INTRODUCTION

 Whole cell immobilization has been defined as "the physical confinement or localization of intact cells to a certain defined region of space with preservation of some desired catalytic activity" (Karel et al., 1985). Many microorganisms own the capability to adhere to different kinds of surfaces in nature on which way are in close proximity to nutrients and easy realize a food supply. Therefore, we can say that these biological systems in their natural state are immobilizad. 
However, many biotechnological processes need to be carried out using immobilization of biocatalysts. Thereby several different techniques and support materials have been proposed for the cell immobilization in vitro. Figure 1 illustrates basic methods for immobilization. These techniques can be divided into four major groups based on the physical mechanism causing immobilization: physical entrapment within a porous matrix, attachment or adsorption to a pre-formed carrier, self agregation by floculation (natural) or crosslinking agents (artificially induced) and cell contained behind barrier (Pilkington et al., 1998). 

All of these methods have the same purpose: to retain high cell concentrations within "a certain defined region of space" such as a bioreactor giving increased volumetric productivity of a system. 
 During the last thirty years a numerous different kinds of cell-supporting materials for immobilization have been developed, such as polymeric matrices (alginate, agar, gelatin, k-karrageenan, chitosan, pectin, polyacrylamide, epoxy resin, silica sol), and porous and non-porous preformed support materials (wood chips, stainless steel, volcanic rock, cotton cloth, porous glass particles, DEAE cellulose, porous silica, porous ceramics, diatomaceous earth). The choice of cell-supporting material for any specific application is related to the following points:
- bioreactor configuration
- high carrier activity
- availability of the carrier in commercial qualities
- low cost of immobilization
- immobilization easy and controllable
- ease of operation scale-up
- excellent mechanical strength during long-time operation
- physiological and environmental safety of the materials used
- low affinity to contaminations
Entrapment in insoluble Ca alginate gel is recognized as a rapid, nontoxic, inexpensive, versalite and the most often used method for immobilization of cells (more than 80% of cell immobilization processes are still carried out using alginate). Alginates make a family of naturally occuring unbranched binary copolymers, which are formed of D-mannuronic and L-guluronic acids linked by 1,4-glycosidic bonds. The monomers are arranged in a pattern of blocks along the chain, with homopolymeric regions (termed M and G blocks) interspersed with regions of alternating structure-MG blocks (Thu et al., 1996, 1996). Alginates are differing among themselves by the monomeric composition and the block structure, and these properties are connected with the type of algae and the tissue they are isolated from. In the presence of monovalent cations alginates are forming water solubile salts, but with polyvalent cations, such as Ca²⁺, Ba²⁺ and Sr²⁺, they are forming inert polymer network by binding the polyvalent cation (ussualy Ca²⁺) to guluronic acid units. This property of alginates is used for entrapping of living cells in the alginate matrix. The procedure consists of mixing of cell suspension with Na-alginate solution and dripping of it into a solution containing divalent cations. This is causing instantaneous gel spheres formation and cell entrapment into a three dimensional lattice structure of ionically cross-linked alginate. Since the conditions for the cells during this procedure are very mild, cells are remaining viable and catalytic active for a long period of time. There are many examples of applications of these systems in different fields of industry, medicine and agriculture. 
Immobilization offers many potential advantages over free cell systems, such as: 

•  Higher cell densities and cell loads
•  Increased volumetric productivity
•  Shorter overall reaction times
•  Smaller fermenter sizes which may lower capital costs
•  The reuse of the same biocatalysts for prolonged period of time due to constant cell regeneration
•  A continuous process which may be performed beyond the nominal washout rate 
•  Improved substrate utilization
•  Reduced risk for microbial contamination
•  Process design simplified
•  Constant product quality
•  Improved tolerance or protection of cells from substrate and end-product inhibition. 

BEER PRODUCTION WITH IMMOBILIZED YEAST CELLS

The production of fermented beverages such as lager-type beer require a fermentation time of 6-7 days as well as large scale fermentation and storage capacities. Present trends in immobilized yeast cell technology nowadays provide the brewing industry with a method of reducing processing time without sacrificing product quality. By increasing the yeast cell concentration in the bioreactor vessel, it is possible to accomplish a faster fermentation and consequently much higher volumetric bioreactor productivity of beer with significant reduction in fermentation costs.
The earliest applications of immobilized cells to brewing were described by works of some authors during '70s (Narzzis and Hellich, 1971, Baker and Kirsop, 1973, White and Portno, 1978). The early packed-bed reactors consisted of mixture of brewer's yeast and diatomaceous earth forming a porous biomass bed (Narzzis and Hellich, 1971, Baker and Kirsop, 1973). Rapidly and efficiently produced beer had the flavour which did not compare well with that of a similar beer made by conventional fermentation. The resultant beer had a high concentration of diacetyl, and low of higher alcohols and esters. Also, the amino acid uptake have been frequently reported to be low (Pardonova et al., 1982). This and many other studies have found that amino acid metabolism is obviously critical to beer quality because the amino acid metabolism is closely linked to production of flavour compounds such as the vicinal diketones, higher alcohols, organic acids, and sulphur compounds (Masschelein et al., 1994). The main reason for this unbalanced metabolic behaviour was altered growth pattern of immobilized cells caused by mass transfer limitations. In terms of improving amino acid utilisation as well as beer quality and fermentation efficiency, bulk mixing of the liquid phase and consequently improved mass transfer is likely to play an important role.
Thereby in beer brewing with immobilized yeast cells reactor design has a leading role not only in system selection but also in system efficiency. Since insufficient mass transfer, i.e. transport of nutrients to yeast cells, and removal of fermentation by-products from the immobilization matrix, has been identified as the main factor that causes the unbalanced flavour profile in beer produced using immobilized yeast packed bed reactors it was important that extensive care should be taken in assessing and improving mass transfer in immobilized cell bioreactors. Significant improvement has been achieved by switching from packed bed to fluidized bed reactors. Liquid fluidized beds are suitable for support particles that are significantly more dense than the fermentation medium, because a less dense particle would be carried upward in these configurations. These systems give much improved contact between the immobilized yeast and the substrate, which undoubtedly increases the productivity and permits shorter residence times. However, mixing may also cause abrasion of the support material, thus affecting the efficiency of the system
Latest developments in brewing processes with immobilized cells clearly indicate that by the use of different reactor and system designs, such as two-stage multi-channel loop reactor system (Andries et al., 1996), fluidized bed reactor with wort recirculation (Shindo et al., 1994), multi-stage reactor system (Inoue, 1995) and gas-lift bioreactor system (Nedovic et al., 1993; Nedovic et al., 1996; Mensour et al., 1996), and a wide range of beads nature, much better results in a quality of produced beer are accomplished, than by the use of classic packed bed reactors. 

GAS-LIFT SYSTEM

 Gas circulating bed reactors can combine the advantages of fluidized beds, such as high loading of solids and good mass transfer, with the efficient mixing and controlled liquid flow which are inherent for gas-lift reactors (Vunjak-Novakovic et al., 1992). The absence of mechanical agitation creates a relatively low shear environment, which make these reactors ideally suitable for the application of shear sensitive cells and solid matrix. The solid phase in external or internal loop bioreactors can be fluidized and kept in suspension due to circulation of liquid phase and gas bubbles. Low dense alginate and carrageenan gel particles are typically used in three-phase gas-lift reactors as carriers of yeast cells in beer fermentations (Nedovic et al., 1996; Mensour et al., 1996). Since Ca alginate is hydrogel, its density is very close to that of water and consequently near that of wort. Mixing requirements for these particles will therefore be relatively low as compared to heavier matrices such as glass or ceramic beads. The other important characteristics of gas-lift bioreactors for biochemical processes’ applications are simple construction, low risk of contamination, easy adjustment and control of the operational parameters and simple capacity enlargement (Nedovic et al., 1997).
With an interest to produce beer of acceptable quality using immobilized yeast cells we have designed and studied during the several last years a three-phase internal loop gas-lift bioreactor used in conjunction with yeast cells (Saccharomyces cerevisiae) entrapped in small sized Ca-alginate beds.
 The experimental unit was the internal loop gas-lift bioreactor with a conical bottom, schematically shown in Figure 2. This reactor consisted of glass column, gas distributor and glass internal draft tube. The fermenting wort in the reactor was divided into two distinct zones where only one of them (internal zone) was spargged by nitrogen. Gas phase was introduced through a central fritted glass sparger at the conical bottom. The different gas hold-up in the gassed and ungassed zones resulted in different bulk densities of the fermenting wort in these regions which caused circulation and mixing of the wort and beads in the reactor by gas-lift action. The part of the reactor containing the gas-wort-beads upflow is the riser and the region containing wort and beads downflow is known as the downcomer. Solid phase was fluidized alginate particles with immobilized yeast cells, produced by a droplet generator (FTM, Belgrade), and gellated in calcium chloride (60 min).
 Series of fermentations of hopped wort were carried out in this bioreactor with the same immobilized cell load. 
 
 

Main results: 
· Primary beer fermentation, which takes 6-7 days in traditional brewing, was drastically shortened to only about 16 hours. 
· Obtained results also showed insignificant differences in chemical composition and sensory characteristics between beers obtained with immobilized cells and conventionally produced beers. The concentrations of higher alcohols and esters were at the same level as those obtained by the fermentation with suspended cell.
· During the 3-5 months period the same immobilized cell load of unchanged viability and activity was utilized. 
· Within the range of superficial gas velocity between 0.03 and 0.2 cm/s fermentation efficiency showed increasing trend and after that further increasing in superficial gas velocity little bit changed the fermentation efficiency.
· Superfficial gas velocity was also the main parameter for amino-acid nitrogen uptake during the fermentation. The increase of superficial gas velocity leads to the increase of amino acid consumption. The final amino-acid concentration was similar to those obtained during the conventional brewing, and even lower than that.
· By comparing the maximum obtained value of fermentation rate in the bioreactor with values obtained in packed bed and fluidized bed reactors one can conclude that internal loop gas-lift bioreactor is much more efficient among similar systems. It can be explained by liquid mixing characteristics of the bioreactor, i.e. excellent liquid-solid and gas-liquid mass transfer within the system, with a balanced flavour profile of finished product, as a consequence.

INDUSTRIAL SCALE APPLICATION AND ECONOMICAL AND ENVIRONMENTAL ASPECTS
 Above all previously mentioned facts there are also several very significant economical and environmental reasons confirmed through practice that clearly indicate advantages of the immobilized yeast cell technology introduction in the industrial beer processing.

- Proposed immobilized cell technology is very simple for introduction in beer processing. Compact continuously operated processes will increase flexibility in production planning, and production losses will be smaller.
- Initial savings could be gained by reduction in investment costs (reduced number and size of the fermentors as well as minimal space requirements), while further savings could be gained in reduced operating costs. 
- There is great variety of different yeast carriers. Some of them are easily reusable and can have a lifetime of more than 10 years.
- The utilization of yeast carriers leads furthermore to the reduction of kieselguhr consumption in filtration, which makes handling easier and cheaper. 
- Application of immobilized cell technology will decrease environmental load, because immobilized yeast reduce waste yeast load, as new biomass is not produced in conventional quantities. Furthermore, reduced product losses, reduced water consumption for planning purposes and smaller cleaning chemicals consumption also have positive environmental effects.
 
 
 
 
 
 

LITERATURE
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17. Nedovic, V., Durieux, A., van Nedervelde, L., Rosseels, P., Vandegans, J., Plaisant, A-M., Simon, J-P., Proceedings of the EC Symposium "Yeast as a Cell Factory", P. Osseweijer and J.P. van Dijken, eds., Vlaardingen, The Netherlands, 188-190, 1998
18. Norton, S., and Vuillemard, J.-C., Critical Reviews in Biotechnology 14(2), 193-224, 1994
19. Nunokawa, Y., Hirotsune, M., Industrial Application of Immobilized Biocatalysts, Tanaka, A., Tetsuya, T., Kobayashi, T., eds., Marcel Dekker, Inc., 235-255, 1993
20. Pardonova, B., Polednikova, M., Sedova, H., Kahler, M., Ludvik, J., Brauwissenschaft 35, 254-258, 1982
21. Pilkington, P.H., Margaritis, A., Mensour, N.A., Russell, I., J. Inst. Brew. 104, 19-31, 1998
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23. Simon, J.-P., Durieux, A., Pinnel, V., Garre, V., Vandegans, J., Rosseels, P., Godan, N., Plaisant, A.-M., Defroyennes, J.-P., and Foroni, G., Progress in Biotechnology 11, Immobilized Cells: Basics and Applications, Wijffels, R.H., Buitelaar, R.M., Bucke, C., Tramper, J., eds., Elsevier Science B.V., 615-622, 1996
24. Sodini, I., Corrieu, G., andLacroix, C., Progress in Biotechnology 11, Immobilized Cells: Basics and Applications, Wijffels, R.H., Buitelaar, R.M., Bucke, C., Tramper, J., eds., Elsevier Science B.V., 687-695, 1996
25. Thu, B., Smidsrod, O., Skjak-Braek, G. Progress in Biotechnology 11, Immobilized Cells: Basics and Applications, Wijffels, R.H., Buitelaar, R.M., Bucke, C., Tramper, J., eds., Elsevier Science B.V., 19-31, 1996
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SHORT CURRICULUM VITAE
 

Ida Leskosek-Cukalovi} B.Sc. Engineering - University of Belgrade, Faculty of Technology and Metallurgy, Belgrade 
Ph. D. Biotechnological Science, Faculty of Agriculture, Belgrade 
Currently: Associated Professor of Brewing Technology, Faculty of Agriculture, University of Belgrade
Specialization: Heriot Watt University, Edinburgh, UK
A member of the Institute of Brewing, UK. A member of the Organizing Committee of the II and III Yugoslav Conference on Food Technology.  A member of the Organizing Committee and invited speaker for the Symposium on Yugoslav Brewing Industry. A member of the Organizing Committee and invited speaker for the Symposium on Yugoslav Fermentation Industry. Founder and head of the Laboratory for Brewing at the University of Belgrade, performing studies in the field of beer science, alcoholfree beer production, membrane separation technique, yeast immobilization. Member of several Yugoslav and international multidisciplinary projects performed by joint efforts University / National Laboratories / Industry, 1979 - 1999. Published and presented over 80 scientific and technical papers. Has been teaching undergraduate and graduate level courses in Brewing Science.

Office address:  Institute of Food Technology and Biochemistry
  Faculty of Agriculture, University of Belgrade
  11080 Beograd-Zemun, Nemanjina 6
  Phone: (381-11) 615-315/ ext.353
  E-mail: i.les@Eunet.YU