom the elements of the immune system, prevents the rejection of the transplantedcells/tissue. Consequently, the necessity of immunosuppressive drugs in allo- andxenotransplantations can be avoided. Recently, intensive research has been focused on thedevelopment of polymer-encapsulated living cell transplants as therapies for human diseasessuch as diabetes (insulin release), Alzheimer's disease (nerve growth factor), haemophilia(Factor VIII), and Parkinson's disease (dopamine). The financial benefit of solving these


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problems is very promising: the potential US market in these areas alone is estimated at morethan $10 billion per year (Goosen, 1993).

3.1 Encapsulation Materials and Methods

The various mechanisms of micro-encapsulation as well as the materials commonly used arelisted in Table 36.

TABLE 36MECHANISMS FOR LIVING CELL MICRO-ENCAPSULATION

Principle Material

Encapsulation by coating Alginate-poly-L-lysine, alginate-poly-L-ornithine,a spherical hydrogel core alginate-poly-L-glutamate, alginate-polyvinylamine,

alginate-protamine sulphate, alginate-poly-L-lysine-alginate, alginate-PLL-alginate-PLL-g-MPEG-alginate,

alginate-poly-L-lysine-polyethyleneimine, alginate-polyethyleneimine-protamine-heparin, alginate-chitosan, alginate-Eudragit

RL, agarose-polyacrylamide- polybisacrylamide,carboxymethylcellulose-poly-L-lysine, κ-carrageenan-chitosan

Encapsulation by coextrusion Eudragit RL polyacrylate, hydroxyethyl methacrylate-and interfacial precipitation methyl methacrylate (HEMA-MMA)

Encapsulation by interfacial Carboxymethylcellulose-chitosan,ionic cross-linking cellulose-poly(dimethyldiallyl-ammonium chloride),

chitosan-alginate, polyacrylate

Encapsulation by interfacial Chitosan, gelatin, nylon, polyethyleneiminepolymerisation

3.1.1 Alginate

(a) Alginate-polyaminoacidEncapsulation using alginate-poly-L-lysine (PLL)-polyethyleneimine (PEI), was first

reported by Lim and co-workers (Lim and Sun, 1980; Lim and Moss, 1981; Lim, 1982;1983). They developed this technique for the immunoisolation of islets of Langerhans. Themethod consisted of suspending unwashed islets uniformly in a 0.6 to 0.8% sodium alginatesolution in physiological saline. Droplets containing islets were produced by syringe pumpextrusion into 1.5% calcium chloride solution. They were then decanted and further treated in


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a polylysine solution (0.02% w/v). The polylysine-alginate micro-capsules were washed with1% CaCl2 solution, resuspended in an aqueous polyethyleimine solution (0.2%, MW 40000-60000), and then washed again with CaCl2 and saline. Finally, they were suspended in anisotonic sodium citrate solution (pH 7.4) in order to liquefy the alginate inside the capsule,and washed with saline. Since then, several modifications to the original protocol have beenmade. For long-term in vivo survival, the polymeric micro-capsule must be biocompatiblewith the body fluids and tissues. It was showed that polyethyleneimine had a poorbiocompatibility which resulted in a rapid rejection of implanted micro-capsules (Sun et al.,1983). The biocompatibility could be improved by adding an additional layer of alginate onthe outside of the capsule (O'Shea et al., 1984). In a later study, the initial contact timebetween PLL and alginate was increased from 3 to 6 min, the PLL concentration wasincreased from 0.02 to 0.05% (w/v), and the PEI was replaced by alginate resulting inalginate-PLL-alginate capsules (Sun and O'Shea, 1985). These changes resulted in a capsulemembrane with a molecular weight cut-off below 150,000 (the molecular weight of IgGantibodies) (King et al., 1987), and in an improvement in capsule biocompatibility to theextent that transplanted islets survived in rats for as much as 2 years. Besides poly-L-lysine,several other polyamino acids have been used, such as poly-L-ornithine and poly-L-glutamate(e.g. Bugarski et al., 1993; Young, 1993).

Considerable flexibility with regard to permeability, properties and size of the alginate-PLL capsules have been reported. Perfectly spherical and smooth micro-capsules could onlybe formed using a highly purified sodium alginate and keeping the viscosity of the sodiumsolution above 30 cps (Goosen et al., 1985). The strength of the capsule membrane can beenhanced by increasing the reaction time as well as the PLL concentration (Goosen et al.,1985). King and co-workers (1987) reported that an increase in the average molecular weight(MW) of PLL produced membranes with lower strength, flexibility, and thickness; whenPLL MW exceeded 1.2 10 5, 10 to 20% of the micro-capsules showed ruptures. In contrast,Schlameus and co-workers (1990) showed that reduction of PLL MW from 3.85 10 4 to 3.30103 increased the severity of alginate-PLL defects. There appeared to be a correlationbetween membrane permeability, physical integrity, and thickness above a certain criticalmembrane polymer MW . Under such conditions, increased membrane strength andthickness, due to the greater compactness, reduced the incidence and/or magnitude ofmembrane defects, and hence also permeability (Okhamafe and Goosen, 1993).

The time required to reach a maximal binding between the Ca-alginate beads and PLLdecreased six times by doubling the PLL concentration from 0.05% to 0.1% (w/v). A furtherincrease of the concentration had a less profound effect on the time to reach maximal binding.Also the amount of PPL bound increased upon the increased PLL concentration by theformation of a thicker membrane (Vandenbossche et al., 1993c). When PLL concentrations of0.2% (w/v) and higher were used, a fraction of the bound PLL released after reachingmaximal binding (Vandenbossche et al., 1993c). In contrast, Goosen et al. (1985) doubled thePLL solution concentration and could not notice an alteration of the membrane thickness,although membrane strength was improved. In a subsequent work (King et al., 1987),however, it was demonstrated that a rise in PLL solution concentration lowered capsulemembrane permeability to carbonic anhydrase, and this was attributed to the increased


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compactness, strength, and thickness of the membrane as a result of greater PLL-ionicinteractions. This result was also confirmed by Schlameus and co-workers (1990).

A frequently used term to characterise the permeability is the micro-capsule membranemolecular weight cut-off. It is defined as the minimum MW of a solute which is totallyexcluded by the capsule membrane. However, it is also defined as that molecular weight atwhich 90% of the (macro)molecular solutes are rejected by the membrane (Mulder, 1993).Modification of the molecular weight cut-off of the micro-capsules can be accomplished byinterrupting the reaction as suggested by Goosen co-workers (1985; 1989), King et al. (1987)and Shimi et al. (1991). However, a reaction time shorter than the time required to reachmaximal binding will be sensitive to small fluctuations, and can have a large influence on theamount of PLL bound. Increasing the reaction time above the time required to reach maximalbinding did not influence the amount of PLL bound (Vandenbossche et al., 1993c). King andco-workers (1987) noticed that the membrane permeability for albumin decreased, while itsstrength increased as the reaction time was raised from 3 to 40 min. Prolonging the reactiontime resulted in the formation of thicker and more compact membranes due to the greaternumber of ionic bonds established between PLL and the alginate core. The time required toreach a maximal binding was related to the reaction temperature. The PLL concentration andthe reaction temperature influenced the density of the capsule membrane: the permeabilitydecreased as the PLL concentration increased and increased at decreased reactiontemperatures (Vandenbossche et al., 1993c). The permeability increased when PLL with ahigher average molecular weight was used (Goosen et al., 1985; 1989; King et al., 1987). Onthe contrary, Shimi and co-workers (1991) found an inverse correlation between the micro-capsule permeability and the molecular weight of the PLL. However, the former results wereobtained by looking at the inward diffusion, whereas the latter relationship resulted fromoutward diffusion experiments. Shimi et al. (1991) recommended to use PLL with a MW of22000 to prepare robust capsules which are relatively impermeable to high MW species suchas immunoglobulins.

The capsule parameters of major significance in permeation control are size, swelling andshape (Okhamafe and Goosen, 1993). Capsule size control can be used as a process tool inmodulating the diffusion of solutes into and out of micro-capsules. When alginate-PLLcapsules were placed into the sodium citrate buffer in order to liquefy the Ca-alginate core,they expanded (Goosen et al., 1985; King et al., 1987). The increase in capsule diameter wasdirectly proportional to the PLL molecular weight. Swelling can have some implications onthe permeation of solutes. Swelling results in a greater surface area and hence a largerpermeability. The distention of the capsule may induce rupture, micro-cracks and/orbroadening of the diffusion pathways. The increase in capsule volume may produce a lowerintracapsular product concentration and as a result the outward membrane transport of theproduct is decreased due to a decrease of the transmembrane concentration gradient. Thedesired capsule shape is a sphere, and any deviation from this shape creates stress locations inthe membrane which constitute potential sites for rupture and other effects, especially duringhandling and capsule swelling. Goosen et al. (1985) observed that a reduction of the purealginate solute concentration to below 1.2% resulted in the formation of alginate-PLLcapsules with tails and striations.


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The mechanical stability of liquid-core alginate-PLL capsules used for the encapsulation ofhybridoma cells can be greatly enhanced by the inclusion of polyethyleneimine in thehardening solution containing calcium chloride (Hsu and Chu, 1992). These capsules couldwithstand severe agitation and aeration in an air-lift reactor over a period of 3 weeks withminimal damage.

Recently, a graft copolymer having PLL as the backbone and monomethoxy poly(ethyleneglycol) (MPEG) as pendent chains was used to form micro-capsules with sodium alginate(Sawhney and Hubbell, 1992). The use of poly(ethylene glycol) (PEG), MPEG andpoly(ethylene oxide) (PEO) to reduce protein adsorption and cell adhesion is welldocumented in literature (e.g. Nagaoka et al., 1984; Miyama et al., 1988; Gombotz et al.,1989; Desai and Hubbell, 1991; Desai et al. 1992; Shoichet et al., 1994). Micro-capsules andmodel surfaces formed with PLL-graft-MPEG demonstrated reduced protein adsorption,complement binding and cell adhesion in vitro compared to materials with unmodified PLL.Micro-capsules with PLL-g-MPEG on the surface were seen to be much more biocompatiblethan alginate-PLL-alginate micro-capsules in a mouse intraperitoneal implant model. Thegraft copolymers demonstrated lower affinity for alginate and increased micro-capsulepermeability (more than PLL). This was corrected by constructing pentalayered alginate-PLL-alginate-PLL-g-MPEG-alginate micro-capsules which showed appropriatepermselectivity and enhanced biocompatibility.

(b) Alginate-chitosanThe cationic polysaccharide chitosan has been used to coat polyanionic alginate beads

(Rha, 1984; Rha et al., 1984; 1985; McKnight et al., 1988; Huguet et al., 1994). Differentencapsulation procedures have been developed. Cells can be immobilised in Ca-alginatebeads which are then coated with chitosan (McKnight et al., 1988; Pandya and Knorr, 1991;Shimi et al., 1991). McKnight and co-workers (1988) coated Ca-alginate beads withchemically modified chitosan using a chitosan-derivative solution which reacted for 20 minunder adequate mixing. Following membrane formation, the micro-capsules were washedwith buffer, CaCl2-solution and saline to prevent clumping of the capsules and to tie up anyunreacted chitosan groups. The exterior of the capsules were then reacted with 0.03% (w/w)sodium alginate and washed with saline. Finally, the capsule cores were reliquefied byreaction with sodium citrate. While unmodified chitosan (MW=12.1 10 5) formed thin andweak micro-capsule membranes, when the MW of the chitosan was reduced to 2.4 10 5, thepolymer exhibited optimum membrane forming characteristics in terms of capsule strengthand flexibility. The degree of deacylation of chitosan varied from 94.3% for the unmodifiedpolymer to 93.2% for the chitosan of MW=1.6 10 5. During encapsulation, it was observedthat the capsules swelled in citrate and saline and it was shown that the swelling increasedwhen the MW of chitosan decreased. Alginate beads (2% w/v) have been coated with water-soluble chitosan salts (1% w/v, hydroglutamate chitosan) and acid-soluble chitosan (1% w/v)(Pandya and Knorr, 1991). The alginate-chitosan hydroglutamate coacervate micro-capsulesshowed higher mechanical strength than those using acid soluble chitosan and had goodspherical shape and size. Studies using these coacervate capsules for plant cell (Daucus


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carota, Apium graveolens) immobilisation showed that the plant cells were more viable in thewater-soluble chitosan salt containing capsules than in alginate-acid soluble chitosan.

Encapsulation can also be performed by preforming biocatalyst-containing capsules byadding the sodium alginate-biocatalyst mixture into a chitosan solution, then gelling theinterior by addition and diffusion of CaCl2; or directly in adding dropwise the sodiumalginate-biocatalyst mixture into a chitosan-CaCl2 solution (Huguet et al., 1994). Membranesformed with chitosan (MW=0.5 106) excluded β-amylase, suggesting a membrane molecularweight cut-off of approximately 200000 (McKnight et al., 1988). Permeability of the capsulemembrane depends on the chitosan molecular weight. It was found that when the chitosanmolecular weight was increased, protein permeability was reduced (Polk, 1990; Huguet et al.,1994). An increase of chitosan solution concentration from 0.25 to 1% (Kim and Rha, 1989)or from 0.2 to 0.8% (w/v) (Huguet et al., 1994) had no significant influence on the membranepermeability. However, Polk (1990) reported a rise in the permeability of bovine serumalbumin entrapped in alginate-chitosan micro-capsules when the chitosan solutionconcentration was doubled from 0.1 to 0.2%. The pH of the chitosan solution has an influenceon the electronic interactions during membrane formation. It was observed that thepermeability of the polycation-polyanion membrane decreased when the pH of the chitosansolution was lowered (Kim and Rha, 1989; Huguet et al., 1994). Studies on the influence ofreaction time on the physicochemical properties of alginate-chitosan micro-capsules indicatethat chitosan membrane permeability is essentially independent of reaction time (Shioya andRha, 1989; Polk, 1990).

(c) Alginate-polyethyleneimineAlginate gel beads have been coated with polyethyleneimine (PEI) and partially

quaternised polyethyleneimine (QPEI) (Tanaka et al., 1984c). QPEI with a degree ofquaternisation of 30 mol%, was obtained by treating the PEI with CH3Br. Membraneformation was achieved by immersing the beads in the polymer solution (1%, pH 5) during afew minutes. Beads coated with QPEI were impermeable for glucoamylase, whereas PEIcoated beads were permeable for glucoamylase but mass transport was seriously hinderedcompared to non coated beads.

As discussed previously, PEI has also been used to coat alginate-poly-L-lysine capsulesand encapsulate islets of Langerhans (Lim and Sun, 1980) but PEI membranes showed a poorbiocompatibility (Sun et al., 1983).

(d) Alginate-polyethyleneimine-protamine-heparinMicro-capsules with a protamine-heparin membrane have been prepared by washing Ca-

alginate beads successively in 0.2% aqueous polyethyleneimine solution, 1% CaCl2, 0.1%protamine sulphate from salmon, distilled water, heparin, and afterwards, several times inbuffered saline (Tatarkiewicz, 1988). The amounts of protamine and heparin were adjusted toneutralise themselves. Next, the micro-capsules were suspended in 0.1% sodium citrate inbuffered saline (pH 7.4) to liquefy the alginate gel inside. Permeabilisation measurementsrevealed that glucose and insulin could easily diffuse into and out of the capsules, but themembrane was not permeable for human albumin and γ-globulins. The protamine-heparine


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membrane is highly biocompatible for islet transplantation as demonstrated by in vitro and invivo experiments (Tatarkiewicz et al., 1994).

(e) Alginate-acrylateErythrocytes have been immobilised in alginate beads coated with the polyacrylate

Eudragit RL (cf. 3.1.2 (a)) (Lamberti and Sefton, 1983). Alginate beads were prepared byextrusion of sodium alginate into a calcium alginate solution and then suspended in a 0.5%Eudragit RL emulsion for a predetermined period of time before transfer to a Ca-containingstorage solution. Eudragit RL emulsions can be prepared when it is added to boiling water.The reinforcement of the alginate core is due to an electrostatic interaction between thepolyanionic alginate and the Eudragit RL emulsion.

Micro-capsules can also be prepared by using graft copolymer emulsions of sodiumalginate with hydroxyalkyl methacrylates (Stevenson and Sefton, 1987). The preparation ofthese micro-capsules is simple: droplets of emulsion (containing cells) are extruded from aneedle tip into a Ca-containing solution. HEMA-alginate graft copolymer emulsions can beprepared by γ-irradiation of the aqueous alginate and the HEMA monomer. It has been shownthat γ-irradiation of alginate results in chain scission and production of radicals on thealginate. It has been suggested that graft copolymer was formed through initiation of chaingrowth from the alginate and the coupling of growing vinyl polymer with the alginate(Stevenson and Sefton, 1987). Graft copolymer emulsions are well tolerated by erythrocytesin both the emulsified and the precipitated state.

3.1.2 Polyacrylate

Polyacrylates are based on the [-CH2-C(R1)OOR2-] repeat unit, where R1 and R2 canrepresent various chemical substances (Stevenson and Sefton, 1993). Because of the availablesubstituents, polyacrylates can have a wide diversity of chemical and physical properties.Some polyacrylates are water insoluble, which is an advantage for maintaining stability of thecapsule in an aqueous environment but a disadvantage for preparing micro-capsules. Theproperties can be adapted for a particular application by copolymerisation of various acrylicmonomers. For example, hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA)copolymer which contains 75 mol % HEMA: this polymer is hydrophilic, swells in water likepolyHEMA but has sufficient PolyMMA character to become tough and elastic.Copolymerisation with ionic monomers (e.g. dimethylaminoethyl methacrylate, DMAEMA)results in capsules which supports the growth of anchorage dependent mammalian cells (e.g.fibroblasts) on the polymer surface. Alternatively, ionic polyacrylates are water soluble. Theycan be used together or in conjunction with other polyelectrolytes to produce capsules bycomplex coacervation without resorting to organic solvents. Also, HEMA-alginate graftcopolymers and polyacrylate emulsions have been used to encapsulate cells in water systems.

(a) Eudragit RL PolyacrylateEudragit RL is a commercially available-insoluble polyacrylate (Röhm Pharma,

Darmstadt, Germany). This polyacrylate is an acrylic ester-methacrylic ester copolymer with


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a low content (5%) of quaternary ammonium groups (MW: ~ 150,000). It was used as a 12%(w/v) solution in diethyl phthalate for encapsulation. The Eudragit RL micro-encapsulationtechnology has been developed by Sefton and co-workers as a model system to demonstratethat interfacial precipitation and organic solvents can be used to encapsulate live mammaliancells (Sefton and Broughton, 1983; Sefton et al. 1987; Boag and Sefton, 1987). Theencapsulation method is based on coextrusion and interfacial precipitation. The polymerdissolved in diethyl phthalate is pumped along the annulus formed from two concentricneedles, while the cell suspension is pumped inside the inner needle. Droplets are blown offthe end of the needle by a coaxial air stream. The droplets are dispersed into a corn oil-mineral oil curing bath to extract the solvent and precipitating the polymer at the surface.Capsules are washed free of oils and solvent in a fractionated plasma that allowed forquantitative transfer of capsules from the oil phase to an aqueous phase. By appropriateadjustment of the coaxial air flow rate, capsule size could be varied from 250 to 1000 µm(Sefton et al., 1987). Eudragit RL membranes have a limited permeability.

Human diploid fibroblasts were encapsulated within Eudragit RL capsules (Boag andSefton, 1987). The anchorage-dependent cells were viable after encapsulation, but grew onlyif collagen was co-encapsulated to act as a growth scaffold for the cells. Chinese hamsterovary (CHO) cells failed to grow inside intact capsules, they grew only in broken capsules(Broughton and Sefton, 1989). It has been showed that islets of Langerhans could beencapsulated in Eudragit RL and the cells preserved their viability in vitro, but the capsulewall was only slightly permeable to insulin (Sugamori and Sefton, 1989). The in vivo use ofEudragit encapsulated islets was also not successful due to the poor biocompatibility of thecapsule.

(b) HEMA-MMAMammalian cells have been successfully immobilised in HEMA-MMA micro-capsules

(which contained ~ 75 mol % HEMA in the monomer mixture). These capsules are soft,elastic and tough. The water uptake is greater than that of Eudragit RL, consistent with theobserved differences in glucose permeability (Douglas and Sefton, 1990). Polyethyleneglycol 200 has been chosen as the solvent to soluble 75% mol % HEMA, since it wastolerated at least to a limited extent by mammalian cells and was water soluble, enabling anaqueous salt solution to be used as the non solvent. Capsules with immobilised CHO cellshave been prepared with a coaxial air stream to control the size of the capsules, but theproduced capsules were relatively large (> 1 mm), had an irregular shape and had a sponge-like interior with little space for cell growth (Dawson et al., 1987). Consequently, a newtechnique (called the "submerged jet extrusion" method) for the HEMA-MMA encapsulationhas been developed based on extrusion with a submerged nozzle leading to droplet formationwithin another liquid phase, i.e. hexadecane (Crooks et al., 1990). The inert liquidhexadecane was used to increase the shear force and to produce well formed smaller capsules.In the submerged jet method, the coextrusion needle is lifted out of a layer hexadecane(which lies above the precipitation solution) thereby shearing the capsule droplet (polymersolution plus cells) off the needle at the hexadecane-air interface. The droplet falls throughthe hexadecane layer into the precipitation solution which consists of PBS with 50 ppm of


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Pluronic L101 surfactant to aid droplet penetration. The PBS extracts the solvent and leavesthe precipitated polymer behind as a capsule wall around the cells. These capsule membranesconsist of a thin outer macroporous layer and a very thick (~90 µm) dense membrane. Thepermeability of HEMA-MMA capsule membranes is extremely low. The molecular weightcut-off has been determined by measuring the overall mass transfer coefficients of glucose,inulin, albumin and alcohol dehydrogenase, and was of the order of 100,000 dal (Stevensonand Sefton, 1993). Recently, a new droplet generation scheme was developed by suspendingthe cell and polymer co-extrusion nozzle in a uniform co-axial fluid jet which enabled theproduction of 300 to 600-µm capsules (Uludag et al., 1994). It was demonstrated that HepG2hepatoma cells in 400-µm-diameter HEMA-MMA capsules were able to retain theirmetabolic activity during and after the encapsulation process.

Experiments with encapsulated islets of Langerhans in HEMA-MMA capsules andimplanted subcutaneously or intraperitoneally in rats or mice demonstrated a very benignresponse and a good biocompatibility (Stevenson and Sefton, 1993). Hapatoma cells, ananchorage-dependent cell line, has been micro-encapsulated in a HEMA-MMA membrane towhich the cells do not adhere (Babensee et al., 1992). Sites for cell attachment were providedby the co-encapsulation of Matrigel (a reconstituted extracellular matrix derived from theEngelbreth-Holm-Swarm mouse tumor basement membrane).

(c) Cationic PolyacrylatesAnchorage dependent cells need substrates with surface charge to grown on. Several

cationic polyacrylates which support the proliferation of human diploid fibroblasts (HDFcells), have been developed. However, the growth of these micro-encapsulated cells on theinside wall was not quite successful due to permeability limitations. It has been shown thatDMAEMA-MMA copolymers with a DMAEMA content of 16% supported the growth ofHDFs (Sefton et al., 1987). Also, the methacrylic acid (MAA) containing terpolymer 17/2.2DMAE-MAA-MMA (17% DMAEMA, 2.2% MAA and remainder MMA) was a goodsubstrate for cell proliferation (Mallabone et al., 1989). HDF cell growth in DMAEMA-MMA capsules was not successful due to diffusional limitations with the capsule wall. Byadding MAA to the copolymer, the water content — and hence the permeability — of thecapsules can be increased. But HDF cells which were encapsulated in 17/2.2 DMAE-MAA-MMA, died although initially some cell growth could be observed. Increasing the MAAcontent in the terpolymer to increase the permeability resulted in copolymers which did notsupport HDF cell proliferation.

(d) Polycation/Polyanion MethodsViable hybridoma cells have been encapsulated within polyacrylate membranes which

were formed as a result of polyelectrolyte complex formation and covalent bonding betweenacrylic copolymers containing ionic and chemically reactive groups (Gharapetian et al.,1986). The encapsulation was performed by consecutively introducing droplets of asuspension of hybridoma cells in a solution of polyanionic acrylic copolymer into aqueoussolutions of three polycationic polymers. A large number of polyanionic copolymers weresynthesised using a variety of combinations of HEMA, methacrylic acid (MMA) or acrylic


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acid (AA), and either MMA or ethylmethacrylate (EMA) or a combination of both. Also, alarge number of polycationic copolymers were synthesised using a variety of combinations ofthe following monomers: HEMA, DMAEMA, EMA and MMA or AA. The capsule-formingproperties of the polymers, such as sinking of the droplets, ionic bond formation, and thephysico-mechanical properties of the membrane (shape, strength, flexibility and smoothness),were directly related to the type of acrylic monomers and their relative concentrations in thecomonomer combinations. Hybridoma cells have been encapsulated to produce monoclonalantibodies into micro-capsules made from a polyanionic copolymer containing 26.8 mol %HEMA, 32.8 mol % AA and 40.4 mol % MMA monomers, and a polycation containing 7.5mol % HEMA, 17.6 mol % DMAEMA, 17.5 mol % DEAEA, 53.3 mol % EMA and 4.2 mol% AA. The growth rate and viability of the cells were not affected in these capsules(Gharapetian et al., 1986).

Due to their good hydrolytic stability, Stevenson and Sefton (1988; 1993) usedmethacrylates to produce micro-capsules by polyelectrolyte complexation. Functionalitycould be introduced through the introduction of either MAA or DMAEMA to the polymerand the hydrophobic-hydrophilic balance in the remainder of the polymer can be adjusted bycopolymerising with MMA, HEMA or hydroxypropyl methacrylate (HPMA). Capsules havebeen prepared based on highly (> 25 mol % ionic functionality) and sparingly (< 25 mol %ionic functionality) charged polymers. In the first category, the best capsules were preparedfrom anionic polymer containing 22 mol % MAA, 53 mol % MMA and 25 mol % HEMA,and with cationic polymer containing 38 mol % DMAEMA, 40 mol % MMA, 22 mol %HEMA. Capsules from this system were featureless and transparent, resisted puncture with aneedle, and remained stable for several months. It was also shown that the initial toxicity ofthe process for red blood cells was low (Wen et al., 1991a). In the second category, the mostpromising system made use of an anionic copolymer containing 8.8 mol % MAA and 91.2mol % HEMA, and a quaternised polybase containing 8.2 mol % DMAEMA with 91.8 mol% HEMA. These capsules resisted puncture by a fine-gauge needle and it was demonstratedthat the initial stress on encapsulated red blood cells was low (Wen et al., 1991b).

3.1.3 Nylon

The preparation of nylon (polyamide) micro-capsules for applications in biotechnology hasbeen firstly reported by Chang (1964). The nylon micro-capsules are formed by interfacialpolycondensation of organic acid dichloride and diamine. The dispersion of the biocatalystsolution is performed by emulsification. The diamine base is solubilised in an aqueous phaseof high pH, ionic strength and osmotic pressure and the dichloride is dispersed in a polarsolvent mixture. The membrane formation involves the transfer of diamine to the organic sideof the membrane, reaction with the dichloride and precipitation of the formed nylon polymer.The limiting process in nylon membrane formation is the transfer of diamine from theaqueous drop to the organic phase. High pH and polar solvents are needed to increase the noncharged diamine fraction (Poncelet et al., 1985; 1990) and its solubility in the organic phase(Morgan and Kwolek, 1959). These conditions create a toxic environment for viable cells.Permeability may be controlled by judicious selection of the monomers (Kondo, 1978), by


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adjusting reaction time and/or polymerisation conditions (Poncelet et al., 1990), or bymembrane coating. At high pH values (11), the nylon membrane thickness is sufficientlylarge (approximately 1 µm) to ensure good mechanical resistance. At lower pH values,however, the membrane is sufficiently thin (200 nm) that compounds such as proteins orpolyethyleneimine are needed to ensure good mechanical resistance. The membrane thicknesswas seen to be strongly dependent on the solvent polarity and control of temperature wasneeded for reproducibility of the membrane thickness. Mean diameters of micro-capsulesranging from 40 to 200 µm have been obtained depending on the impeller type, rotationalspeed and emulsifier concentration (Poncelet De Smet et al., 1990). It was shown that theprimary process which determined the micro-capsule breakage in a turbine reactor was theshear effect (Poncelet and Neufeld, 1989). The conditions for nylon membrane encapsulationare not well suited for live cell immobilisation due to the reactive nature of the membraneformation process (elevated levels of pH, high ionic strength and osmotic pressure, and toxicnature of solvents and reagents). Lactococcus lactis cells were non viable after encapsulationwithin nylon membranes (Larisch et al., 1994). However, an interfacial polymerisationprocess for cell encapsulation has been patented (Tice and Meyer, 1985). It was suggested toincorporate cross-linking proteins (e.g. casein, collagen, soy protein and gluten) into themembrane which could subsequently be degraded enzymatically to attain the desiredmembrane properties.

3.1.4 Polyethyleneimine

A micro-encapsulation technique to immobilise viable cells based on the cross-linking ofpolyethyleneimine (PEI) with acid dichloride, has been developed by Poncelet et al. (1994a).PEI membranes are formed by a polycondensation reaction between the PEI in bufferedaqueous solution at an initial pH of 8.0-9.5 and sebacoyl chloride in the organic solvent(cyclohexane). PEI membrane formation is slightly different from nylon formation. PEI is apolar compound which is insoluble in the organic phase (Horn, 1980) and the acid dichlorideis hydrolysed in water. The reaction takes place at the organic-aqueous interface, probablymore extensive on the aqueous phase due to the hydrophilic properties of cross-linked PEI.The membrane mass was found to be independent of the reaction time and PEI concentration,and proportional to the concentration of the cross-linking agent (sebacoyl chloride). Sincemembrane formation is performed at pH 8 in a non polar solvent, conditions for micro-encapsulation are much milder and better suited for the encapsulation of viable cells thanthose for nylon encapsulation. Encapsulation of L. lactis in cross-linked PEI capsules resultedin viable but non active cells (Larisch et al., 1994).

3.1.5 Chitosan

(a) ChitosanMicro-encapsulation within cross-linked chitosan membranes can be performed by an

emulsification and interfacial polymerisation technique (Groboillot et al., 1993).Encapsulation of living cells requires biocompatible conditions. Mineral and vegetal oils


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which are non toxic in contact with living cells, have been used as the continuous organicphase. Chitosan cross-linking with hexamethylene diisocyanate or glutaraldehyde resulted instrong membranes with a narrow size distribution about a mean diameter of 150 µm. Thestrength of the membrane is related to the chitosan concentration and to the type of cross-linking agent. Cell viability and activity of encapsulated L. lactis was demonstrated by theacidification of milk. Loss of acidification activity during micro-encapsulation (due to thetoxic cross-linking agent) was recovered in subsequent fermentations to levels similar to thatof free cell fermentations.

(b) Chitosan-alginateCapsules have been prepared by dropwise addition of a 3% (w/v) chitosan solution

containing 450 mM CaCl2 to a stirred sodium alginate solution or a mixture of sodiumalginate and esterified alginate solution (0.75% w/v) containing 2.25% (w/v) glucose (Knorrand Daly, 1988). After washing with distilled water, the capsules were stored for three hoursin 3 different buffer solutions containing CaCl2 and either borax, phthalate or Trizma Base.The interphasic membrane is formed by cross-linking polycationic chitosan and polyanionicalginate. CaCl2 diffusing through the interphasic membrane and subsequently CaCl2 presentin the buffer solution provides additional Ca-alginate layers. Cross-linking of chitosan presentin the liquid core could occur along the interphasic membrane because of precipitation ofchitosan above pH 6.5 due to a pH gradient in the liquid core. Buffer treatment and partialsubstitution of alginate with propylene glycol alginate affected capsule wall propertiesincluding mechanical stability and membrane thickness, allowing tailoring of such properties.

3.1.6 Cellulose

(a) Carboxymethylcellulose-chitosanChitosan is a polycation, derived by deacetylation of chitin (cf. Chapter 2) and it may form

strong polymerised membranes as a result of the reaction of chitosan with polyanions(Shiotani et al., 1986; Uragami et al., 1986). Carboxymethylcellulose (CMC)-chitosan micro-capsules have been constructed by interfacial ionic cross-linking to encapsulate mammaliancells: micro-capsules were formed in one step by placing a drop of cell suspension mixedwith the negatively charged carboxymethylcellulose into the positively charged chitosansolution (Yoshioka et al., 1990). These capsules had a mean diameter of about 1.5 mm and awall thickness of 3 µm. Considerable amounts of unreacted CMC existed within the capsuleswhich resulted in a drastic swelling of the capsules after incubation in the medium. Thevolume of the capsules attained about the three-fold of the initial one; still there was noappreciable breakage of the capsules. Hybridoma cells have been grown in these capsules toproduce monoclonal antibodies. A maximum cell density of 1 107 cells/ml was reached insidethe capsules, which was 10 times higher than the free cell culture concentration.

(b) Carboxymethylcellulose/alginate-poly-L-lysinePLL-coated carboxymethylcellulose liquid core capsules have been prepared for the

encapsulation and culture of hybridoma cells (Hsu and Chu, 1992). The core material of the


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capsules contained 1.2% CMC and 0.2% alginate. The core material solution was mixed withthe cell suspension and added dropwise to the hardening solution composed of CaCl2 andPEI. PLL-coating of the gel beads was performed by immersing the water-washed beads in asolution containing PLL (0.05% w/v). The low level of alginate present was meant to assist inthe bead formation of the liquid coacervation capsules. At this low level, Ca-alginate wasgradually degraded in the phosphate-containing medium and no citrate treatment wasnecessary to liquefy the capsule core. Due to the high mechanical stability of these capsules,they could withstand severe agitation and aeration in an air-lift reactor over a period of 3weeks with minimal damage. These micro-capsules have been used for the cultivation ofmurein hybridoma cells and to produce IgG monoclonal antibodies.

(b) Cellulose NitrateCellulose nitrate (collodion) micro-capsules have been prepared by dispersing an aqueous

solution of the biocatalyst (haemoglobin, urease) in a water-saturated-ether organic phase,with the aid of an emulsifier (Span 85) to yield a water-in-oil emulsion (Chang, 1977;Arbeloa et al., 1986; Poncelet De Smet et al., 1989). This step was followed by coacervationof a collodion membrane on the surface of the aqueous droplets. Finally, transfer of themicro-capsules from the organic phase into the aqueous phase was performed using asurfactant (Tween 20), and the aqueous suspension was washed several times. The control ofmean diameter and size distribution was achieved by varying the conditions of emulsification:reactor and impeller geometry, rotational speed and duration, emulsifier concentration, andphase ratio (Poncelet De Smet et al., 1989).

(c) Cellulose sulphate/poly(dimethyldiallylammonium chloride)Mechanically stable polyelectrolyte-complex (PEC) micro-capsules have been formed by

the interaction of the polyanion cellulose sulphate (CS) and the polycationpoly(dimethyldiallylammonium chloride) (PDMDAAC) to encapsulate living cells. Cells tobe encapsulated were distributed in a highly viscous solution of CS and dropped into theprecipitation bath of PDMDAAC, which had a concentration between 0.5% and 2%.Immediately after immersion of the drop into the precipitation bath at the phase boundary, apolysalt membrane was formed enclosing the cells in the interior of the drops.

The encapsulation of the filamentous yeast Yarrowia lipolytica required an adaptation ofthis method (Förster et al., 1994). The yeast cells were aggregated before being suspended inthe cellulose sulphate solution (pre-immobilisation). This was accomplished by the formationof PEC coprecipitates: the cells are suspended in a 0.25% (w/v) CS solution and a 0.25%(w/v) PDMDAAC solution was added until the iso-ionic point was reached. The formedpolysalts sedimented easily and could be collected, and homogeneously mixed with thehighly viscous (2.4% w/v) CS solution. This suspension was dropped into the PDMDAACprecipitation bath for subsequent formation of micro-capsules (diameter of 2.5-3.5 mm).

A variety of applications using the CS/PDMDAAC encapsulation method has beenpublished, ranging from the protection of bovine embryos during fertilisation (Torner et al.,1986), liver microsomes for extracorporal detoxification (Stange et al., 1990), pancreatic


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islets for in vitro production of insulin (Braun et al., 1985) to Y. lipolytica yeast cells tosynthesise citric acid from glucose in a continuous fermentation process (Förster et al., 1994).

3.1.7 Agarose-acrylamide

Mammalian living cells have been encapsulated in agarose beads which were coated withpolyacrylamide polybisacrylamide polymer (Dupuy et al., 1988). The micro-encapsulationmethod consisted of two separate stages: initial embedding in agarose followed by an in situpolymerisation. Micro-capsules were produced from an arrangement of two coaxial nozzles.The living cells were mixed with the agarose solution and passed through the central nozzle,while a hydrophobic medium (paraffin oil) was passed through the external one, and extrudedinto a paraffin oil bath at 4°C for rapid gelling. This procedure resulted into spherical beadswith a relatively homogeneous size distribution. The beads were then subjected to a secondembedding procedure using the polymerisable solution. The beads were passed through acapillary which was connected to a T-piece. A mixture composed of the solution ofmonomers (30% acrylamide and 1.5% bisacrylamide), a sensitiser (riboflavin) and a catalyst(TEMED) was pumped into the side arm of the T-piece. The extension of the capillary wascoiled around a glass column cooled with water and containing a mercury lamp, which wasused to initiate the photo-chemical reaction. The dye-sensitised photo-polymerisation of themixture around the agarose beads is the consequence of the presence of the free radicalsproduced during the primary photo-reduction step of the riboflavin and subsequent oxidationof the reduction products by molecular oxygen. This oxido-reduction initiation reactionenabled the process to be carried out at laboratory temperature. Riboflavin absorbs light in thevisible part of the spectrum, so that the harmful effects of infrared rays and ultravioletradiation can be avoided. The presence of the oil medium significantly reduced the toxicity ofthe acrylamide. At the end of the polymerisation reaction, the micro-capsules were rinsed in alarge volume of culture medium to remove secondary reaction products. The authorsdemonstrated a lack of cytotoxicity for encapsulated islets of Langerhans by measuring theinsulin secretion at the end of the different steps.

The second embedding procedure has been adapted for the encapsulation of pituitary cells(Dupuy et al., 1991). Part of the acrylamide monomers was prepolymerised inside a micro-emulsion, resulting in micro-latex beads of homogenous size. Micro-emulsions were preparedby addition under stirring of the aqueous phase (water plus acrylamide/bisacrylamide pluselectrolyte) to the oil phase (blend of Isopar M (isoparaffinic mixture) plus surfactant). Themicro-latex was aggregated by dilution of the micro-emulsion in acrylamide solutions. Theaggregates were then coagulated by polymerisation at the interfaces of the agarose beadscirculating in the capillary tube containing paraffin oil. Secretion assays performed with theencapsulated pituitary cells showed that this method permitted the preservation of goodactivity.


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3.1.8 Gelatin

Using the emulsification and interfacial polymerisation technique, living bacterial cellshave been micro-encapsulated within gelatin membranes cross-linked with toluene-2,4-diisocyanate at an oil/water interface (Hyndman et al., 1993). Reagent toxicity was avoidedby using vegetable or silicone oil as a dispersant, and by minimising cell exposure to thewater insoluble cross-linking agent during membrane formation. Contact with the cross-linking agent was minimised by a reduction in the reaction agent concentration and byminimising the surface to volume ratio of the micro-capsules. A lower viscosity oil (silicone)used in the dispersion step reduced the surface to volume ratio by 45%, yielding a 44%increase in activity over capsules produced in a higher viscosity oil (sunflower seed oil).Nevertheless, a substantial reduction in viable cell count was observed after the encapsulationprocess. Growth of L. lactis cells within the gelatin micro-capsules was observed duringfermentation and acidification activity of the encapsulated cells increased with subsequentreuse.

3.1.9 Carrageenan-chitosan

Micro-capsules with a good mechanical strength, composed of a κ-carrageenan core andcoated with water-soluble chitosan salts or acid-soluble chitosan, have been constructed(Pandya and Knorr, 1991). The formation of coacervate capsules was carried out by extrudingan aqueous 4% (w/v) solution of κ-carrageenan into 1% (w/v) potassium chloride. Thecounterion solution consisted of either one of 1% (w/v) chitosans (acid-soluble chitosan,hydroglutamate chitosan or chitosan lactate). Capsules were agitated to harden in thecounterion solution for 30 min before washing and dispersing into the growth medium. Thepermeability of these coacervate capsules could be controlled either altering the type ofchitosan and/or the counterion. In the capsules utilising water-soluble chitosan, plant cells(Apium graveolens) could successfully be immobilised over a period of 2 weeks without anydetrimental damage to the cells. The use of chitosan salt for plant cell entrapment offerssignificant advantages as the permeabilisation of whole cells results in the release ofbiochemicals while maintaining high cell viability.

3.2 Applications

3.2.1 Micro-encapsulation of Islets of Langerhans

Diabetes mellitus is a heterogeneous group of disorders in which the regulatorymechanism of blood glucose is impaired. The most severe form (insulin-dependent diabetesmellitus) is caused by the autoimmune destruction of the majority of the insulin producingbeta cells. The non-insulin dependent form is caused by diminished rates of secretion ofinsulin by the beta cells of the islets of Langerhans. The islets of Langerhans and the acini,which secrete digestive juices into the duodenum, are the two major types of tissues of thepancreas. The islets contain three major types of cells: the alpha, beta and delta cells. The


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beta cells secrete insulin, the alpha cells secrete glucagon and the delta cells secretesomatostatin.

Diabetic patients can be treated by exogenous insulin injection. But, the daily injection ofinsulin has not been able to prevent the common secondary complications of the disease, suchas nephropathy, retinopathy and neuropathy (Krolewski et al., 1987; Hernandez, 1989).Alternatively, transplantation of islet tissue, as a whole (Sutherland and Moudry, 1987;American Diabetes Association, 1992) or as isolated islets (Hering et al., 1988; Sharp et al.,1990; Warnock et al., 1992), can provide near-normal blood glucose control and has thepotential to prevent diabetic complications (Kennedy and Lyons, 1989; Lacy, 1993; Pipeleerset al., 1994). The future use of allografts does not appear hopeful because of donor scarcityand the need for recipient immunosuppression to prevent both allograft rejection andautoimmune disease recurrence (Sutherland et al., 1984; 1989; Sutherland and Moudry,1987).

Recently, islet encapsulation is being pursued as a novel therapeutic modality for thetreatment of diabetes: pancreatic islets (allogeneic as well as xenogeneic) may beencapsulated in semipermeable membranes and implanted to replace or supplement theinsulin secretory function of the pancreas. Islets are protected from the surroundings by theimmunoisolation membrane which excludes antibodies that would otherwise cause rejectionor destruction of the transplanted cells and allows the diffusion of glucose, oxygen, othernutrients to the cells, and insulin and waste products from the islets. Differentimmunoisolation devices are classified as intravascular and extravascular; and the latter canbe subdivided in macro-capsules (sheaths, rods, discs) and micro-capsules. The critical issuesfor the creation of a bioartificial pancreas include: (i) membrane biocompatibility (fibrosis forextravascular devices and blood clotting for intravascular devices), (ii) diffusion limitation(access of encapsulated tissue to the vascular system for nutrition, gas exchange andelimination of waste products), (iii) dynamics of insulin release to changing glucose levels,(iv) mechanical stability, (v) device retrievability (Mikos et al., 1994). Although preliminaryresults with intravascular devices (Sullivan et al., 1991) and macro-capsules (Lacy et al.,1991; Lanza et al., 1992; 1993) seem promising, most research efforts have been focused onmicro-encapsulation. The small scale of micro-capsules compared to macro-capsules providesfor a reduction of mass transfer limitation. As a result, the cell viability is increased and theresponse time of insulin release is decreased. The mechanical stability of micro-capsules issuperior over the one of macro-capsules where membrane breakage due to bending is one ofthe major problems. The major disadvantage of micro-capsules is their poor retrievability,e.g. 80% (Lum et al., 1992) and 72 - 80% (Fritschy et al., 1994).

Various micro-encapsulation methods have been applied as listed in Table 37.Encapsulation materials include agarose gels, agarose-polyacrylamide-polybisacrylamide,alginate-PLL-alginate, alginate-PLL-polyethyleneimine, alginate-polyornithine-alginate,Ba(or Ca)-alginate, photo-cross-linked poly(ethylene glycol), polyelectrolyte complexes ofcellulose sulphate, protamine-heparin, and copolymers and terpolymers of 2-hydroxyethylmethacrylate, methyl methacrylate, dimethylamino-methacrylate, and cellulose-poly(dimethyldiallylammonium chloride).


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Encapsulation in alginate-poly(L-lysine)-alginate is a widely used technique, whichalready led to human clinical testing for allograft transplantation (Soon-Shiong et al., 1994).This encapsulation method evolved from the method of Lim and Sun (1980). They performedthe first experiments in islet micro-encapsulation using alginate-PLL-polyethyleneimine. Butit was shown that the use of polyethyleneimine as outer layer resulted in a poorbiocompatibility. By using alginate as outer layer the biocompatibility could be substantiallyimproved (O'Shea et al., 1984; Vandenbossche et al., 1993a). It has been shown that failure ofalginate-PLL-alginate grafts was associated with a dense pericapsular infiltrate resulting innecrosis of the encapsulated islets (Wijsman et al., 1992). Also, empty capsules were found toinduce a similar pericapsular infiltrate which was identical in composition to that foundaround encapsulated islets. It has been demonstrated that the alginate used in the outeralginate layer should be rich in guluronic acid to prevent capsular fibrosis (Shoon-Shiong etal., 1991). In contrast, Clayton et al. (1991; 1992b) demonstrated that alginate capsules withan outer layer with a high mannuronic acid content provoked a weaker fibrotic response thanalginate with a high guluronic acid content. As noted by Zimmermann et al. (1992) andClayton and co-workers (1992b; 1993), the degree of purity of the alginate preparations (andnot the chemical composition) could be a major factor affecting these results. Vandenbosscheet al. (1993a) found also a significant reduction in host reaction when alginate-PLL capsulescoated with Pronova MVG alginate were injected intraperitoneally into mice compared withcapsules coated with Manucol DH. It was suggested that the lower host reaction could beexplained by differences in charge density (guluronic acid has a higher charge density thanmannuronic acid) and differences in molecular weight. Characterisation of the alginatesshowed a molecular weight distribution of 180-350 kDa range for Pronova MVG and a 280-360 kDa range for Manucol DH; and the ratio guluronic acid was 63/37 for Pronova MVGand 29/71 for Manucol DH. Also, the endotoxin content of Manucol DH dispersion was 6times larger than in Pronova MVG dispersions. Not only the higher guluronic content ofPronova MVG but also the lower endotoxin content as compared to Manucol DH, bothinduced a lower interleukin-1 and tumour necrosis factor (TNF) - α release from themacrophages, and therefore stimulated fibroblast proliferation in a less pronounced way(Soon-Shiong et al., 1991). A significant lower host reaction (evaluated by leucocytecounting and histology of recovered capsules) was found in mice injected with emptyalginate-PLL-alginate micro-capsules or encapsulated MO cells as compared to tumourogenicMO4 cells, which was ascribed to soluble factors released from the tumourogenic MO4 cells(Vandenbossche et al., 1993b). Recently, it has been shown that empty Ba-alginate micro-beads as well as macro-capsules (polysulphone hollow fibre) induced lymphoid proliferationas a result of mitogenic impurities in the encapsulation materials themselves (Zekorn et al.,1993). In the same donor-recipient combination in which successful transplantation wasdemonstrated, it was found that at least the sensibilisation arm of the immune system wasactivated, i.e. the encapsulated islets were recognised by the recipient's immune system. Themechanisms of lymphoid activation are unknown, but macrophages play a central role (Coleet al., 1992; Pueyo et al., 1992; Vandenbossche et al., 1993a). It has also been recentlydemonstrated by immuno-histological staining that the pericapsular infiltrates (PCI) onalginate-PLL-alginate capsules showed no differences in the types of cells in the PCI on


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capsules with or without islets; and it was concluded that the early PCI was a capsule-inducedforeign body reaction that was not influenced by MHC incompatibility or the presence ofautoimmune diabetes (Fritschy et al., 1994). All these observations suggest that thebiocompatibility of the alginate-PLL-alginate micro-capsules should be further improved.

Weber and co-workers (1991) found that UV-B irradiated donor islets encapsulated inalginate-PLL-alginate and xenografted intraperitoneally into diabetic NOD mice provokedonly a mild cellular reaction. They hypothesised that antigen(s) released from encapsulatedislets are responsible, at least in part, for the NOD cellular reaction to the islet xenograft, anda characteristic of this (these) antigen(s) is a sensitivity to UV-B exposure. Horcher et al.(1994) supposed also that alloantigens could be released from Ba-alginate encapsulated isletsand could induce fibrosis. Soon-Shiong et al. (1990) demonstrated that the alginate-PLL-alginate micro-capsule system can prevent cytotoxic T-lymphocyte (CTL) and natural killercell-mediated cytotoxicity. However, as noted by Zekorn et al. (1993), these researchers mayhave circumvented a variety of immunological interactions as they performed theexperiments with selected subsets of the effector cells only. It has been demonstrated that theNOD mice reaction to alginate-PLL-alginate micro-encapsulated xenogeneic islets (rat ordog) is helper T cell-dependent, and it was stated that the target of this reaction is not themicro-capsule itself but the donor cells within (Weber et al., 1990b). Various biomedicalpolymers have been shown to activate macrophages involved in foreign body reactions andstimulate interleukin-1β production. It has been found that interleukin-1β (Sandler et al.,1987; Palmer et al., 1989) and other cytokines, including the combination of TNF-α andinterferon-γ (Campbell et al., 1988), were toxic to islets. In vitro experiments showed that thealginate-PLL membrane does not exclude interleukin-1β (Cole et al., 1992). It is wellestablished that a number of cytokines and monokines alter or damage islet cells. It has beenshown that IFN-γ mediates Ia (class II MHC) antigen expression on the beta cells (Wright etal., 1986; Pujol-Borell et al., 1987; Varey et al., 1988). Koivisto et al. (1989) have shown thatinterferon impairs glucose tolerance and insulin sensitivity in man. Interleukin-1, which maybe induced by IFN-γ, has been shown to inhibit insulin secretion from isolated rat islets(Zawalich and Diaz, 1986) and to be toxic to murine beta cells (Mandrup-Poulsen et al.,1986). Moreover, interleukin-2 is known to activate lymphoid (NK) cells cytotoxic for BB/ratislet cells (Rikel et al., 1987); and it is possible that lymphokine-secreting cells may injuredonor islets via these factors (Weber et al., 1990b). In vitro experiments have demonstratedthat nitric oxide (NO) secreted by activated macrophages is able to destroy islets despiteencapsulation in Ca-alginate, and that both, inhibition of NO formation and scavenging offormed NO, protect encapsulated islets from destruction (Wiegand et al., 1993). It has alsobeen found that fibroblast secrete NO when activated (Werner-Felmayer et al., 1990). Sincethe most prominent cell types in the foreign body-type reaction are macrophages andfibroblasts, NO radicals from these activated cells of the inflammatory overgrowth-coveringmicro-capsules may have a great role in encapsulated graft failure (Wiegand et al, 1993).

In vitro and in vivo experiments have been performed to evaluate encapsulated islets (cf.Table 37). The in vitro viability of encapsulated islets has usually been assessed by eitherstatic incubation or perifusion experiments. The latter experiment is dynamic which givesinformation on the response time of glucose and insulin for diffusion. However, the


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perifusion system may encourage insulin release from islets due to the solvent drag effect(Chicheportiche et al., 1988). In contrast, a static incubation system more closely mimics theenvironment of an encapsulated islet following transplantation into the peritoneal cavity.Transplantation of syngeneic, allogeneic and xenogeneic islet tissue into diabetic recipientshave shown that long-term graft function can be accomplished as shown in Table 37.

3.2.2 Production by and Growth of Micro-encapsulated Cells

The micro-encapsulation technology, originally developed for islets of Langerhans, canalso be used for the transplantation of other cells and tissues. Potential applications includefunctional replacement of the major organs (e.g. liver) as well as transplantation ofengineered cells for gene therapy purposes.

Micro-encapsulation is also an effective technique to achieve high cell densities andproduct concentrations in vitro, and permit cost-effective large-scale production withenhanced product recovery.


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106


107


108


109


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3.2.2.1 Mammalian CellsThe encapsulation of mammalian cells within micro-capsules is a novel way for the in vivo

controlled release of therapeutic agents. The in vitro retention of cell viability and secretionof several bioactive agents have been demonstrated (cf. Table 38). As noted, some in vivoexperiments have already been performed.

Neurological diseases (such as Parkinson's disease, Alzheimer's disease or chronic pain)may be improved by the transplantation of encapsulated cells which release neuroactivemolecules such as neurotransmitters, neurotropic factors or enzymes (Aebischer et al., 1993;Bellamkonda and Aebischer, 1994). Parkinson's disease is characterised by a loss ofdopamine-producing cells in the substantia nigra that project to the striatum. Its symptomshave been alleviated in experimental models by implants of dopamine-producing cells in thestriatum (Winn et al., 1991b). PC12 cells (a cell line derived from a rat pheochromocytomawhich secretes large amounts of dopamine (Greene and Tischler, 1976) encapsulated inalginate-PLL-alginate alleviated the symptoms for 4 weeks after implantation in the striatumof a rat (Winn et al., 1991b). Micro-encapsulated bovine adrenal chromaffin cells (a primarycell type that releases dopamine, epinephrine and norepinephrine) have also shown efficacyin the rodent model of Parkinson's disease for up to 4 weeks (Winn et al., 1991a). A rodentmodel of Alzheimer's disease is induced by lesioning the fimbria-fornix in rats, leading to adecrease of choline acetyltransferase (ChAT) expression in septal cells. The transplantation ofunencapsulated rat fibroblasts engineered to secrete nerve growth factor was shown todecrease the loss of ChAT expression (Rosenberg et al., 1988). However, potentially lethaltumours had developed two weeks after implantation. Recently, it has been demonstrated thatencapsulation prevented the cells from forming a tumour, while preventing the loss of ChATexpression (Hoffman et al., 1991; Maysinger et al., 1993).

Gene therapy involves the genetic modification of the patient's own cells ex vivo which arethen implanted autologously to avoid immune rejection (Miller, 1992). To provide a more-effective method for somatic gene therapy, an alternative strategy of nonautologous somaticgene therapy has been proposed (Chang et al., 1993): a standard established cell line isengineered to secrete a desired gene product and enclosed in a immuno-protective capsule toprevent immune rejection. The growth of genetically engineered mouse fibroblasts inalginate-PLL-alginate micro-capsules and their secretion of the human growth hormone havebeen demonstrated in vitro (Chang et al., 1994) (cf. Table 38).

Micro-encapsulation is also an effective technology to produce high-value products invitro (cf. Table 38). Fragile mammalian cells are protected by the outer membrane layer andhigh cell densities can be obtained. The micro-encapsulated cells grow inside the capsuleswithout breaking and leaking out, whereas cell growth inside gel beads results in thedestruction of the gel structure. Products with a molecular weight which is higher than theexclusion limit ("cut-off" value), can be concentrated inside the capsule. Thispreconcentration of products within the capsule has been commercially exploited for theproduction of monoclonal antibodies. High intracapsular antibody concentration and puritygreatly facilitate downstream processing and purification. Damon Biotech (Needham Heights,MA, USA) has used the alginate-PLL method (patented as Encapcel™ process in 1978) forthe production of interferon (Jarvis and Grdina, 1983) and the large-scale production of both


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human and murine monoclonal antibodies of high purity and activity (Duff, 1985; Posillico,1986). Since then, other encapsulation methods have been used to produce monoclonalantibodies, such as alginate-PLL-alginate, alginate(/CMC)-PEI-PLL, alginate-chitosan,polyacrylate capsules, CMC-chitosan and collagen (cf. Table 38).

Recently, insect cell culture has received an increased amount of attention since these cellsare hosts for a class of viruses, the baculoviruses, which has been shown to be an excellentvector for genetic engineering (Luckow and Summers, 1987; Agathos et al., 1990; Shuler etal., 1990; Betenbaugh et al., 1992). Micro-encapsulation technology has been used to increasethe insect cell density and product concentration, and to facilitate product purification fromgrowth media. Spodoptera frugiperda insects cells, infected with a temperature-sensitivemutant of the Autographa californica nuclear polyhedrosis virus (AcNPV), were cultured inalginate-PLL-alginate micro-capsules (King et al., 1989; Goosen et al., 1993). The micro-capsules had a controlled membrane molecular weight cut-off and a low intracapsularalginate concentration. Cell culture experiments indicated that the intracapsular alginateconcentration appears to be a key factor in achieving good cell growth. It was possible toobtain intracapsular cell densities of 8 107 cells/ml capsules and virus concentrations to 109

IFU/ml capsules, and it was demonstrated that virtually all of the virus was retained withinthe capsules.

TABLE 38PRODUCTION BY MICRO-ENCAPSULATED ANIMAL CELLS

Product Cell Type Encapsulation ReferenceMethod

α1-acid glyco- Human hepatoma cells HEMA-MMA Uludag and Sefton, 1993a; protein (HepG2) Uludag et al., 1993a, b; 1994α1-antitrypsin Human hepatoma cells HEMA-MMA Uludag and Sefton, 1993a;

(HepG2) Uludag et al., 1993a, b; 1994Dopamine Rat pheochromocytoma Alginate-PLL- Winn et al., 1991a

cells (PC12) alginatePC12 (in vivo) Alginate-PLL- Winn et al., 1991b

alginatePC12 HEMA-MMA Sefton et al., 1992;

Uludag et al., 1993aEpidermal FR3T3 HEMA-MMA Uludag et al., 1993a, b growth factor (genetically engineered)Erythropoietin Rat kidney cells Alginate-PLL- Koo and Chang, 1993

alginateFibrinogen Human hepatoma cells HEMA-MMA Uludag and Sefton, 1993a;

(HepG2) Uludag et al., 1993a, b; 1994Haptaglobin Human hepatoma cells HEMA-MMA Uludag and Sefton, 1993a;

(HepG2) Uludag et al., 1993a, b


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Hepatic-stimu- Rat hepatocytes Alginate-PLL- Kashani and Chang, 1988; lating factor (in vivo) alginate 1991; Chang, 1990; 1993Hepatitis B sur- Fibroblast BHS 1 Alginate-PLL Yuan et al., 1990 face antigen (genetically engineered)Human growth Recombinant fibroblast Alginate-PLL- Chang et al., 1994 hormone alginateIgA Hybridoma (mouse, rat) Alginate-PLL Posillico, 1986IgG Hybridoma (mouse, rat) Alginate-PLL Posillico, 1986

Hybridoma (mouse Bugarski et al., 1989αAcNPV-II20)Hybridoma, CHO Collagen Ray et al., 1990a, bHybridoma Alginate-PEI- Hsu and Chu, 1992(murein, CT04) PLL;CMC/algi-

nate-PEI-PLLIgM Hybridoma (mouse, rat) Alginate-PLL Posillico, 1986

Hybridoma M2 Polyacrylate Gharapetian et al., 1986α-Interferon Lymphoblastoid cells Alginate-PLL Jarvis and Grdina, 1983β-Interferon Diploid fibroblast Alginate-PLL Jarvis and Grdina, 1983

(FS-4)Interleukin 2 Gibbon lymphoma HEMA-MMA Sefton et al., 1992;

(MLA-144) Uludag et al., 1993aMonoclonal Hybridoma Alginate-PLL Duff, 1985 antibody Hybridoma Alginate-PLL- King et al., 1987

(mouse AcVI-II20) alginateHybridoma (rat-mouse+ Alginate-PLL- Edmunds et al., 1989mouse-mouse) alginateHybridoma HP-6 Alginate-chitosan Overgaard et al., 1991Hybridoma HB-8852 CMC-chitosan Yoshioka et al., 1990

Nerve growth Rat fibroblasts Alginate-PLL Maysinger et al., 1993 factor (genetically engineered) alginateNuclear Polyhe- Spodoptera frugiperda Alginate-PLL- King et al., 1989 drosis Virus alginate (AcNPV ts-10)Prolactin Rat pituitary cells Agarose-acryla- Dupuy et al., 1991

mideUDP-glucoro- Hepatocytes (Wistar, Alginate-PLL Bruni and Chang, 1991 nyltransferase Sprague-Dawley, Buf-

falo rats; and Guinea pig)


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Animal cells have been grown in micro-capsules to evaluate new encapsulation materialsand methods (cf. Table 39). Optimisation of new methods is performed by evaluating theviability and growth of the encapsulated cells. In addition to the maintenance of viability, theencapsulated cells obviously have to retain their functional or differentiated state, i.e.synthesise and secrete specific biomolecules.

The majority of mammalian cell types, with the exception of some transformed tissueculture lines and blood-borne cells, do not grow in suspension but require attachment to asubstratum as a condition for growth (anchorage-dependent growth). Micro-carriers havebeen used for the growth of anchorage-dependent cells, even on a large scale. However, thisculture system is very susceptible to damage of the fragile cells. The use of porous micro-carriers represent a major improvement over conventional non porous micro-carriers, but stilldo not offer complete protection of the cells. Total protection of fragile cells can be obtainedby encapsulation in micro-capsules. Anchorage-dependent cells have been encapsulated inalginate-PLL or -PLO capsules (Young et al., 1989; Young, 1993). Substantial growth wasonly observed when attachment substratum (i.e. gelatin shards) was co-encapsulated with thecells (cf. Table 39).

Micro-encapsulation of semen has been used for artificial insemination in animals (e.g.Jöchle, 1993; Nebel et al. 1985; 1986; 1993; Watson, 1993; Nebel and Saacke, 1994).Successful artificial insemination in cattle depends on the correct identification of onset ofestrus; and correct timing and placing of sufficient numbers of viable sperm in the femalereproductive tract. There would also be an enhanced expectation of conception followingartificial insemination in non-domestic animals, because the detection of the receptive periodis still a major difficulty in many species. Encapsulated sperm is less susceptible to retrofluxaction of the uterus and to phagocytosis by leucocytes following insemination. Micro-encapsulation enable the slow release over an extended period of time, so that timing ofinsemination for high fertility is less stringent. Micro-encapsulation of sperm can also allowsperm to be sequentially exposed to different environments without the necessity ofpotentially injurious centrifugations and resuspensions. Several encapsulation methods havebeen evaluated as listed in Table 39. Sperm is immobilised in alginate with the addition of anextender (successful extenders include Cornell University Extender (CUE), CAPROGEN andegg yolk-citrate glycerol), to maintain spermatozoal viability (Nebel et al., 1993; Nebel andSaacke, 1994). These beads are then covered with poly-L-lysine, polyvinylamine orprotamine sulphate membranes. The alginate gel core is liquefied using sodium citrate.Encapsulation was successful with capsules ranging in size from 0.75 to 1.5 mm, and withsperm concentrations from 46 to 180 106 cell/ml. Capsule fragility (ability to rupture underageing and physical stress) is negatively related to membrane thickness which ranges from1.92 to 5.32 µm (depending on the concentration of polymer used) and positively related tothe encapsulated sperm concentration. Heterospermic studies have shown that encapsulatedsperm is capable of fertilisation in vivo, but is at a disadvantage to unencapsulated spermwhen cows are inseminated at conventional times. Uterine retention of inseminates isfavoured by capsules having a "sticky" polyvinylamine membrane. However, use ofpolyvinylamine as a capsule membrane is discouraged because it is not biodegradable. Using


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current procedures, preliminary homospermic fertility studies indicated that spermencapsulated with poly-L-lysine or protamine sulphate may achieve normal fertility.

TABLE 39GROWTH OF ANIMAL CELLS IN MICRO-CAPSULES

Cell Type Encapsulation ReferenceMethod

CHO fibroblast HEMA-MMA Uludag and Sefton, 1992; 1993bCHO-K1 Alginate-PLL/PLO + Young et al., 1989

gelatin shardsHaK Alginate-PLL/PLO + Young et al., 1989

gelatin shardsHepatocytes (rat, pig) CS/PDMDAACa Dautzenberg et al., 1995Human diploid fibroblast Eudragit RL Boag and Sefton, 1987Human hematopoietic cells Alginate-PLL-alginate Levee et al., 1994Human tumour cell lines Alginate-PLL/PLO Gorelik et al., 1987MDCK-ras-e Alginate-PLL-alginate Vandenbossche et al., 1994Tumourogenic MO4 and Alginate-PLL-alginate Vandenbossche et al., 1993b non-tumourogenic MO cellsSpermatozoa Alginate -PLL Nebel et al., 1985; Nebel and Saacke,

1994Alginate-polyvinyl- Nebel et al., 1986; Nebel and Saacke, amine 1994Alginate-protamine Munkittrick et al., 1992sulphate

a cellulose sulphate/poly(dimethyldiallylammonium chloride) polyelectrolyte complex

Micro-encapsulation technology has also been used as a tool in cancer research. Thegrowth of encapsulated established human tumour cell lines in the peritoneal cavity ofimmunocompetent animals has been demonstrated and this system was proposed as a modelfor the testing of chemotherapeutic agents in vivo (Gorelik et al., 1987; Chen et al., 1988).Recently, the role of E-cadherin in cancer development has been investigated using micro-encapsulation technology (Vandenbossche et al., 1994). The invasion-suppressor molecule E-cadherin mediates Ca2+-dependent cell aggregation and prevents invasion. E-cadherin-positive Madin-Darby canine kidney (MDCK) cells which were non-invasive in vitro, lost theE-cadherin invasion-suppressor function in vivo and formed invasive tumours uponintraperitoneal injection in mice. Such differences between cells in vitro and cells in vivoindicate that the expression is at least partly determined by the micro-ecosystem of which thecells are part (Mareel et al., 1990; Van Roy and Mareel, 1992). The elements of the micro-


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ecosystem modulating the expression and function of E-cadherin are unknown. To determinewhether the immediate contact between tumour cells and structural elements from the hostwould be needed for modulation of E-cadherin, MDCK-ras-e cells were micro-encapsulatedin alginate-PLL-alginate (Vandenbossche et al., 1994). Micro-encapsulation of intraperitonealinjected cells prevented the loss of the E-cadherin invasion-suppressor function, which led tothe conclusion that this loss in vivo was dependent upon immediate contacts between tumourcells and host cells or upon host factors that could cross the capsule membrane.

3.2.2.2 Microbial and Plant CellsMicro-encapsulation technology has only recently been evaluated as an alternative

immobilisation method using microbial or plant cells, as listed in Table 40. Nevertheless, theuse of micro-capsules instead of gel beads can be beneficial in certain applications. Micro-encapsulation prevents cell leakage, and immobilised cells can be used in media containingchemical agents detrimental for the mechanical stability of certain gel types, e.g. it has beendemonstrated that alginate gels containing lactic acid bacteria tend to be liquefied by lacticacid (Roy et al., 1987). Cell release makes downstream processing more laborious and isparticularly undesirable when the product is directly destined for consumption, as indenitrification of drinking water or dairy fermentations.

TABLE 40PRODUCTION OF BIOCHEMICALS BY MICRO-ENCAPSULATED MICROBIAL AND PLANT CELLS


Microbial cells:Citric acid Yarrowia lipolytica CS/PDMDAACa Förster et al., 1994

Y. lipolytica coimmobi- CS/PDMDAACa Mansfeld et al., 1995lised with invertase

Gluconic acid Gluconobacter oxydans Alginate-Eudragit Hartmeier and Heinrichs,coimmobilised with RL 1986catalase

Lactic acid Lactococcus lactis Alginate-PLL Larisch et al., 1994Chitosan Groboillot et al., 1993Gelatin Hyndman et al., 1993Nylon Larisch et al., 1994Polyethyleneimine Larisch et al., 1994

Reduced 1-phenyl- Yeast cells polyamide Green et al., 1995 1,2-propanedionePlant cells:Artificial seed Brassica napus Alginate-chitosan Tay et al., 1993Phenolics Nicotiana tabacum Alginate-PLL Haigh and Linden, 1989a cellulose sulphate/poly(dimethyldiallylammonium chloride) polyelectrolyte complex


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Encapsulation techniques based on interfacial cross-linking have been developed toimmobilise lactic acid bacteria (cf. Table 40). In the acidification of cream for the productionof cottage cheese dressing and in the manufacture of cultured butter, loss of cells from theimmobilised starter may result in continued acidification during storage (Champagne andCôté, 1987). Other applications include the synthesis of citrate from glucose byCS/PDMDAAC encapsulated Yarrowia lipolytica, where cell leakage of the filamentousyeast was strongly reduced (Förster et al., 1994); the production of phenolics by alginate-PLLencapsulated tobacco cells (Haigh and Linden, 1989); and artificial seed production byencapsulating Brassica napus embryoids in alginate-chitosan capsules (Tay et al., 1993).


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4. MASS TRANSFER AND MODELLING

The analysis of the influence of mass transfer on the reactor performance in immobilised-cellreactors is an important topic since the effectiveness of these reactors may often be reducedby the rate of transport of reactants to and products from the immobilised cell system(external mass transfer limitation), and by the rate of transport inside the immobilised cellsystem (internal mass transfer limitation). External mass transfer limitations can be reducedor eliminated by a proper design of the reactor and immobilised cell system. Severalphenomena are involved such as axial dispersion, convective flow, and macro- and micro-mixing. More information about these topics can be found in various (bio)chemicalengineering books (see Table 11 in Chapter 1 for a book list) and several reviews (e.g. Moo-Young and Blanch, 1981; Radovich, 1985b). Internal mass transfer limitations are often moredifficult to eliminate and their knowledge is a prerequisite to analyse the performance of theimmobilised cell system. Therefore, in this chapter, emphasis will be on diffusion which isthe major internal mass transfer mechanism in gel entrapment and micro-encapsulation.

Mathematical modelling of immobilised cell systems have been used to analyseexperimental data to get a better understanding of these systems (to obtain intrinsicparameters), to reduce and guide experimental work. These first principle models are basedon the mass and heat balances. Hydrodynamics, mass and heat transport, and kinetics andstoichiometry are needed to construct the mass balances. The discussion will be limited to theimmobilised cell system. The heat balance may often be ignored on the level of the cellaggregate since the aqueous phase in biological systems has a large heat capacity, goodconductivity and the heat generation is low. The hydrodynamics plays a role on the level ofthe bioreactor and is similar to what occurs in heterogeneous chemical reactors. Since themass transport can have a serious impact upon the overall kinetics, attention will be paid onthe interpretation of kinetic data. An overview of dynamic, pseudo-steady-state and steady-state models is presented.

4.1 Diffusion in Immobilised Cell Systems

4.1.1 Definitions of Diffusion Coefficients

Mass transport by molecular diffusion is defined by Fick's law, i.e. the rate of transfer ofthe diffusing substance through a unit area is proportional to the concentration gradientmeasured normal to the section:

J = −D∂C∂x

(1)

where J is the mass transfer rate per unit area of section, C the concentration of diffusingsubstance (amount per total volume of the system), x the space co-ordinate, and D is calledthe diffusion coefficient. It is general practice to use an effective diffusion coefficient (De),which can be readily used in the expression for the Thiele modulus and for the determination


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of the efficiency factor of a porous biocatalyst. This effective diffusion coefficient can bedefined as

J = −De∂CL∂x

(2)

where CL is the amount of solute per unit volume of the liquid void phase. Concentration Cmay be correlated with CL by using the void fraction (ε) which is the accessible fraction ofthe porous particle to the diffusion solute as

C = εCL (3)

Hence, the relationship between the effective diffusion coefficient and the diffusioncoefficient can be written as

De = εD (4)

In the (bio)chemical engineering literature however, alternative definitions of the termeffective diffusion coefficients have been introduced (e.g. Furusaki and Seki, 1985; Chresandet al., 1988; Libicki et al., 1988; Pu and Yang, 1988; Sakaki et al., 1988; Scott et al., 1989).

4.1.2 Diffusion through Support Materials

The effective diffusion coefficient through a porous support material (matrix) is lower thanthe corresponding diffusion coefficient in the aqueous phase (Da) due to the exclusion andobstruction effect. By the presence of the support, a fraction of the total volume (1-ε) isexcluded for the diffusing solute. The impermeable support material obstructs the movementof the solute and results in a longer diffusional path length which can be represented by atortuosity factor (τ). The influence of both effects on the effective diffusion coefficient can berepresented by

De =ετDa (5)

This equation holds as long as there is no specific interaction of the diffusion species with theporous carrier. In the case of gel matrices, predictions using the polymer volume fractions arerecommended, since ε nor τ can be measured for a gel in a simple way (Mackie and Meares,1955; Brown and Johnsen, 1981; Muhr and Blanshard, 1982):

D =(1 − φ p)2

(1 + φ p)2 Da (6)


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where φp is the polymer volume fraction. For low molecular weight solutes in cell-free gels,an approximate measure of ε can be given as

ε = 1 − φ p (7)

De can also be expressed as a function φp by combining Equation (4) and (7) (Mackie andMeares, 1953; Westrin and Axelsson, 1991; Beuling et al., 1995) as

De =(1− φ p )3

(1+ φp )2 Da (8)

4.1.2.1 Methods of MeasurementBoth steady-state and transient methods for measurements of diffusion coefficients are

used, sometimes in combination as in the lag-time method. A few researchers have usedindirect methods, such as the use of a reaction-diffusion model, to determine the diffusioncoefficient of gel immobilised cells (Hiemstra et al., 1983; Adlercreutz, 1986; Kurosawa etal., 1989a). Westrin and co-workers (1994) have recently reviewed in depth the methods formeasuring diffusion coefficients. They compared different meyhods (including holographiclaser interferometry and nuclear magnetic resonance) with regard to accuracy, reproducibility,time, cost and limitations.

The equation which describes the transient diffusion can be readily derived by writing themass balance over the system:

ε∂CL∂ t

= x −n ∂∂x

(xnDe∂CL∂x

) (9)

where n is a shape factor which is 1 for planar, 2 for cylindrical or 3 for spherical geometry.The four most popular transient methods can be described as (1) "diffusion out of a matrixinto an infinite solution", (2) "diffusion into a matrix from a finite solution", (3)"diffusioninto a matrix from an infinite solution", and (4) "lag-time method using a two chamberdevice". Other methods used are "measurements using an electrode covered by the supportingmaterial" (e.g. polymeric gel) (Renneberg et al., 1988), "measurements using a massspectrometric membrane inlet covered by the matrix material" (Willaert, 1993) and the use ofmicro-electrodes to measure in the gel phase or biofilm (Beuling et al., 1995; Ottengraf andvan den Heuvel, 1996).

Experimentally, a concentration disturbance is applied over the system and the change ofthe concentration as a function of time is followed until the steady-state is reached. Diffusioncoefficients are determined by fitting the theoretical curve to the experimental one, which isobtained after solving Equation (9) using the appropriate initial and boundary conditions.Analytical solutions for simple cases are described by Crank (1975). Crank gives also thesolution in terms of Mt, the amount of solute in the matrix at time t, as a fraction of M∞, theequilibrium amount, which is practically more accessible.


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Steady-state measurements can be performed by using a "diaphragm-diffusion cell". Twoliquid-filled well-stirred compartments are separated by a membrane under investigation,through which a steady-state diffusional flux is set up (Hannoun and Stephanopoulos, 1986;Axelsson and Persson, 1988; Sun et al., 1989; Mignot and Junter, 1990a, b; Axelsson andWestrin, 1991). In the steady-state method, a constant upstream and different downstreamsolute concentration is employed. Whereas in the pseudo-steady-state method, a change insolute concentration in the compartments is allowed.

If the diffusion coefficient is measured by the steady-state measurement, the value of theequilibrium partition coefficient (K) should be known . Another approach is to determinedirectly the product of K and D, which equals the effective diffusion coefficient (Westrin,1990). Sun and co-workers (1989) used a non steady-state method to determine immediatelythe product of K and De for oxygen in cell-free and cell-containing Ca-alginate and PVA-SbQ gels. The partition coefficient is defined as

K =CmCL

(10)

where Cm is the solute concentration in the matrix (amount per volume matrix) and CL thesolute concentration in the free (surrounding) liquid phase. In the absence of specificadsorption phenomena, the partition coefficient for cell-containing matrices can be predictedby the following equation (Axelsson and Persson, 1988; Westrin 1990; Westrin andAxelsson, 1991)

K = 1 − (αφc + φp ) (11)

where α represents the volume fraction of the individual cell that is not accessible to thesolute. If the outer cell membrane totally excludes the solute, the value of α will be unity.Axelsson and Persson (1988) found a value of 1 for glucose, lactose, galactose and ethanol(Axelsson, 1990) for alginate gels containing yeast cells deactivated by iodoacetic acid.Chresand and co-workers (1988) measured the partitioning coefficient of labelled sodiumacetate between 1% (w/w) agar gel containing mammalian cells and the surrounding solution,and found a value which was unity. This result corresponds with a value of zero for α . It canalso be assumed that the solute is excluded from a certain volume fraction of the individualcell: 0 < α < 1. This volume fraction may, e.g., be approximately equal to the dry weightfraction of the cells, which can be assumed to be equal to 0.25 (Westrin and Axelsson, 1991).If there are no immobilised cells present, Equation (11) is reduced to 1-φp which equals ε (cf.Equation 7). Values of experimentally determined partition coefficients for cell-free gels wereclose to unity; e.g. between 0.97 and 1.02 for glucose in 2% Ca-alginate (Merchant et al.,1987); 0.98 for lactose in 2.75%/0.25% (w/w) κ-carrageenan/locust bean gum (Arnaud andLacroix, 1991); 1.2 for lactose, 1.3 for glucose, 1.1 for galactose and 1.32 for ethanol in 2.4-2.8% (w/w) alginate, the higher value for ethanol was explained as adsorption of ethanol onthe alginate gel; 1.14 for catechol, 0.99 for L-DOPA and 2.03 for dopamine in cross-linkedgelatin (10% w/v) beads at pH 7.1 and 35˚C (Anderson et al., 1992).


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In measuring solute diffusivities, it is important to ensure that the external overall masstransfer resistance is negligible. In the case of permeable spheres, the effect of the externalmass transfer resistance on the overall uptake and/or release rate by the beads may bequantitatively evaluated by calculating the time constant for the external film (τe), and tocompare it to the time constant for diffusion in the sphere (τi) (Willaert, 1993). The internaltime constant can be calculated as (Glueckauf, 1955)

τi =R2

15De (12)

where R is the radius of the bead. Alternatively, according to the film theory, the filmthickness can be estimated (see e.g. Nguyen and Luong, 1986). The external mass transferresistance can also be neglected if the Biot number (Bi) is much larger than one (Arnaud andLacroix, 1991). Bi for beads is defined as

Bi = ksRDe

(13)

An estimation of the external mass transfer coefficient (ks) is required to calculate τe, Bi orthe film thickness. The value of ks can be calculated by a procedure recommended by Harriot(Sherwood et al., 1975). Merchant and co-workers (1987) determined Bi for a rotating sphere.Using the empirical correlation of Noordsij and Rotte (1967), ks could be estimated:

Sh = 10 + 0.43Rer1/2 Sc1/3 (14)

where Sh is the Sherwood number, Rer the rotational number of Reynold and Sc the Schmidtnumber. In the case of diffusion through a membrane or thin disc, Bi can also be calculated.An estimation of ks can be calculated if the stirred chambers have the shape of flat cylinders(De Backer et al., 1992) using the following correlation (Sherwood et al., 1975)

ks = 0.62Da2/3ν−1/6ω 1/2 (15)

where ν is the cinematic viscosity and ω the rotational speed of the stirrer (in rad/s). Othercorrelations can be adapted from heat transfer correlations (Axelsson and Westrin, 1991). Onthe other hand, the external mass transfer limitation can be experimentally investigated byobserving the concentration-time profile at different rotation speeds of the stirrer (Sun et al.,1989).

4.1.2.2 Diffusion in Cell-containing MatricesThe presence of the immobilised cells in the immobilisation matrix can have a substantial

effect on the effective diffusion coefficient, especially at high biomass concentrations. Also,the effective diffusion coefficient decreases as a function of time in growing immobilisedsystems because the cell volume fraction increases. Diffusion in gels containing immobilised


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cells has been recently reviewed by Westrin and Axelsson (1991). The influence of the cellson the effective diffusion coefficient can be described as

De = f (φc ,Dc )De0 (16)

where Dc is the effective diffusion coefficient within the cells and De0 the effective diffusioncoefficient in a cell free matrix. Two theoretical approaches have been developed: (1) thecells are impermeable to the diffusing solute or (2) the solute diffuses in the cells with a verylow De. Suitable expressions have been derived for f(φc, Dc) which were based onheterogeneous media (e.g. Neale and Nader, 1973; Akanni et al., 1987). Expressions could beclassified in five categories:

(1) The exclusion model; assuming Dc to be zero. The presence of the impermeablecells reduces the volume available for diffusion. Equation (16) can now be written as

De = (1− φc )De0 (17)

Using this equation, effective diffusion coefficients have been determined for substratediffusion in calcium alginate gel with entrapped deactivated yeast cells (Axelsson andPersson, 1988; De Backer et al., 1992).

(2) Models of suspended impermeable spheres, regarding the cells as impermeablespheres (Dc equals zero) which are suspended in a continuum. The expression for this modelhas been adapted from the expression derived by Maxwell (1892) for effective conductivityin a composite medium of periodically spaced spheres:

De =1− φc

1+ (φc / 2)De0 (18)

This equation has been used to correlate experimental diffusion coefficients with immobilisedmammalian cells in agar gel (Chresand et al., 1988) and in artificial biofilms (agar containinginert polystyrene particles of the same size as bacteria) (Beuling et al., 1995).

(3) Models of suspended permeable spheres, assuming permeable cells (Dc > 0). Inthis case, a more complex expression (adapted from Maxwell, 1881) has been derived:

De =2 / Dc + 1 / De0 − 2φc (1/ Dc − 1 / De0)2 / Dc + 1 / De0 + φc (1 / Dc − 1 / De0)

De0 (19)

Values for Dc/Da have been determined to be 0.31, 0.30 and 0.20 for fermentation media ofSaccharomyces cerevisiae, Escherichia coli and Penicillium chrysogenum, respectively (Hoand Ju, 1988). Equation (19) has been used to correlate experimental diffusion coefficients incell-containing gels (Chresand et al., 1988) and dense cell suspensions (Ho and Ju, 1988;Libicki et al., 1988).

(4) Capillary models, which have been developed to quantify the influence of thetortuosity on the effective diffusion coefficient, i.e. the model of Kozeny (e.g. Coulson andRichardson, 1978) and the improved random-pore model (Wakao and Smith, 1962). This


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model has been adapted to immobilised cell systems where cells are considered asimpermeable (i.e. Dc = 0):

De = (1− φc )2De0 (20)

Several investigators have used this equation to correlate the effective diffusion coefficientwith the immobilised cell concentration (Klein and Schara, 1981; Furusaki and Seki, 1985;Sakaki et al., 1988; Sun et al., 1989).

(5) Empirical models. Prediction equations have been obtained by fitting somearbitrary function to experimental data (e.g. Klein and Manecke, 1982; Scott et al., 1989).

Some researchers have reported that the diffusivity was not influenced by the presence ofcells. Kurosawa and co-workers (1989a) found that the oxygen diffusivity in Ca- and Ba-alginate was not influenced by the presence of immobilised yeast cells up to a cell density of30 gDW/l. Estapé et al. (1992) studied the influence of counter-diffusion of ethanol andglucose on the diffusion coefficient of glucose and ethanol respectively: there was nosignificant decrease of the diffusion coefficients in the presence of yeast cells when counter-diffusion was involved; without counter-diffusion the presence of cells resulted in asignificant decrease of the diffusion coefficient for ethanol, but in contrast an increase wasobserved for the glucose diffusion coefficient.

In the determination of the effective diffusion coefficient in cell-containing matrices bymethods based solely on diffusion, the solute may not react, or erroneous results would beobtained. In the literature, different methods have been described to deactivate living cells.Cells have been deactivated using ultraviolet radiation (Matson and Characklis, 1976); heat(Matsunaga et al., 1980a; Dibdin, 1981; Tatevossian, 1985a, b; Sun et al., 1989; Mignot andJunter, 1990a; Cronenberg, 1994); organic solvents like ethanol (Hannoun andStephanopoulos, 1986; De Backer et al., 1992), chloroform (Cronenberg, 1994) andglutaraldehyde (McNee et al., 1982; Chresand et al., 1988; Cronenberg, 1994); organic acidslike iodoacetic acid (Axelsson and Persson, 1988); toxins or inhibitors like mercuric chloride(Matson and Characklis, 1976; Cronenberg, 1994; Beuling et al., 1995), dimethyl sulfoxide(Pu and Yang, 1988) and sodium azide (Arnaud and Lacroix; 1991); detergents, e.g. TritonX-100 (Libicki et al., 1988). Some researchers have investigated the effect of the deactivatingagents on the effective diffusion coefficient. Matson (1975) found that the deactivationprocess by mercuric chloride or heat had no effect on the diffusion process. The results ofDibdin (1981) and McNee and co-workers (1982) suggest that neither heat treatment norglutaraldehyde fixation have significant effect on the rate of diffusion through dental plaque.In contrast, Tatevossian (1985a, b) found a significant effect by using heat deactivation.Libicki and co-workers (1988) found that the mass transport of nitrous oxide in aggregates ofE. coli prepared from cells treated with detergent or disrupted by dehydration and grindingdiffered only slightly from the values obtained for aggregates formed from treated cells. Puand Yang (1988) observed that the permeabilisation of apple cells with dimethyl sulfoxide ledto an increase in effective diffusivity. Beuling et al. (1995) showed that glucose was excluded


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by deactivated (with mercuric chloride) Micrococcus luteus cells entrapped in agar, while thecells were somewhat permeable for oxygen.

Another strategy is to select a solute that will not be consumed by the micro-organism, e.g.nitrous oxide (Libicki et al., 1986; 1988), inert gases (H2, He, CH4, C2H2,N2O, CHClF2, SF6)(Kawashiro et al., 1975), yohimbine which is a secondary metabolite of plant cells (Pu andYang, 1988); potassium chloride (Mignot and Junter, 1990b), galactose which is notmetabolised by Zymomonas mobilis (Sakaki et al., 1988).

4.1.2.3 Diffusion in GelsA lot of research has been performed on the diffusion in polymeric gels with and without

encapsulated cells. Results of measurements of diffusion coefficients in cell-free and cell-containing gels are presented in Table 41.

Size-exclusion chromatography (SEC) has been used to study the porosity of alginate gels(Klein et al., 1983; Smidsrød, 1974; Stewart and Swaisgood, 1993). Smidsrød (1974) studiedthe porosity of alginate gels using scanning electron microscopy and found that there was avery broad distribution in pore diameter ranging from 5 to 200 nm. Globular proteins withradii of gyration of approximately 3 nm and with net negative charges should therefore not betrapped in the alginate network, but be able to diffuse at a rate dependent upon their sizes(Martinsen et al., 1989). In contrast, Klein et al. (1983) showed, using SEC and dextrans ofknown size, that the pore diameters in Ca-alginate gels were fairly uniform. The exclusionvolumes for standard macromolecules were recorded and values of 6.8, 14.1 and 16.6 nmwere estimated for 3 different alginates. SEC with 2% Ca-alginate beads using proteins ofknown Stokes radii indicated a pore diameter of 8-10 nm when eluents of 30 and 150 mMCaCl2 were used at pH 6.2 (Stewart and Swaisgood, 1993). The whey proteins α -lactalbumine and β-lactoglobulin both easily penetrated the alginate pores and no proteinshad complete access to the total internal volume of the matrix.

Polakovic (1995) used SEC to evaluate the pore size distribution of 5% Ca-pectate gel andpredicted — using a cylindrical and slit-pore model — the pore diameters of 50 and 35 nmrespectively.

From pore-size distribution studies using electron microscopy (Andresen et al., 1977), itwas suggested that there is a more constricted network on the bead surface than in the gelcore. Skjåk-Bræk and co-workers (1989) found that the alginate structure is governed notonly by the concentration and chemical structure of the gel material, but also by the kineticsof the gel formation. They showed that gels with varying degrees of anisotropy(heterogeneity) can be prepared by controlling the kinetics of the process. Therefore, it isdifficult to compare the different diffusion coefficients reported by different workers becauseof the considerable variations in the experimental conditions of preparing alginate gels. Whengels were formed in the presence of anti-gelling cation, such as Na+ or Mg2+, isogeneous Ca-alginate gels were obtained (Skjåk-Bræk et al., 1986a). Such beads were mechanicallystronger and had higher porosity than those formed in the absence of anti-gelling ions. Kleinand co-workers (1983) have reported that glucoamylase is retained for many days in ferric-alginate gel, although it is lost from Ca-alginate or aluminium alginate beads. Gray and


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Dowsett (1988) showed that insulin may be entrapped in zinc alginate and zinc-calciumalginate gels.

Studies on the diffusion of NAD and hemoglobin in calcium and barium alginate gelsshowed that NAD diffusion characteristics are unaffected by alginate (from 2.5% to 4% w/v)and calcium chloride (from 0.125 to 0.5 M) concentrations; however, hemoglobin diffusionwas affected by the alginate concentration (Kierstan et al., 1982). Hannoun andStephanopoulos (1986) demonstrated that D for ethanol and glucose decreased when thealginate concentration was increased from 1 to 4 % (w/v). Itamunoala (1987) showed that byincreasing the concentration of either the calcium chloride (1-4 % w/v) or sodium alginate (2-8% w/v) component used in calcium alginate formation substantially decreased De, but theeffect of the alginate was the greater of the two. A slight reduction of the product of K and Defor oxygen was found when the alginate concentration was increased from 2 to 4% (w/v) bySun and co-workers (1989). In contrast, Tanaka et al. (1984d) demonstrated that the diffusionof solutes with a molecular weight less than 2 104 (glucose, L-tryptophan and α-lactoalbumin) was not disturbed by increasing the alginate concentration (2-4% w/v) andCaCl2 concentration used in the gel preparation; and diffusion coefficients comparable withthose in water were found. For larger molecules such as albumin, γ-globulins and fibrinogen,the diffusion in the gel was retarded to an extent which depended upon the concentration ofalginate and calcium chloride concentration. Moreover, these proteins could diffuse out of,but not into the beads. Therefore, it was suggested that the structure of Ca-alginate gel formedin the presence of large protein molecules was different from that of the gels formed in theirabsence. Axelsson and Persson (1988) found also that the dependence of De on the alginateconcentration (in the range 1.4-3.8% w/w) for glucose, lactose, galactose and ethanol wasnegligible. Hulst and co-workers (1989) examined the influence of the concentration of Ca-alginate, gellan gum, κ-carrageenan, agarose and agar on De for oxygen. For agarose andagar, De decreased when the gel concentration was increased from 2 to 8 % (w/v). Gellan gumand alginate showed a remarkable maximum value for the diffusion coefficient with respectto the gel concentration: at 1 and 2% (w/v) respectively. κ-Carrageenan had a maximum at 5% (w/v) in the concentration range of 1 to 5% (w/v). The diffusivity of Cu2+ in Ca-alginateincreased by a factor of two when the gel concentration was increased from 2 to 5% (w/v)(Lewandowski and Roe, 1994; Jang, 1994).

The diffusivity of glucose in κ-carrageenan was affected by the presence of other solutes inthe glucose solution (Nguyen and Luong, 1986): electrolytes such as ammonium sulphate,potassium chloride and calcium chloride were observed to enhance the diffusion coefficient.Estapé et al. (1992) demonstrated that de glucose and ethanol effective diffusion coefficientswere significantly reduced in the presence of counter-diffusion of ethanol and glucose,respectively.

Renneberg and co-workers (1988) investigated the influence of different variables on theoxygen diffusivity in gels derived from prepolymers. A series of ENT-type (prepared fromhydroxyethylacrylate) hydrophilic polymers showed a steady increase in water content anddiffusivity with increasing chain length of the prepolymers from 10 nm to 60 nm. The slightlyhigher diffusivity of the anionic ENT-type polymer compared with the cationic seemed to bedue to the lower water content rather than to the electronic charges. The polyvinyl-alcohol


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stilbazolium (PVA-SbQ) gels have a higher water content and also a higher diffusivity thanthe polymers of ENT-type; the polymers with 22 nm distance between the photo-functionalgroups showed a higher O2 diffusion than the polymers with 6.6 and 6.5 nm distance. ThePVA-SbQ 1800-100 with the highest polymerisation degree (1800) and the highestsaponification (100%) had the highest water content (94.2%) and the highest O2 diffusivity.

The influence of the temperature on the glucose diffusion in 3% (w/v) κ-carrageenan hasbeen investigated in a temperature interval between 10 and 33˚C (Nguyen and Luong, 1986).The diffusivity remained unchanged in the interval from 10 to 25˚C; from 25 to 32˚C, thediffusivity increased linearly from 3.73 10-10 to 6.10 10-10 m2/s. This behaviour wasexplained by the fact that the diffusion in the gel system is governed by the bead pore size aswell as by the viscosity of the gel. The κ-carrageenan gel viscosity remains unchanged attemperatures below 20˚C, and the viscosity decreases when the temperature increases above25˚C. The diffusion coefficient of glucose, lactate, hydroquinone and urea in 2% collagen and1% agar increased when the temperature was raised from 25˚C to 37˚C (Chresand et al.,1988). The temperature dependence of glucose in 3% (w/v) Ca-alginate followed theArrhenius relation with an activation energy of 4350 J/mol (Teo et al., 1986). Martinsen et al.(1992) calculated the activation energy as 23.5 kJ/mol for the Arrhenius temperaturedependence of the diffusion of albumin in 4% (w/v) Ca-alginate.

The diffusion of solutes in alginate gel is also affected by the chemical composition of thealginate. Martinsen and co-workers (1992) showed that the diffusion coefficient of albumin in4% Laminaria digitata, Macrocyctis pyrifera and L. hyperborea Ca-alginate gels decreasedwith decreasing content of guluronic acid and with any decrease in the average length of theguluronic acid (GG) blocks. Also, Itamunoala (1987) found a glucose diffusion coefficientwhich was slightly higher for Manutex RS gel compared to Manutex RH gel.

Because the alginate gel matrix is negatively charged, the pH influences the diffusion ofcharged substrates and products. Most proteins are negatively charged at pH 7 and will noteasily diffuse into the gel matrix. However, when immobilised in the gel, they tend to leakout more rapidly than would be expected from their free molecular diffusion. The rate ofdiffusion of bovine serum albumin out of alginate beads increased with increasing pH, due tothe increased negative charge of the protein (Martinsen at el., 1992).

Teo et al. (1986) found that the effective diffusion coefficient of glucose in 3% (w/v) Ca-alginate is independent of the glucose concentration over the range from 10 to 150 g/l.Concentrations of glucose in the range 2-100 g/l and ethanol in the range of 10-80 g/l did notaffect their diffusion coefficients in Ca-alginate (Hannoun and Stephanopoulos, 1986). Incontrast, Itamunoala (1987) showed that only at low glucose concentrations was the glucosediffusivity in Ca-alginate higher. The diffusion of glucose in κ-carrageenan decreased whenthe glucose concentration was increased from 6.5 to 65 g/l (Nguyen and Luong, 1986).

Most of the reported effects can be explained on the basis of: change in tortuosity τ orporosity ε; change in partition coefficient; interaction of large molecules with the polymernetwork of the matrix; interaction between different diffusing solutes; and effect of thetemperature.


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4.1.2.4 Diffusion in Micro-capsulesThe permeable membrane of a micro-capsule introduces a supplementary mass transfer

barrier compared to gel beads. The core of the micro-capsule can be composed of cells in aliquid solution, cells in gel phase, or a combination of both. In the latter case, three masstransfer layers are present. A model for the transient diffusion of proteins from a bulk solutioninto micro-capsules has been recently developed (Kwok et al., 1991; Yuet et al., 1993). It wasused to describe the experimental concentration profiles and to determine the membranediffusion coefficient of alginate PLL-alginate capsules. By introducing an effective volumefraction as parameter in the model, micro-capsules with different sizes of impermeable gelcores could be studied. It was found that the ability of proteins to diffuse into the capsule wasnot only controlled by the membrane diffusivity, but also by the amount of Ca-alginate coreremaining in the micro-capsule.

The permeability of the capsule membrane is often characterised by the molecular weightcut-off value (MW cut-off). Application of micro-capsules for immunoisolation required amembrane with a MW cut-off of around 50000 to prevent the permeability of cytotoxicantibodies (Christenson et al., 1993). The permeability of a membrane for a certain compoundis not only dependent on the pore size and structure but also on other physical characteristicssuch as the hydrophobicity/hydrophylicity and charge, and the nature of the solute molecule(e.g. electrostatic interactions between proteins and synthetic polymers).

By manipulating the parameters involved in membrane manufacturing such as themolecular weight and concentration of the membrane forming molecules, the reaction time,polymerisation conditions (e.g. temperature, pH, ionic strength), addition of (an) additionallayer(s) at the outside of the capsule (usually to improve the biocompatibility). Permeationcan also be influenced by changing size, swelling and shape of the capsules (Okhamafe andGoosen, 1993). These parameters were reviewed in Chapter 3 by the discussion of the variousencapsulation materials and methods.

4.2 Modelling

4.2.1 Gel Immobilised Cell Systems

4.2.1.1 IntroductionFundamental engineering techniques and formalisms developed in the chemical

engineering literature for describing combined reaction and mass transfer have been appliedto immobilised cell systems to analyse their performance and behaviour. The ability to predictthe behaviour of these systems is required for the understanding, the design and optimisationof immobilised cell reactors. Several aspects are involved in modelling fermentationprocesses (cf. Figure 2). It is necessary to consider both the bioreactor performance and themicrobial kinetics (Nielsen and Villadsen, 1992). Description of the bioreactor performanceinvolves modelling of mass transfer effects and the flow pattern in both gas and liquid phaseswhile microbial kinetics deals both with the kinetics on the individual cell level and on thelevel on the whole cell population. The single cell kinetics can be described either with an


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unstructured model (no intracellular components considered) or with a structured model(intracellular components considered). The population model may either be unstructured (allcells in the whole population assumed to be identical, i.e. only one morphological form), ormorphologically structured (with an infinite number of morphological forms the termsegregated population model is often used (Bailey and Ollis, 1986)).

MODELLING OF MICROBIAL PROCESSES

BIOREACTOR PERFORMANCE

MICROBIAL KINETICS

MASS TRANSFER

Unstructured

MorphologicallyStructured

CELL MODEL POPULATION MODEL

Structured

MorphologicallyUnstructured

FLOW PATTERN

Fig. 2: Aspects to consider in modelling of fermentation processes (Nielsen and Villadsen,1992).

The models describing immobilised cell behaviour are normally of the unstructured type.However, Monbouquette and Ollis (1986, 1988b) have constructed a structured model tosimulate steady-state substrate diffusion and consumption in porous, cell-laden matrices. Anappropriate two-compartment structural representation of biomass was incorporated in orderto predict the intracellular RNA concentration with depth in a cell support. The modelmaintained the distinction between abiotic and biotic volumes in its description of effectivediffusivity and limiting substrate uptake, and explicitly described phenomena observedexperimentally such as the thickness of a metabolically active cell layer and leakage ofbiomass from a support at a rate commensurate with immobilised microbial growth.Nevertheless, the predictions of the structured model were not experimentally verified.

Initially, models which described immobilised cell kinetics were based on the steady-statemodels for immobilised enzymes. Steady-state models can give valuable information fordesign purposes, but fail to describe transient phenomena (like the start-up dynamics andresponse to changing conditions in the reactor) encountered in growing immobilised cellsystems. Therefore, dynamic models have been developed to simulate the transient behaviourof growing immobilised cells.

In general, gel immobilised cell systems are considered as effective continua. However, ithas been observed that when gel beads are inoculated with a low cell concentration, eachgrowing cell will be the origin of a micro-colony and growth results in the formation ofexpanding micro-colonies (e.g. Bailliez et al., 1985; Salmon and Robertson, 1987; Salmon,


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1989; Wijffels and Tramper, 1989; Willaert and Baron, 1993). Due to mass transportlimitations in the gel, colonies near the surface of the support material grow faster than thecolonies near the centre. Further growth of large spherical micro-colonies give rise tostretched micro-colonies (egg shaped) due to mass transfer limitations in the colonies.Moreover, Hilge-Rotmann and Rehm (1990) have shown that S. cerevisiae cells grown in theform of micro-colonies in alginate beads possessed increased specific hexokinase andphosphofructokinase activities compared to immobilised single and free-living cells. Arigorous modelling approach of this micro-colonies system requires consideration of themicro-structure of the immobilised cell system: diffusion in the gel phase, and reaction anddiffusion in the micro-colony ("two-phase" system).

4.2.1.2 Intrinsic KineticsIntrinsic kinetics describe the growth and product formation rates of cells in the

immobilised (or free) state as a function of the local concentrations. A typically simpleunstructured model of microbial kinetics for growth on a single substrate can be described bythe following 3 equations:

µ = f (s) (growth of biomass) (21)

qs =1

YX /Sµ +ms (consumption of substrate) (22)

qp =1

YX/Pµ +mp (formation of metabolic product) (23)

where µ is the specific growth rate of the cells (gDW formed/gDW/h); qs is the specificsubstrate utilisation rate (g substrate used/gDW/h); qp is the specific product formation rate (gproduct formed/gDW/h); YX/S (gDW/g substrate) and YX/P (gDW/g product) are the yieldcoefficients; ms (g substrate/gDW/h) and mp (g product/gDW/h) are the specific maintenancerates for substrate and product, respectively. In some cases, these maintenance coefficientsmay be omitted or combined with a "cell death coefficient". µ = f (s ) is a function of thesubstrate concentration and is usually of the Monod kinetics form:

µ = µmaxCS

KS + CS (24)

where µmax is the maximum specific growth rate, Ks the so-called Monod constant and CS thesubstrate concentration. The model can also be extended to include growth inhibition by theproduct (and biomass), and f(s) becomes some function of substrate and product (andbiomass) (for a review of kinetic models for inhibition, cf. e.g. Han and Levenspiel, 1988).The Monod equation is bound by zero order (at high substrate concentrations relative to KS)and first order (at vanishingly small substrate concentrations) kinetics. The solutions ofreaction-diffusion problems with these two simple rate equations are valuable in that they can


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be applied as lower or upper bounds to the general problem without requiring detailedknowledge of the rate expressions and thus considerably facilitating the calculations.

In the interpretation of kinetic data for immobilised cells, it is important to assess thesignificance of mass transfer limitations. If negligible mass transfer limitation is present, theexternally observed kinetics are the intrinsic cell kinetics. Any external or internal masstransfer limitation will lead to externally observed lower conversion rates. Mass transferlimitations may appear either in the external film around the support matrix or within the gelmatrix, or in both.

A variety of claims have been made regarding changes in the intrinsic growth rate ofimmobilised cells, primarily regarding cells adhering to a surface. It has been asserted that thegrowth rate for immobilised cells is much higher than that for free-living cells (Karel andRobertson, 1989a, b). For gel immobilised cell systems, it has been observed that themetabolic rates of gel immobilised cells depend only on the local solution concentrations andare identical to those for free cells if diffusional limitations are absent; although some reportsshowed a decreased growth rate upon entrapment. As can be noticed in Table 42, someresearchers found no significant difference between the maximum specific growth rates forimmobilised yeast and bacteria, and those for free cells. Also, specific growth rates (over apH range from 4.3 to 5.5) of Lactobacillus helveticus entrapped in κ-carrageenan-locust beangum beads were close to those determined in free-cell batch fermentations (Norton et al.,1994a, b). On the contrary, a significant decrease has been noted by other researchers for thesame micro-organisms. This change of metabolic activity upon immobilisation may be due todiffusional limitations or a change of cellular physiology (cf. Chapter 2: 2.2 Physiology ofGel Immobilised Cells).

4.2.1.3 ModellingBy the entrapment of cells in gel matrices, an additional barrier to mass transfer relative to

free cells is introduced. This tends to lower the overall reaction rate, as well as creating aspecific micro-environment around the cells. Immobilised cells can grow in the gel matrixand the mass transfer limitations on substrate delivery and product removal lead to time-dependent spatial variations in growth rates and biomass densities, which may beaccompanied by alterations in cellular physiology and biocatalytic activity. Since the localeffective diffusion coefficient depends on the local biomass density, this non homogeneousgrowth will influence the local diffusive rates. The existence of chemical environmentalgradients in immobilised cell systems has been verified experimentally with variousmicroprobe techniques.

Many researchers have also investigated qualitatively the development of resultant spatialvariations in cell loading through microscopic examination of the biocatalyst support crosssections (cf. Chapter 2: 2.2.1 Physiology and Experimental Techniques). These studiesdemonstrate the eventual formation of a densely packed cell layer adjacent to the immobilisedcell-bulk liquid interface. This characteristic layer arises due to mass transfer limitationswhich restrict the growth of deeper lying cells. The global kinetics of substrate consumptionand product formation, the leakage of cells from the immobilisation matrix are all dependenton the spatial and temporal dynamics of the immobilised cell population.


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TABLE 42GROWTH RATES OF GEL IMMOBILISED CELLS COMPARED TO THOSE OF FREE CELLS

Micro-organism Gel System Specific Growth Rate Reference(% w/v) µI (h-1) µS (h-1)

Escherichia coli Carrageenan (2%) 2.04a 2.08a Marin-Iniesta et al. ,19881.69b 1.63b

E. coli B/pTG201 Carrageenan (2%) 0.24c 0.30 Hooijmans et al., 1990a0.18d

Carrageenan (2%) 0.18 0.36 Huang et al., 1990Saccharomyces Ca-alginate (1.5%) 0.115e 0.126e Agrawal and Jain ,1986 cerevisiae Carrageenan (2.5%) 0.100e

Gelatine (25-30%) 0.28f 0.51f Doran and Bailey ,1986Ca-alginate (2%) 0.25f 0.41f Galazzo and Bailey, 1990aCa-alginate (2%) 0.24f 0.31f Hannoun and

Stephanopoulos, 1990Ca-alginate (2 %) 0.46e 0.5e Vives et al., 1993Ca-alginate (2%) 0.30g 0.31f Willaert and Baron, 1993

0.27hThiospaera Agarose (5%) 0.45i 0.45 Hooijmans et al., 1990b pantotropha 0.58j

µI, specific growth rate of immobilised cells; µS, specific growth rate of suspended cellsa Supply of 21 % oxygen f anaerobiosis (by continuous bubbling ofb Supply of 100 % oxygen nitrogen into the medium)c Growth rate determined in gel slabs g Single immobilised cellsd Growth rate determined in gel beads h Cells in a micro-colonye probably anaerobic, not mentioned how i Experiment in a stirred tank reactor anaerobiosis was maintained j As for i but in a Kluyver flask

(a) Dynamic ModellingConsiderable effort has been exerted in applying the mathematical theory of reaction and

diffusion in porous media to gel immobilised cell systems. A general dynamic model, whichdescribes the growth of the immobilised cells and the resulting time dependent spatialvariation of substrate and product in the gel, can be constructed by writing the mass balancesover the immobilisation matrix, when it is assumed that diffusion in gels is governed byFick's law, the cells are initially distributed homogeneously over the carrier and there is nodeformation of the matrix due to cell growth or gas production:

∂∂ t(εβCi ) = z−n ∂

∂zzn De,i

∂Ci∂z

± εβri (25)


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where ε is the ratio of the volume of the pores to the total volume; β is the ratio of the volumeof the pores minus the volume of the cells to the volume of the pores in the matrix; Ci is thesubstrate (i=S) or product (i=P) concentration (expressed per volume available for substrate;n is a shape factor of value 0 for planar, 1 for cylindrical or 2 for spherical geometry; and De,iis the effective diffusion coefficient for species i. The substrate consumption rate (rs) and theproduct formation rate (rp) are linked to the growth rate (rx) by the following equations:

∂∂ t(εCX ) = ε rx = εµ CX (26)

rs =1

YX/Srx and rp =

1YX/P

rx (27)

where Cx is the biomass concentration expressed per volume available for the cells. Since ε isconstant with time, the left hand side of Equation (25) can be written as

∂∂ t(εβCi ) = εβ

∂Ci∂t

+ εCi∂β∂t

(28)

In general for cell entrapment in gels, the second term on the right hand side of Equation (28)can be neglected. Based on the same principles, the continuum equation for biomass can bewritten as

∂CX∂ t

= µCX − z−n ∂∂z

zn JX( ) (29)

where Jx is the radial flux of biomass. In a typical start-up phase of a immobilised cellfermentation, it is observed that the system is characterised by a rapid cell growth and anegligible cell concentration in the bioreactor effluent. Many generations later, viablebiomass in the reactor effluent is suddenly observed. At this time, the biomass concentrationin the edge of the gel particle has reached the maximum attainable biomass concentration(CXmax) and is released in the bulk liquid. So, two dynamic phases can be distinguished: (1)CX < CXmax: the biomass accumulates with no radial movement (i.e. Jx=0 and ∂CX/∂t = µCX);(2) CX = CXmax: the matrix is fully packed and ∂CX/∂t = 0. The net biomass flux from the gelmatrix can be calculated after integration of Equation (29) over the applicable region(Salmon, 1989; Monbouquette et al., 1990):

JX =1zbn znrxza

zb∫ dz at z = zb (30)

where za and zb are the co-ordinates of the active cell layer in which CX = CXmax. If one isonly interested in the internal growth dynamics and the model is not linked to the reactor


146

balance equations, this growth model can be simplified by not taking into account thebiomass flux when CX reaches CXmax (e.g. Willaert and Baron, 1994).

These equations have to be solved (usually together with the reactor model) using thecorrect initial and boundary conditions. Initial conditions (no substrate and productconcentration in the gel, and a given immobilised cell concentration) can be expressed asCS=CP=0 and CX=CX0. In the case of cylindrical or spherical geometry, the boundarycondition for substrate and product in the centre of the particle can be written as

De,idCidz

= 0 at z = 0 (31)

The boundary condition at the edge of the particle when external mass transfer limitation istaken into account can be expressed as

De,idCidz

= ki (Ci − Cib ) at z = z b

(32)

where ki is the external mass transfer coefficient and Cib the bulk concentration. When theexternal film resistance is negligible, Equation (32) is reduced to Ci = Cib.

Dynamic modelling of gel immobilised cell systems has only recently been started assummarised in Table 43. The dynamic behaviour (growth on glucose) of S. cerevisiaeentrapped in a Ca-alginate bead at the start-up phase of an anaerobic fermentation isillustrated in Figures 3 and 4 (Willaert and Baron, 1994)(see Table 43 for more details of theemployed model). Figure 3 shows the simulation of the substrate (glucose) concentrationprofile as a function of time. After immersing an immobilised gel bead in a fermentationmedium, the substrate diffuses relatively rapidly into the bead and a concentration gradientdevelops. This gradient becomes larger when the reaction proceeds. For large fermentationtimes, the centre concentration drops to zero and substrate is only available in a thin outerlayer of the bead. The yeast cell concentration is initially homogeneously distributed (cf.Figure 4). During the first hours of the fermentation, this distribution remains unchangedbecause the substrate concentration at each position in the bead is much larger than theMonod constant. After a fermentation time of approximately ten hours, the yeast cells in thecentre of the bead stop growing while the cells at the edge still grow exponentially.Ultimately, the maximum attainable biomass concentration is reached at the edge, and thenthis densely packed region grows inward.

(b) Pseudo-Steady-State ModellingUnder certain conditions the full dynamic modelling to describe transient behaviour can be

simplified to "pseudo-steady-state" modelling. Therefore, the biomass growth and thesubstrate consumption rate and/or product formation rate are treated separately. Thisapproach is valid as long as the time scale for growth is much larger than the time scale forconsumption and product formation. Hence, a pseudo-steady-state substrate/productdistribution is assumed at each instant. As a result, the system of partial differential equations


147

is reduced to a system of ordinary differential equations which facilitates the numericalsolution. Table 44 summarises the pseudo-steady-state models which have been used tosimulate transient behaviour in gel immobilised cell systems.

00.75

1.52.25

33.75

4.55.25

66.75

7.58.25

99.75

10.511.3

1212.8

13.514.3

1515.8

16.5

0 0.26 0.370.45 0.52

0.58 0.630.68 0.73

0.77 0.820.86 0.89

0.93 0.971

0

0.2

0.4

0.6

0.8

1

Substrate

Time (h)

Position (-)

(-)

Fig. 3: Simulation of glucose concentration profile in a Ca-alginate bead with entrapped S.cerevisiae (Willaert and Baron, 1994).

00.75

1.52.25

33.75

4.55.25

66.75

7.58.25

99.75

10.511.25

1212.75

13.514.25

1515.75

16.5

00.37

0.520.63

0.730.82

0.890.97

0

50

100

150

200

250

Biomass

Time (h)

Position (-)

(gDW/l)

Fig. 4: Simulation of biomass concentration profile in a Ca-alginate bead with entrapped S.cerevisiae (Willaert and Baron, 1994).


148


149


150


151


152

TABLE 44PSEUDO-STEADY-STATE MODELLING OF GROWING CELLS ENTRAPPED IN GELS

Immobilised Cell Microbial Kinetics De Commentsa ReferenceSystem

Escherichia coli Monod A, C, D Salmon, 1989 in Ca-alginate beads: a) continuum model X independent b) colony expansion Decol

Da= 0. 25

model

Nitrobacter agilis Combination of growth De0 1−CXCXmax

A, C de Gooijer et

al., in κ-carrageenan models of Pirt and 1991;Wijffels et beads Herbert (as described by al., 1991;

Beeftink et al. (1990)) Wijffels, 1994Nitrosomas europaea Combination of growth Decol

De0= 0. 25 A, C de Gooijer et

al., in κ-carrageenan models of Pirt and 1992; Wijffels, beads: colony Herbert 1994; Wijffels expansion model et al., 1995Zymomonas mobilis Monod + substrate and De0 a1(1 − a2CX )

2 A, B Seki et al., in Ca-alginate beads product inhibition 1990; Furusaki

and Seki, 1992a A, Comparison to experimental data; B, Internal mass transfer resistance only; C, Internal andexternal mass transfer resistance; D, Includes overall reactor performance modela1 constant (dimensionless) Da diffusion coefficient in watera2 constant X biomass

TABLE 45STEADY-STATE REACTION-DIFFUSION MODELS FOR GEL IMMOBILISED CELL SYSTEMS

Immobilised Cell Microbial Kinetics Commentsa ReferenceSystem

Corynebacterium Michaelis Menten B, C Ogbonna et al., 1991a, b glutamicum in Sr-alginate beadsDaucus carota Zero order C, F, H Hulst et al., 1985a in gel beads


153



Denitrifying bacterial Michaelis Menten A, C, G de Beer and Sweerts, 1989; population in agar de Beer et al., 1990 beadsEscherichia coli Zero order A, C, F Hooijmans et al., 1990a in κ-carrageenan beadsGluconobacter oxydans Michaelis Menten A, B, D, G Adlercreutz, 1986 in Ca-alginate beadsHansenula polymorpha Michaelis Menten A,B, C, G Hiemstra et al., 1983 in Ba-alginate beadsMycobacterium sp. Product inhibition A, B, D, G Brink and Tramper, 1986b in Ca-alginate beadsSaccharomyces cerevi- First order B, D, E, Ramakrishna et al., 1991 siae in alginate beads F, HS. cerevisiae in Ca-alginate gel: a) continuum model Monod B, C, G, H Willaert, 1993 b) micro-colony model Zero order B, C, G, H Willaert, 1993; Willaert

and Baron, 1993Thiosphaera pantotro- Zero order A, D, F Hooijmans et al., 1990b, c pha in agarose beadsTrichosporon cutaneum Michaelis Menten B, C, G Chen and Huang, 1988 in Ca-alginate beadsZymomonas mobilis Non-competitive B, C, G, H Luong, 1985 in κ-carrageenan beads substrate and product

inhibitionZymomonas mobilis Monod modified for A, B, C, E, Melick et al., 1987 in Ca-alginate beads product inhibition G, HZymomonas mobilis Zero and first order A, B, C, Sakaki et al., 1988 in Ca-alginate beads F, HZymomonas mobilis General rate expression B, D, G, H Webb et al., 1990 in gels (slab, cylinder, (function of substrate and sphere) product concentration)

General models:Gas production by Zero order C, F, E, H Krouwel and Kossen, whole cells (slab, 1980; 1981 cylinder and sphere)


154



Review of models for e.g. Michaelis Menten, A, B, D, E, Kasche, 1983 enzymes and cells without and with G, H

substrate and product inhibition

Review: engineering General, e.g. Michaelis B, D, F, Karel et al., 1985 principles of immobi- Menten G, H lised whole cellsModel to evaluate the Monod B, C, F, Dalili and Chau, 1987 centre concentration G, H (slab, cylinder, sphere)Model for gel beads Zero and first order B, C, F, Salmon and Robertson, containing micro- G, H 1987 coloniesModel to estimate Zero order B, D, A, H Chang and Moo-Young, oxygen penetration 1988a, b depth (slab, cylinder and sphere)Model to estimate criti- Zero and first order C, F Chen and Humphrey, cal bead diameter 1988Instrinsic structured Monod B, C, G, H Monbouquette and Ollis, model of immobilised 1986; 1988b cell kinetics and RNA contentModel for immobilised Michaelis Menten, Non- B, D, F, Vos et al., 1990 non-growing biocata- competitive substrate G, H lysts (sphere and slab) and product inhibition,

competitive productinhibition, reversibleMichaelis Menten

Model for growth of First-order growth B, D, C, F Van Ede et al., 1992; biomass films immo- inhibition by one of 1993 bilised in or around the products carriersa A, Comparison to experimental data; B, Effectiveness factor calculation; C, Internal mass transferresistance only; D, Internal and external mass transfer resistance; E, Includes overall reactorperformance model; F, Analytical solution; G, Numerical solution; H, Thiele modulus calculation


155

(c) Steady-State ModellingProvided cell mass does not vary rapidly, or is fairly uniform, the concentration profiles in

a gel matrix with entrapped cells can be simulated using a steady-state model at any point intime. These models can give valuable information for design purposes or can be combinedwith experimental in situ measurements (e.g. microelectrodes). In this case, mathematicalcalculations can be very simple and straightforward analytical solutions can be obtained forsimple reaction kinetics (e.g. zero or first order kinetics). A list of steady-state reaction-diffusion models (specific and general models) is tabulated in Table 45.

(d) Effectiveness FactorIn both the chemical and biochemical engineering literature, the effectiveness factor (η)

has been adopted as a numerical measure of the influence of mass transfer on the reactionrate. The effectiveness factor is defined physically as (Bailey and Ollis, 1986)

η =observed reaction rate

ratewhich would be obtainedwithout masstransfer resistance (33)

Effectiveness factor calculations for immobilised enzyme catalysts based on the chemicalengineering literature are well documented. Approximate expressions of effectiveness factorfor Michaelis-Menten kinetics have been derived for membranes (Moo-Young andKobayashi, 1972), rods and spherical biocatalysts (Kobayashi et al., 1976), incorporatingexternal mass transfer resistance (Yamané, 1981) and the partition coefficient for gelimmobilisation (Yamané et al., 1981). Numerical algorithms have been developed to solvethe problem of enzyme effectiveness factors for Michaelis-Menten reaction (Fink et al., 1973;Ramachandran, 1975; Ghim and Chang, 1983). The effectiveness factor calculationmethodology for immobilised enzymes has been applied to biocatalysts with immobilisedliving microbial cells (Kasche, 1983; Luong, 1983; Radovich, 1985a) and extended forbiofilms around particles (Vos et al., 1990; Van Ede et al., 1992, 1993). These calculationsare based on steady-state reaction-diffusion models with the assumption of a homogeneousdistribution of cells over the carrier. In the case of growing immobilised cells, the globalkinetics of substrate consumption and product formation are all dependent on the spatial andtemporal dynamics of the immobilised cell population. Accordingly, a rigorous calculation ofthe effectiveness factor has to be coupled to a transient reaction-diffusion model, which leadsto a time dependent effectiveness factor (Willaert and Baron, 1994).

The effectiveness factor for substrate consumption can mathematically be expressed as thevolume averaged reaction rate relative to the rate at bulk phase concentration:

η = (n' +1)

DedCS'

dz'

z'=1

rs(1) (34)

or


156

η = (n'+1)z'n' rs(CS

' )dz'01∫

rs(1) (35)

where n' is a shape factor (0 for planar, 1 for cylindrical or 2 for spherical geometry) and z' isthe dimensionless position co-ordinate; rs is the substrate reaction rate which is a function ofthe dimensionless substrate concentration (CS') (and also of the position in the case oftransient effectiveness factor).

4.2.2 Micro-capsules

Only a few models including mass transfer and reaction kinetics, have been developed.Steady-state as well as transient models, with or without cell growth within the capsules, havebeen constructed.

4.2.2.1 Models without Cell Growth(a) Steady-State ModellingHeath and Belfort (1987) constructed a steady-state model to simulate glucose and oxygen

concentration profiles in micro-capsules. Assumptions included homogeneous cellsuspension, Fickian diffusion, and kinetics described by the zero and first order limits of theMonod equation. Furthermore, it was assumed that the capsule membrane provides negligibleresistance to diffusion compared to the resistance of mass transfer in the cell suspension;there is no convective fluid motion inside the particles (the validity of this assumptiondepends on the rate of stirring of the solution and the permeability and flexibility of thecapsules); diffusion within the spheres is a geometrical function of radius only; and the bulksolution is well mixed (no external mass transfer limitation) and is maintained at a constantsubstrate concentration. The simulated concentration profiles indicated the possibility of anecrotic core due to insufficient substrate in those cases where diffusion is low and/or uptakeis high. The model equations provided a means of estimating the maximum capsule radiuswhich would allow adequate diffusion of nutrients to all contained cells.

Willaert and Baron (1995) constructed a model which describes the oxygen diffusion andconsumption by micro-encapsulated islets of Langerhans. The model deals with the situationwhere the islets of Langerhans in the micro-capsule core are embedded in a gel. The influenceof the oxygen mass transfer on the oxygen consumption is evaluated by calculatingconcentration profiles and Thiele modulus versus effectiveness factor plots.

(b) Dynamic ModellingMorvan and Jaffrin (1989) have constructed a bioartificial pancreas model, constituted by

a micro-encapsulated islet of Langerhans implanted in the peritoneal cavity, to study theeffect of different physical parameters upon the glucose and insulin kinetics during theirtransfer between an implanted islet and an arteriole. For each solute (glucose and insulin), themass conservation equations inside and outside the micro-capsule were reduced to a set ofcoupled integral equations which were solved numerically (boundary element method


157

associated with subregions formulation). The insulin release localised on the islet ofLangerhans was controlled by the local value of glycemia. The analysis was made for a two-dimensional geometry. The numerical experiments demonstrated that the insulin quantitydiffusing through the arteriole wall depends on the resistance to diffusion of the mediumlocated between the islet and arteriole wall. While the kinetics of glucose did not seem to beaffected by a small modification to the transfer conditions between the islet and the arteriole,the kinetics of insulin were extremely sensitive to such conditions. The insulin available fordiffusion in the vascular system was considerably reduced as the islet to arteriole walldistances increased, or when the diffusion coefficient between the islet and the arterioledecreased.

4.2.2.2 Models with Cell GrowthRecently, a transient mathematical model describing animal cell growth in micro-capsules

has been developed and compared to experimental data (Yuet et al., 1993; 1995; Goosen,1995). The modelling study was based on the scenario in which micro-capsules with fluidintra-capsular liquid are used in stationary culture, i.e. cells initially settle to the bottom of thecapsules and the cell population expands from the bottom up during the culture period. Themodel described (a) material transport from the bulk to the interior phase of the micro-capsuleand (b) mass transfer and cell growth kinetics in the intra-capsular region. An unstructuredmodel was used to describe animal cell growth:

dCXdt

= u(t − tlag) µCX + λ CX (r)dr0t∫

(36)

where

u(t − tlag) =1if t > tlag0if t < tlag

(37)

and λ is the death constant and tlag is the lag time which has to be determined experimentally.The specific growth rate (µ) depended on the rate limiting substrate concentrations (Ci)according to a Monod relation and the cell density was controlled according to a contact-inhibition model developed originally by Frame and Hu (1988). The complete expression forµ was therefore proposed as follows

µ = µmaxCi

KM,i + Ci

i=1

m∏ 1 − exp −B

CXmax − CXCX

(38)

where CXmax is the maximum density physically allowable in a micro-capsule and B is anadjustable parameter. The numerical "control-volume" method was used to solve the modelequations. This approach involves setting up one or more criteria. If these criteria are met in acertain control volume, the neighbouring control volumes will be initialised with a certain celldensity. Cell growth in the neighbouring control volumes will then be based on these initial


158

cell densities. The distribution of cells and nutrients in the micro-capsule clearly illustratedthe delicate balance between the supply and demand of nutrients and oxygen in the micro-capsule system. The highest growth rate was found in the boundary region at the top of thecell mass. This region received the most abundant supply of nutrients and oxygen across themembrane and from the upper half of the capsule. The cells at the bottom of the capsule closeto the membrane had virtually stopped growing since the maximum cell density was reachedand no more space for cell division was left. The cells at the central region of the populationwere probably suffering from a lack of nutrients and oxygen and therefore had only a verylow specific growth rate. The simulation results agreed quite well with experimental data.


159

5. SUMMARY AND CONCLUSIONS

From a laboratory curiosity, the immobilisation of living cells in gel matrices and micro-capsules has evolved to an established technique with numerous applications in fields such asindustrial microbiology, pharmaceuticals, food and beverages, biomedical engineering,agriculture, or waste treatment. The techniques for analysis and design of these systems nowgradually move from trial and error to a more systematic use of accurate descriptions of masstransport and conversion with the help of mathematical modelling.

In a first section, "whole cell immobilisation" and "immobilised cell system" are defined.The advantages of these systems are compared to immobilised enzyme systems and ordinarysuspension culture systems and their specific requirements are discussed. A classification ofcell immobilisation in four categories - i.e. surface attachment, entrapment within porousmatrices, containment behind a barrier and self aggregation systems - is presented, withattention for the materials used in each of these methods. A brief discussion of thecharacteristics of each category, and some major pros and cons are presented, which can beused as guidelines to choose the optimal system for a particular living cell or application.

In the second section, gel immobilisation of living cells is treated in great detail. Gelentrapment has become one of the most popular immobilisation techniques, resulting in theuse of various materials and numerous applications in different fields. Natural(polysaccharides and proteins) and synthetic polymers are described concomitantly with theirmethods for cell entrapment. It is shown that various parameters have to be optimised in orderto obtain a gel system with suitable characteristics. Gel systems have usually a limited area ofapplication and the characteristics of the various systems should be compared in order tomake a proper choice for the construction of a new system. Although natural polymers havemany advantages such as mild immobilisation conditions, synthetic polymers have theadvantage that adequate properties can be more precisely and reproducibly designed. Thephysiology of gel immobilised cells has been studied for different cell types. Non invasiveexperimental techniques have been used to obtain in situ information. The results obtainedwith these techniques are discussed. Additional physiological information of gel immobilisedbacteria, fungi, algae, plant and animal cells are also given. This section is concluded with areview of applications using microbial, plant and animal cells. Microbial cells are used for theproduction of various biochemicals such as amino acids, organic acids (bioconversions andde novo synthesis), antibiotics, steroids, proteins (including production by geneticallyengineered micro-organisms), alcohols, and sugars. The number of applications for food andbeverages, agriculture, waste treatment and energy production are also rapidly growing. Plantcells entrapped in gels have been used for the production of compounds by biotransformation,salvage and de novo synthesis. If industrial or large scale use of many of these systems is stilllimited and will take more time to be accepted, some applications were introduced almostovernight due to their exceptional advantages. Good examples are the construction ofartificial seeds using gels and immobilised animal cells for the production of high valueproteins. A number of experiments where the growth of animal cells was studied, is alsogiven in detail. Also, a detailed discussion of the intrinsic problems with this method helps inexplaining why some earlier attempts of industrial use failed, both technically and


160

economically. Evidently, expensive and delicate immobilisation techniques are not veryappropriate for production of low added value commodity chemicals that can more efficientlybe produced by free cell fermentation or even chemical processes. Successful applicationsare mainly to be found where products have high added value or when the process constitutesan excellent and economically sound alternative to current technology.

The third section deals with micro-encapsulation of living cells and an overview of thevarious materials to encapsulate living cells is given. Four mechanisms can be distinguished:encapsulation by coating of a spherical hydrogel core, by coextrusion and interfacialprecipitation, by interfacial ionic cross-linking, and by interfacial polymerisation. Importantcharacteristics are discussed to guide the construction of micro-capsules for a particularapplication. The micro-encapsulation of islets of Langerhans as a novel cell therapy for thetreatment of diabetes as a case study is discussed in detail. These techniques are now widelyused in the construction of other bio-artificial organs as well as the large-scale production oftherapeutic proteins by encapsulated animal cells. The few recently developed applicationsusing encapsulation of microbial and plant cells reported in the literature are also described.

In gel or micro-capsule systems, hindered transport of nutrients, products, and especiallylarger molecules (e.g. proteins) from the surrounding bulk fluid can limit the growth of orproduction by immobilised cells. A good understanding of these phenomena and how toinfluence them, is essential in the correct design and use of these systems. This is the topic ofthe fourth section where mass transfer in and modelling of the resulting reaction-diffusionsystem in gel immobilised systems and micro-capsules is reviewed. The discussion of masstransfer is limited to internal mass transport, essentially by diffusion. Diffusion throughsupport materials, in cell-containing matrices in general and in gels and micro-capsules inparticular as well as methods of measurement are reviewed and a vast collection of datatabulated as a guide in design. The link between transport properties and structure of thematerials and molecules is also discussed.

Diffusion transport will be limiting if the conversion rates are higher than the transportrates of certain components. Microbial, yeast or fungi based systems have high needs fornutrients and pose more problems than animal or plant cells. This is the main reason for moresuccessful use of these systems in the latter cases.

Another important factor is the growth in the matrix, necessary up to a certain level for theregeneration of the system, it can become a problem when it leads to degradation or completedestruction of the matrix. Cell retention is no longer guaranteed in this case and then thesystem becomes also less attractive as it loses one of its major advantages over otherimmobilisation methods. Unless growth can be controlled or avoided by clever manipulationof the media or even genetic modification of the micro-organism, gel and micro-capsuleimmobilisation are not suitable methods of long term immobilisation. In that case externalattachment or porous preformed supports are to be preferred, obviously with loss of theabsolute cell containment property.

Mathematical models based on mass balances to simulate the behaviour of immobilisedcells, dynamic, pseudo-steady-state as well as steady-state models are available. A majorproblem however is the poor knowledge of the intrinsic kinetics for most immobilised cellsystems. These often can only be determined from models by fitting or by a few in situ


161

methods of analysis. Still, even though our knowledge is incomplete, one can now use thesemethods in the design and analysis of gel and micro-capsule systems. One can not onlyidentify diffusion limitations or "physiological changes" due to the resulting modified micro-environment of the cells in the matrix, but even predict the breakdown of the system whenexcessive growth occurs in the matrix. Several case studies are given in some detail as well asrules for the use of modelling in different applications.

Biotechnology, and certainly this subfield of cell immobilisation, is still rapidly evolvingand very remote from mature technology. However, the number and economic impact of themany applications of gel and micro-capsule immobilisation techniques is now so large thatboth engineers and bioprocess scientists should have a sound understanding of these systems.We hope to have filled a gap in the existing literature with this unifying review.


162

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