Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Immobilized yeast bioreactor systems for brewing – recent achievements

Nedovic V.A., Leskosek-Cukalovic I., and Vunjak-Novakovic G. (1996) Short-time fermentation of beer in an immobilised yeast air-lift bioreactor, in J. Harvey (ed.), Proc. 24th Conv. Inst. Brew., Singapore, Winetitles, Adelaide, p. 245

Nedovic, V.A., Leskosek-Cukalovic, I., Milosevic, V., and Vunjak-Novakovic, G. (1997a) Flavour formation during beer fermentation with immobilised Saccharomyces cerevisiae in a gas-lift bioreactor, in F.

Godia and D. Poncelet (eds.) Proc. Int. Workshop Bioencapsulation VI: “ From fundamentals to industrial applications” , Barcelona, Spain, T5.3, pp. 1-4

Nedovic, V.A., Pesic, R., Leskosek-Cukalovic, I., Laketic, D., and Vunjak-Novakovic, G (1997b) Analysis of liquid axial dispersion in an internal loop gas-lift bioreactor for beer fermentation with immobilised yeast cells, in M. Olazar and M.J.San Jose (eds.), Proc. II European Conference on FWIDIZATION, The

University of the Basque Country Press Service, Bilbao, pp. 627-635

Nedovic, V.A. (1999a) Immobilised cell systems in beer fermentation, Monograph (in Serbian), Andrejevic

Foundation, Belgrade.

Nedovic, V.A., Trifunovic, O., Pesic, R., Leskosek-Cukalovic, I., Bugarski, B. (1999b) Production of microbeads containing immobilised yeast cells for continuous beer fermentation by electrostatic droplet generation, in G. Skjåk-Braek and D, Poncelet (eds.), Proc. Int. Workshop Bioencapsulation VIII:

“Recent Progress in Research and Technology”, Trondheim, Norway, P-4, pp. 1-5

Nedovic, V.A., Durieux, A., Van Nedervelde, L., Rosseels, P., Vandegans, J., Plaisant, A.-M., and Simon, J.-P. (2000a) Continuous cider fermentation with co-immobilised yeast and Leuconostoc oenos cells,

Em. Microb. Technol. 26, 834-839.

Nedovic, V., Obradovic, B., Leskosek-Cukalovic, I., Trifunovic, O., Pesic, R., and Bugarski, B. (2000b) Electrostatic generation of alginate microbeads loaded with brewing yeast, Submitted to Proc. Biochem.

Obradovic, B., Dudukovic, A., Vunjak-Novakovic, G. (1994) Local and overall mixing characteristics of the Onaka, T., Nakanishi, K., Inoue, T., and Kubo, S. (1985) Beer brewing with immobilised yeast,

Pajunen E., Gronqvist A. and Ranta B. (1991) Immobilised yest reactor application in continuous secondary fermentation in industrial scale operation, in Proc. Congr. Eur. Brew. Conv., Lisbon, ILR Press, Oxford, pp. 361-368

Pajunen, E. (1996) Immobilised yeast lager beer maturation: DEAE-cellulose at Sinebrychoff, in

Immobilised yeast applications in the brewing industry, Monograph XXIV, Verlag Hans Carl Getranke-

Fachverlag, Nurnberg, pp. 24-34

Pardonova, B., Polednikova, M., Sedova, H., Kahler, M., and Ludvik, J. (1982) Biokatalysator fur die bierherstellung, Brauwissenschaft 35, 254-258.

Pilkington H., Margaritis A., Mensour N.A., and Russell I. (1998) Fundamentals of immobilised yeast cells for continuous beer fermentation: A review, J. Inst. Brew. 104, 19-31.

Pilkington, P.H., Margaritis, A., Mensour, N.A., Sobczak, J., Hancock, I., Heggart, H., and Russell, I. (1999)

The use of kappa-carrageenan gel to immobilize lager brewing yeast, in G. Skjak-Brek and D. Poncelet

(eds.), Proc. Int. Workshop Bioencapsulation VIII: "Recent Progress in Research and Technology",

Trondheim, Norway, P-17, pp. 1-4.

Poncelet, D., Dulieu, C., and Neufeld, R.J. (1997) Fundamentals of the dispersion into encapsulation technology, in F. Godia and D. Poncelet (eds.) Proc. Int. Workshop Bioencapsulation VI: "From fundamentals to industrial applications", Barcelona, Spain, T1.1, pp. 1-4

Prusse, U., Bruske, F., Breford, J., and Vorlop, K.-D. (1998) Improvement of the jet cutting method for the preparation of spherical particles from viscous polymer solutions, Chem. Eng. Technol. 21, 153-157.

Prilsse, U., Dalluhn, J., Sonnenburg, J., Breford, J., and Vorlop, K.-D. (1999) Scale-up strategies for bead production by jet cutting, in G. Skjåk-Braek and D. Poncelet (eds.), Proc. Int. Workshop Bioencapsulation VIII: “Recent Progress in Research and Technology”, Trondheim, Norway, O-6, pp.

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Scott, J.A. and O’Reilly A.M. (1995) Use of a flexible sponge matrix to immobilize yeast for beer fermentation, J. Am. Soc. Brew. Chem. 53, 67-71.

Shindo S and Kamimura, M. (1990) Immobilisation of yeast with hollow PVA gel beads, J. Perm. Bioeng. 70, 232-234.

Shindo, S., Sahara H., and Koshino S. (1994a) Suppression of α -acetolactate formation in brewing with immobilised yeast, J. Inst. Brew. 100, 69-72.

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Shindo S, Sahara H., Watanabe N., and Koshino S. (1994b) Main fermentation with immobilised yeast using fluidised-bed reactor, in Proc. 23rd Conv. Insl. Brew., Sydney, pp. 109-113

Stratton, M., Campbell, S., and Banks, D. (1994) Process developments in the continuous fermentation of beer, in Proc 23rctConv. Insl. Brew., Sydney, pp. 96-100

Tata, M., Bower, P., Bromberg, S., Buncombe, D., Fehring, J., Lau, V., Ryder, D., and Stassi, P. (1999) Immobilised yeast bioreactor systems for continuous beer fermentation, Biotechnol. Prog. 15, 105-113.

Taidi, B. (1995) Beer production with immobilised yeast, in Abstracts of the I7th ISSY “Yeast Growth and Differentiation: Biotechnological, Biochemical and Genelic Aspects”, Edinburgh, UK, O21

Umemoto S., Mitani Y., and Shinotsuka K. (1998) Primary fermentation with immobilised yeast in a fluidised bed reactor, MBAA Tech. Quart. 35(2), 58-61.

Van de Winkel, L. (1995) Design and optimisation of multipurpose immobilised yeast bioreactor system for brewery fermentations, Cerevisia 20, 77-80.

Vunjak-Novakovic, G., Jovanovic, G., Kundakovic, Lj., and Obradovic, B. (1992) Flow regimes and liquid mixing in a draft tube gas-liquid-solid fluidised bed, Chem. Eng. Sci. 47, 3451-3458.

Vunjak-Novakovic G., Vunjak N., Kundakovic Lj., Obradovic B., Nedovic V., Sajc L., and Bugarski B.

(1998) Air-Lift Reactors: Research and Potential Applications, Trends Chem. Eng. 5, 159-172.

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Wittlich, P., Jahnz, U., PrUsse, U, and Vorlop, K.-D. (1999-2000) Jet cutting method as a new process for the production of spherical beads from highly viscous polymer solution, in M. Hofman (ed.), ECB9 CD

ROM Proceedings, Branche Beige de la Societe de Chimie Industrielle, ECB9/2762

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Yamauchi Y., Okamoto, T., M., Murayama, H., Nagara, A., Kashihara T, and Nakanishi, K. (1994b) Beer brewing using an immobilised yeast biorector, J. Perm. Bioeng. 78, 443-449.

Yamauchi Y. and Kashihara T. (1996) Kirin immobilised system, in Immobilised yeast applications in the brewing industry, Monograph XXIV, Verlag Hans Carl Getranke-Fachverlag, Nurnberg, pp. 99-117 Yokotsuka, K., Yajima, M., and Matsudo, T. (1997) Production of bottle-fermented sparkling wine using

yeast immobilised in double-layer gel beads or strands, Am. J. Enol. Vitic. 48, 471-481.

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Different immobilisation methods are demonstrated in figure 1. From the variety of available techniques, these widely used are on the one hand the adsorption and on the other hand the encapsulation of biocatalysts (Cougghlan and Kierstan, 1987; Vorlop and Klein 1985; Kennedy and Medo, 1990). Encapsulation is further classified into microencapsulation and entrapment. In contrast to adsorption, the method of encapsulation offers better protection of the biocatalyst what an important factor when immobilising sensitive cells. Here, of course, the used polymers should guarantee lowest toxicity against biocatalysts while having sufficient mechanical, chemical, and biological stability.

When enzymes have to be entrapped, cut-off of the applied immobilisation material has to be taken into consideration. Often enzymes have to be cross-linked or bound to a carrier for enlargement before they can be encapsulated successfully.

1.2. SHORT OVERVIEW OF SUITABLE MATERIALS FOR ENCAPSULATION

The material used most often for immobilisation is the naturally occurring polymer alginate that can easily be solidified by means of ionotrophic gelation. Gel formation by temperature change can be used for agarose or gelatine among others. Most methods that apply biopolymers offer gentle conditions for the biocatalyst.

An increased stability and lower biodegradability of polymers can be achieved when employing synthetic gels (Leenen et al., 1996; Muscat et al., 1996). These can consist of gels based on chemically bound polymers like polyurethanes or acrylate-co- polymers, or on gels formed by means of hydrogen bonds like polyvinylalcohol hydrogels.

1.3. SHAPES OF PARTICLES WITH IMMOBILISED BIOCATALYSTS

The size and the shape of the immobilised biocatalyst both have a strong influence on its properties of stability, diffusion, and retention in the production process.

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Spherical shapes have been used for long time since many immobilisation techniques result in beads. Furthermore this allows diffusion effects to easily be described with mathematical models which help to understand and optimise the overall process.

If beads are too large in diameter, the biocatalysts in the core of the bead probably will suffer from diffusional limitations that will result in sub-optimal specific activity of the immobilised biocatalyst. Only the outer shell of large beads will be catalytically active. Moreover large particles are susceptible to a ready deterioration caused by stirring facilities and other mechanical charges when employed in stirred reactors. In contrast too small beads cause a higher drop of pressure and often tend to clog outlet lines in continuous processes and in general evoke problems when they have to be retained.

In fact, very often the method that is applied to immobilise the catalyst, i.e. the kind of polymer and the machines used for the bead production, determines the size of the immobilised biocatalyst. A stable matrix means a high content of polymer with high viscosity in the polymer solution. Since it was not possible to work with high viscous solutions in the past, often the stability of beads was not as good as required.

Many more or less useful approaches where made to overcome the problem of producing particles from different materials. For example Tanaka et al. (1996) cut large blocks of polymer with encapsulated biocatalyst into cubes of about 3 mm. Apart from diffusional problem this could result in insufficient mechanical stability especially at the edges of the cubes. Use of particles was completely circumvented by application of biocatalysts entrapped in a layer that is used to coat surfaces within the reactor, e.g. inside a static mixer. This technique unfortunately results in comparatively small surface of the immobilisation layer that is connected to low specific activity. The same is true for polymers shaped like filaments or cells and enzymes enclosed in membrane systems. The latter cause even more problems due to membrane fouling effects.

Of course, the requirements for an immobilised particle depend on the type of reactor to be used. The material in a stirred reactor has to be more stable and withstand especially attrition than that used for a packed bed, but often the reactor hardware cannot be changed since it is already installed in a production line.

Finding the optimal method for immobilising biological matter for a special application requires two decisions to be made: Selecting the suitable matrix, i.e. the polymer or material for encapsulation, and finding the immobilisation device to create appropriate size, shape, and amount of particles.

2. Techniques for bead production

Several techniques have been developed to produce beads from viscous fluids. The following aspects have to be considered when evaluating an immobilisation apparatus:

•What is the range of viscosity that can be handled? Many polymers dissolved in water show high viscosity even at low concentrations.

•Have the beads produced a reasonable diameter and how narrow is the distribution of bead diameters? To have homogenous conditions throughout the reactor one has to apply beads as identical to each other as possible. This is especially true when

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the bead is processed further, e.g. by a drying step, since beads varying in diameter will be affected differently.

•Does the process require sterile conditions during bead production and how can this be achieved?

•Which production scale of beads is possible? Immobilisation is of particular interest to enhance profitability of biotechnical processes for production of bulkchemicals. This demands the availability of immobilised material in industrial scale. It is important from the outset to consider that a method working under laboratory conditions will have to be upscaled at some point.

When a liquid flows slowly out of a vertical nozzle, the surface tension causes the formation of an orb at the tip of the nozzle until it is released due to gravitation. Since using smaller nozzles, e.g. cannulas, has only little influence on the size of the bead the

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detachment of the drop of liquid has to be forced to make smaller beads. Three main mechanisms are recently in use:

•a real cutting of the liquid.

The principle of these methods is shown in the upper row of figure 2. In addition, this figure presents various atomiser principles. Atomisers were developed for large scale production of particles but they in general produce particles with a rather broad particle size distribution.

2.1. BLOW-OFF-DEVICES

As mentioned above, one possibility to release small beads is to add the force of a continuous flow of air to gravitation (Vorlop and Klein, 1983). This can be achieved by sheathing the cannula for the liquid with a larger cannula that is connected to compressed air. By applying a laminar flow of air and regulating the mass flow of the liquid, the size of the produced beads can be controlled. The described technique demands only little technical equipment but has drawbacks: the throughput is very limited even with multi-nozzle systems, and only fluids with low viscosity can be handled. Due to the outer flow of air the processed material tends to dry at the tip of the cannula and thus causes problems in long-term operation. However, the method is appropriate for most initial experiments under lab conditions.

2.2. VIBRATION

The formation of single droplets from a continuous flow of liquid can also be achieved by applying a vibration either to the outlet nozzle or to the liquid itself. The flowing liquid expresses the shape of the obtruded vibration and is laced at the troughs and thus forms individual droplets (Brandenberger and Widmer, 1997). By altering the frequency of the vibration, the size of the resulting particles can be controlled. The distribution of particle sizes is very uniform and higher throughput is often achieved by using multiple nozzles in parallel. Devices based on this principle have been commercialised by several companies. However, beads with diameters below 1 mm can only be made from fluids with viscosity of up to 300 to 500 mPa· that limits the use of the technique.

2.3. ATOMIZERS

Atomisers use high energy dissipation to form droplets from fluids. The force can either be applied by using a nozzle and compressed air, or by spraying the fluid onto a rotating disk or using similar techniques. Although these devices have a large throughput the bead diameter is only insufficiently controlled and the distribution of particle size is accordingly broad. In addition atomisers only work with low viscous fluids and thus cannot be applied for every task.

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2.4. JETCUTTING

All techniques discussed so far lack the possibility of processing fluids with high viscosity and most also suffer from problems when they have to be scaled up. To counteract these problems we invented the method of JetCutting that works with fluid viscosity up to several Pa· (Vorlop and Breford, 1994).

A continuous jet of fluid is pressed out of a nozzle at high speed and cut into cylindrical segments by means of a fast moving cutting tool. This cutting tool is most often realised as a group of thin wires connected to a rotating device (see figure 3). The cylindrical segments produced by the cutting event form beads due to surface tension while they continue to fall down towards hardening.

The amount of liquid which is slung away during the cutting process depends on several parameters: On the one hand the wire should be kept as thin as possible, on the other hand it is advantageous to use rather a thin jet of fluid which is cut into longer cylinders than to work with a thicker jet divided into short segments.

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As can be seen from figure 4 it is also advisable to incline the plane of cutting in ratio to the direction of the jet, since both elements are subject to a continuous motion (Prüße et al., 1998).

Only with an inclination are the resulting fragments of the jet indeed true cylinders without fraying ends at the top and bottom. These fraying tips tend to spray away and thus increase the amount of recyclable loss.

When using wires of 50 in diameter and setting the optimal inclination the amount of liquid that is slung away is drastically reduced and is below 2% of the processed material.

Since the velocity of the jet of fluid is constant when using a pulsation free pump, and the cutting tool rotates electronically controlled with constant speed, all segments are the same size and resulting beads are monodisperse (see figure 5). The lower limit for beads is approx. at the moment, the upper limit is about 2.5 to 3 mm (depending on the properties of the fluid).

The production rate by JetCutting depends on the flow rate of the liquid and the speed of the cutting tool. The upper limit at the moment is 15,000 beads per second and nozzle, but some 25,000 beads will be possible in the future. Table 1 lists the resulting throughput, i.e. the bead production rate, depending on the bead diameter and the frequency of bead generation.

Depending on the material, special precautions have to be taken to allow a smooth transition into the bath were the beads are collected. Due to the enormous number of beads produced and the high speed of the beads (up to 30 m/s), just taking a stirred bath is not enough for many applications. In these cases, equipment for giving the assimilating fluid a flow like in a geyser, a maelstrom or in a wide gutter have to be used.

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Materials processed so far include the biopolymers alginate, chitosan, carrageenan, gelatine and synthetic materials like polyvinylalcohol and silicon. Applications were the entrapment of microorganisms or enzyme preparations, or the formulation of fragrances, vitamins, and other ingredients for food and pharmaceutical industries on a large scale. In addition, sol-gel materials for preparation of inorganic carriers, or molten waxes for entrapping pharmaceutically active substances have been tested.

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