Journal of Biotechnology 65 (1998) 85 98

Review article

Recent advances in the malting and brewing industry1

Matti Linko *, Auli Haikara, Anneli Ritala, Merja Penttila
VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland Received 28 November 1997; received in revised form 26 February 1998; accepted 2 March 1998

Abstract Brewing is often mentioned as a typical example of traditional or old biotechnology, because of its extremely long history. However, the modern malting and brewing industry applies a whole spectrum of new technical, biochemical, microbiological and genetic inventions. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Malted barley contains all the enzymes needed for all-malt brews. Exogenous microbial enzymes may be needed when using high amounts of cereal adjuncts. Transgenic barleys and proper starter cultures in malting offer interesting new possibilities to ensure balanced enzyme activities and to avoid harmful Fusarium contaminations. High gravity brewing, automated mash lters and hop extracts produced with super-critical or liquid carbon dioxide have been adopted by the industry. Continuous bioreactors with immobilized yeast are already used for maturation of beer. The residence time in the bioreactor is only 2 h, whereas several weeks are needed for traditional lagering. Continuous main fermentation with immobilized yeast is the next step. Several genetically modied brewers yeasts have been constructed, e.g. yeasts encoding h-acetolactate decarboxylase and super-occulating yeasts. The brewing industry is now waiting to be assured of consumer approval. 1998 Elsevier Science B.V. All rights reserved. Keywords: Brewing; Malting; Transgenic barleys; Lactic starters; Genetically modied yeasts; Immobilization

1. Introduction
* Corresponding author. Fax: +358 9 4552028. 1 Based on a lecture held at the symposium Biotechnology in advanced food and feed processing, at the 8th European Congress on Biotechnology (ECB8) in Budapest, Hungary, August 1997.

Beer was produced without any knowledge of microorganisms or enzymes thousands of years ago. Because of this extremely long history brewing is often mentioned as a typical example of traditional or old biotechnology. It is true, of

0168-1656/98/$ - see front matter 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00135-7

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In fact, this traditional industry uses similar methods as those industries referred to as new biotechnology. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Some of the new possibilities opened up have not yet been commercialised, often simply because of a desire to maintain a traditional image and also because of uncertainty about consumers attitudes towards, for example, genetic modication of malting barley or brewers yeast.

2. The brewing process The conventional brewing process is outlined in Fig. 1. The scheme is somewhat simplied in order to keep it clear. This scheme illustrates the production of so-called lager beer with bottomfermenting yeast. There are many variations of the brewing process depending on the specic type of beer. For example, ale and stout are produced with top-fermenting yeasts and the process differs from the production of lager beer. However, malted barley is almost always the main source of starch and enzymes. The whole production procedure consists of four stages: (1) malting (based on germination of barley); (2) wort production (mashing, i.e. extraction and hydrolysis of the components of malt and possibly other cereals, followed by separation of non-soluble components and boiling with hops or hop extracts); (3) fermentation (in most cases divided into primary or main fermentation and lagering or secondary fermentation); and (4) down-stream processing (ltration, stabilization, bottling, etc.).

Fig. 1. Simplied scheme for brewing.

course, that brewing has old traditions, but it is also true that brewing has been and still is in the forefront of biotechnological development. It can be said that Louis Pasteur, the author of Etude sur la biere, built the basement of modern biotechnology. Several other scientists working with brewing have affected the development of biotechnology. A good example is Emil Christian Hansen, who introduced the use of pure yeast cultures. Today the malting and brewing industry applies a whole spectrum of novel technical, biochemical, microbiological and genetic inventions.

3. Barley and malt Malting barley should give high yields on the eld, it should give high extract yields in the brewhouse, it should give suitable activities of many enzymes such as h-amylase, i-amylase, proteases and i-glucanase, it should be as free as possible of harmful microorganisms such as Fusarium fungi, etc. The breeders have been quite

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successful in developing barley cultivars meeting fairly well many of the wanted qualications. Conventional breeding will be important also in the future, but in addition transgenic barleys offer new possibilities. For example, genes encoding certain wanted enzymes may be integrated into the barley genome. Lactic acid starter cultures are commonly used in several elds of food industry. In malting they seem to offer new possibilities for controlling the microora, by preventing the growth of harmful microorganisms and by favoring benecial ones.

3.1. Transgenic barley

Transformation of barley has been rather difcult compared with some other cereals. Cell and tissue culture are needed for successful transformation. Thus the development of gene transfer techniques and of tissue culture systems have been parallel in the case of barley. The rst reports covered procedures for cell and tissue cultures, isolation and regeneration of protoplasts and both transient and stable transformation of tissue cultures (Luhrs and Lorz, 1987, 1988; Kartha et al., 1989; Mendel et al., 1989; Creissen et al., 1990; Salmenkallio et al., 1990; Yan et al., 1990; Chibbar et al., 1991; Jahne et al., 1991; Knudsen and Muller, 1991; Lazzeri et al., 1991; Lee et al., 1991; Luhrs and Nilesen, 1992; Ritala et al., 1993). These works veried that also barley is transformable and that it was possible to study the behaviour of foreign genes in the barley genome. The rst transgenic barley plants were produced by particle bombardment (Potrykus, 1990; King and Kasha, 1993; Jahne et al., 1994; Ritala et al., 1994; Wan and Lemaux, 1994; Hagio et al., 1995; Koprek et al., 1996). Later also protoplasts derived from microspore culture and suspension culture gave transgenic plants by electroporation and PEG-mediated gene transfer (Funatsuki et al., 1995; Salmenkallio-Marttila and Kauppinen, 1995; Salmenkallio-Marttila et al., 1995). In transformation of malting barley the main interest has been to insert a thermostable i-glucanase gene into the genome. Transformation of thermotolerant bacterial (Olsen et al., 1991) and fungal (Mannonen et al., 1993) genes or engineered

native barley genes (Fincher, 1994) has been reported. The fungal gene inserted into the barley genome produced heat-stable i-glucanase, which reduced the viscosity of the wort in mashing experiments (Mannonen, 1993; Aspegren et al., 1995). The rst barley plants expressing thermostable i-glucanase were reported in 1996 (Jensen et al., 1996; Mannonen et al., 1996). The transformation with the bombardment method described by Wan and Lemaux (1994) is strongly dependent on the barley variety. Experiments with Golden promise were quite successful, whereas the Finnish variety Kymppi was difcult: by bombarding 13000 embryos only one transgenic line was produced. However, the i-glucanase produced by transgenic seeds during germination was thermostable (Mannonen et al., 1997). The enzyme retained its activity during 2 h incubation at 65C. The amount of thermotolerant i-glucanase was approximately 0.025% of soluble protein, and this amount was earlier shown to reduce the viscosity of wort (Mannonen, 1993). The fertility of the transgenic line was reduced. This was not observed with an earlier transgenic Kymppi barley expressing neomycinphosphotransferase II (Ritala et al., 1995; Salmenkallio-Marttila et al., 1995). With the i-glucanase line the production of homozygous plants through anther culture failed. The amount of regenerated homozygous transgenic plants was very low: approximately 1% instead of the expected 50%. Furthermore, the homozygous plants were sterile. Recently several transgenic barley plants encoding i-glucanase have been produced from the variety Golden promise in cooperation between the United States Department of Agriculture (USDA) and the Technical Research Centre of Finland (VTT) (unpublished results). An interesting strategy is to cross the transgenic Golden promise lines with other malting barley varieties. By backcrossing assisted with genetic markers the unwanted genome will be washed away. Recently Agrobacterium-mediated transformation has again gained attention. Experiments with rice and maize showed that new Agrobacterium strains and so-called supervirulent vectors were the key prerequisites for successful transformation of

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these monocots (Chan et al., 1993; Hansen et al., 1994; Hiei et al., 1994; Adelmita and Hodges, 1996; Ishida et al., 1996). Now also barley has been shown to be transformable by Agrobacterium. Tingay et al. (1997) reported a very efcient system resulting in a transformation frequency as high as 4% of the treated barley embryos of the variety Golden promise. The same group repeated the experiments with another variety, Schooner (Matthews et al., 1997). This system is very promising.

3.2. Lactic acid starter cultures in malting

Barley and malt give sometimes problems for the brewer. A heavy Fusarium contamination of malting barley may lead to formation of deoxynivalenol (DON) and other mycotoxins (Haikara, 1983; Flannigan et al., 1985; Schwarz et al., 1995; Munar and Sebree, 1997). As a water soluble-compound DON is washed out during steeping of barley, but during germination DON is produced by Fusarium fungi. DON is not removed or destroyed during brewing process. Fusarium contamination may also lead to the so-called gushing of beer, which means quick uncontrolled spontaneous over-foaming immediately when opening the bottle or can (Amaha and Kitabatake, 1981). Fusarium graminearum, Fusarium culmorum and Fusarium poe are active gushing inducers (Haikara, 1983; Niessen et al., 1992; Vaag et al., 1993; Schwarz et al., 1996; Munar and Sebree, 1997). The production of mycotoxins may parallel production of the components responsible for gushing. Strict control of incoming barley lots is, of course, vitally important. However, sometimes in some areas there is simply not enough high quality barley available. Microora management in such a way that harmful organisms are discouraged and neutral or benecial organisms are favoured could minimize the risk caused by microbial contamination of barley. A novel method is to use lactic acid bacteria or Geotrichum candidum as starter cultures in malting to reduce the fungal contamination and to improve the malt quality (Boivin and Malanda, 1993; Haikara et al., 1993; Haikara and Laitila, 1995; Boivin and Malanda, 1997a,b). Ad-

dition of starter cultures ensures high quality of malt regardless of the natural variation of the microora of barley. The effect of lactic acid starter cultures on the malting and brewing processes is based on the microbicidic compounds produced and also on their other characteristics such as enzyme activities. Certain Lactobacillus plantarum and Pediococcus pentosaceus strains are especially efcient when added to the steeping waters of barley at the level of about 107 cells g 1. The whole cultures are needed for the restriction of harmful microorganisms, because the effect of lactic acid bacteria is essentially based on the microbicidic compounds present in the medium (Haikara et al., 1993). The addition of starter cultures in the early stage of malting is important due to the intensive growth of Fusaria during the very rst hours of steeping (Fig. 2). The effect of lactic acid bacteria depends on the composition of the ora and on the contamination level of the barley. However, numerous laboratory and pilot experiments using barley crops from different years as well as industrial scale trials have conrmed the fungicidic effect of starter cultures. Fig. 2 demonstrates the clear reduction in the occurrence of Fusarium contamination during malting when L. plantarum has been applied. Lactic acid starter cultures also restrict the growth of harmful Gram-negative and -positive bacteria, which compete with grain tissue for dissolved oxygen and may retard mash ltration (Haikara and Home, 1991; Doran and Briggs, 1993). A marked reduction in aerobic bacterial ora has been observed throughout the malting process when starter cultures have been applied (Haikara et al., 1993; Haikara and Laitila, 1995). Pseudomonas species are especially sensitive. The use of lactic acid bacteria in malting has also led to signicant improvements in the physical and chemical quality of malt (Haikara et al., 1993; Haikara and Laitila, 1995). i-glucans are known to cause a number of problems during the brewing process. These polysaccharides may cause slow lautering, poor beer ltration, and formation of haze during storage of packaged beer (Home, 1993; Narzi, 1993; Bamforth, 1994). A signicant advantage of the use of lactic starters is

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Fig. 2. Effect of lactic acid starter culture (Lactobacillus plantarum) on the occurrence of Fusarium fungi during malting of barley. The vertical lines delimit the three stages of the malting process, steeping, germination and kilning. The lactic cultures were added 1 and 20 h after steeping in.

improved mash lterability (lautering performance). This is important, because lautering is often the bottleneck of brewhouse operations. Fig. 3 shows the increased ltration rates (volumes) measured by the Buchner ltration test (Sjoholm et al., 1994). The contribution of lactic starter cultures to

Fig. 3. Effect of lactic starter cultures (Lactobacillus plantarum, Pediococcus pentosaceus) on the mash lterability.

lautering performance can be partly explained by their own enzyme activity, partly by inuencing the microbial i-glucanase activity present in the grains. High activities of i-glucanase at 60C were found in treated malt indicating the contribution of thermostable microbial i-glucanase in the starter malts (Haikara and Laitila, 1995). In addition, characterization of the molecular mass distribution of i-glucans by gel permeation chromatography revealed a lower molecular size for the wort i-glucans when starter cultures were applied to the rst and second steeping waters of barley (Suortti, 1993). The molecular size of these polysaccharides seemed to be closely related to the ltration performance. The xylanase activities of the starter malts were higher than those of the control malts, possibly indicating enhanced modication of the barley kernels. The malt yield was higher due to inhibition of rootlet growth. Extract yield was also somewhat higher than that of the control malts. The effects of lactic acid bacteria on mycotoxin production are not well documented. Some reports indicate that aatoxigenic moulds produce smaller amounts of aatoxin in the presence of lactic acid bacteria (Karunaratne et al., 1990; Gourama and Bullerman, 1995). Addition of lactic acid bacteria into the steeping waters retarded

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the formation of DON during the malting process (Laitila et al., 1997). In the presence of Lactobacillus plantarum or L. acidophilus starter cultures the DON content of malt was decreased by up to 70%. Also zearalenon formation was diminished by up to 50% when using L. plantarum in malting of heavily (articially) contaminated barley (Laitila et al., 1997). Because wide variation exists in toxigenic potential between individual strains of particular Fusarium species as well as in their growth inhibition caused by lactic acid bacteria more data are needed for the verication of retarded mycotoxin production in starter maltings. The high quality of beer must be ensured when new techniques are applied to the malting and brewing processes. Numerous pilot brewing trials have shown that the aroma proles of the starter beers are within normal variations. Starter beers have compared well organoleptically with control beers and their avour stability has been better (Haikara and Laitila, 1995).

4. The brewhouse Wort is produced in the brewhouse in several successive unit operations and unit processes. After grinding the malt is mixed with water, the temperature is slowly increased for extraction and enzymatic hydrolysis of malt constituents, primarily starch but also other components, such as proteins and i-glucan. The non-soluble parts are separated either in an automatic mash lter or in a so-called lauter tun, in which the non-soluble constituents themselves form a lter layer. The wort is then cooked with hops, the precipitate is removed in a wort cyclon and nally cooled. Production of wort is still a series of batch processes and operations. Some attempts towards continuous wort production were made already a long time ago, but without much success. However, a continuously operated brewhouse would be attractive especially if the subsequent stages, main fermentation and maturation (secondary fermentation) were continuous. A combination of batch and continuous stages is often problematic. It may require large buffer tanks, which in turn

decrease the advantages and create additional problems in terms of microbial contaminations. Wort is quite vulnerable before the addition of yeast. It is only logical that adopting continuous fermentation brings construction of a continuous brewhouse into focus again. Sterile wort would then be pumped without delay to the fermenters. During recent years perhaps the most important technical novelty in the brewhouse operations has been the use of high gravity or very high gravity brewing (DAmore et al., 1991). This means that the wort is produced and subsequently fermented at a substantially higher concentration than needed for the nal beer. After fermentation the beer is adjusted to the desired concentration with oxygen-free water. The increase in capacity is very signicant. Very high gravity worts, i.e. over 20P, are achieved by adding some kind of syrup to the wort kettle (McCaig et al., 1992). High gravity brewing is quite demanding in terms of the capacity of the lauter tun or mash lter. Automated mash lters make a cycle of 2 h possible (Jaaskelainen and Ranta, 1996). Lauter tuns with huge diameters are catching up with the capacity of these lters. The pitching rate in high gravity brewing has also been studied (Suihko et al., 1993). The quality of hop products is, of course, also important for the avour of the nal beer. Baled hops have mostly been replaced by various concentrates, pellets etc. Instead of organic solvents, super-critical carbon dioxide or liquid carbon dioxide is used for extracting hops. This guarantees high quality of the extract without any solvent residues (Laws et al., 1979).

5. Yeast and fermentation Ethanolic fermentation in itself is technically relatively simple. However, when producing beer or wine the most important aim is a balanced avour, not only efcient fermentation and high ethanol yield. It is not an easy task to reach the desired avour composed of numerous compounds. It is wise to remember that good beer can be produced following the old traditions and that there is or at least has been a good reason for all

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the details included. There has been plenty of time for trial-and-error development. On the other hand, in the old times many details were dictated by limited technical possibilities. For example, cooling of large amounts of beer in lager tanks took a long time, whereas a sharp drop in temperature is easy with modern heat exchangers. Most of the avour compounds are produced during the main (primary) fermentation. Maturation (secondary fermentation, lagering) is necessary and important, but relatively few changes occur during this stage. However, in the traditional brewing process lagering takes a long time, several weeks or even months. Time is money, very literally, because this means huge cellars with numerous large tanks full of maturing beer. This leads a biotechnologist to a logical question: is it really necessary to do it this way? Is there not any possibility to shorten the fermentation without changes in the quality of the nal beer? The answer to this question requires detailed knowledge of complicated metabolic pathways. A lot more is happening simultaneously with the wellknown Embden Meyerhof Parnas pathway from sugar to ethanol (Gancedo, 1994). A key compound in maturation is diacetyl, which is always formed as a byproduct in main fermentation. Its formation is closely connected with amino acid metabolism. This compound is an essential component of butter avour, but in lager beer it is unpleasant and its concentration should be decreased below the taste threshold. Unfortunately the taste threshold is very low, 0.05 mg l 1 or less. At the end of the main fermentation the concentration of diacetyl is way above the taste threshold. During the traditional long lagering at a low temperature it decreases slowly, ending up below the taste threshold, and the beer is matured. The formation of diacetyl from h-acetolactate is a slow non-enzymatic reaction. The subsequent reactions are enzymatic: reduction of diacetyl to acetoin and further to 2,3-butanediol (see Fig. 4). There is also an enzymatic way directly to acetoin from h-acetolactate, but unfortunately brewers yeast lacks the enzyme h-acetolactate decarboxylase (Fig. 4). Several species of bacteria produce this enzyme. The taste threshold of acetoin is

Fig. 4. Diacetyl is a key compound in maturation. Brewers yeast lacks the enzyme h-acetolactate decarboxylase (hALDC), whereas several bacteria produce this enzyme capable of metabolizing h-acetolactate directly to acetoin (dotted line).

much higher than that of diacetyl. By proceeding directly to acetoin the off-avour problem is solved. Looking at this series of reactions, what are the options to speed up the maturation of beer? The missing enzyme, h-acetolactate decarboxylase, can be produced separately and added during the main fermentation to make the short-cut from h-acetolactate to acetoin (Rostgaard-Jensen et al., 1987). In fact, this enzyme is now commercially available.

5.1. Genetically modied brewers yeasts

Instead of adding an exogenous enzyme during fermentation the gene encoding h-acetolactate decarboxylase can be integrated into the yeast genome. The genes of Klebsiella terrigena, Enterobacter aerogenes (Suihko et al., 1989, 1990; Blomqvist et al., 1991; Blomqvist, 1992) and Acetobacter xylinum (Yamano et al., 1995) have been used. The promoter has been PGK or ADH. The use of a modied alcohol dehydrogenase (ADH 1) has recently been reported (Onnela et al., 1996). Another approach towards low diacetyl has been the mutation of ILV2 and ILV5 (Gjermansen et al., 1988).

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Pilot scale studies have conrmed that with the yeasts encoding h-acetolactate decarboxylase the diacetyl concentration can be kept low, even below the taste threshold at the end of the main fermentation (Kronlof and Linko, 1992; Linko et al., 1993). The technical and economical advantage is very signicant. However, these yeasts are not yet used in the brewing industry simply because there is some uncertainty about consumers attitudes. The opinions of the consumers should not be overlooked, not even if a possible negative reaction would be based only on prejudice and lack of information. The consumers probable reaction is being investigated at present (Frewer, 1997). In addition to yeasts that keep diacetyl low, some other interesting genetically modied yeasts have been developed. Glucanolytic yeasts have been developed to facilitate the ltration of beer (Enari et al., 1987; Penttila et al., 1987; Suihko et al., 1991). Amylolytic yeasts have been constructed for production of low carbohydrate beers and for maximizing fermentation efciency (Vakeria et al., 1996). Super-occulent yeasts may be valuable, e.g. in continuous fermentation (Watari et al., 1994). Progress in developing genetically modied brewers yeasts has recently been reviewed (Hammond, 1993, 1995).

by yeast. This is the basic concept of maturation with immobilized yeast in a continuously operated bioreactor. The process scheme is outlined in Fig. 5 (Pajunen, 1995). When replacing the traditional lagering with a completely different maturation system it is essential to do this without any change in the avour of the nal beer. Indeed, this has been successfully done in full scale (Pajunen et al., 1991; Pajunen, 1995). There is no need to change anything in the main fermentation prior to the new maturation procedure. However, some precautions are important. To avoid formation of off-avours and technical difculties the yeast must be almost completely removed from the green beer before

5.2. Maturation with immobilized yeast

There is also another way to cope with the diacetyl problem, in addition to the use of h-acetolactate decarboxylase. The bottleneck in maturation is the slow spontaneous (non-enzymatic) formation of diacetyl from h-acetolactate. This reaction, as all reactions, proceeds more slowly at low temperatures. The traditional lagering temperature is close to 0C. At high temperatures diacetyl is formed within a few minutes from h-acetolactate (Baker and Kirsop, 1973). For example, a heat treatment for 10 min at 90C is sufcient to convert all available h-acetolactate to diacetyl and partly directly to acetoin (Pajunen, 1995). Some diacetyl seems to be formed even at the highest temperatures used (Yamauchi et al., 1995). The diacetyl can then be reduced to acetoin

Fig. 5. Process scheme for maturation with immobilized yeast (Pajunen, 1995).

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entering the heat exchanger. Any entering of air into the separator must be prevented. Oxidation during the subsequent heat treatment would lead to formation of carbonyl compounds (Gronqvist et al., 1993). The temperature in the bioreactor with immobilized yeast is typically 10 15C. A residence time of 2 h or even less is sufcient to reduce all diacetyl into acetoin (Gronqvist et al., 1989). Down-stream processing after the bioreactor is conventional, actually somewhat easier than normally, because there is less growth of yeast. This means also some savings in raw materials. Several carriers have been tested for immobilization. At least two carriers are at present used in full scale: DEAE-cellulose with additions of titanium dioxide and polystyrene (Pajunen, 1995) and porous glass (Hyttinen et al., 1995). Reactors are operated by either downow or upow. One of the options is the use of wood chips, not in the same way as in traditional lagering, but in the form of small chips similarly to any other carrier in continuous bioreactors (Linko et al., 1997). Another option is to use tubular silicon carbide units (Andries et al., 1995).

5.3. Continuous main fermentation

Continuous processes are favoured in many elds of industry, for obvious economic reasons. The same trend is true also in biotechnology, but the endeavors towards continuous production have not always been successful. The main difculties encountered are keeping the system aseptic during a period of at least several months and the complicated relations between growth of the microorganism and product formation. If the product is beer or wine there is an additional and most crucial question: how can the desired traditional avour, based on a balance of numerous compounds, be achieved? Some 30 years ago several groups in different countries studied continuous fermentation. In most cases the outcome was disappointment and abandonment although some projects reached the industrial stage. After decades of experience the version developed in New Zealand seems to be successful (Dunbar et al., 1990).

Then, after the many disappointments, the new magic word immobilization reached the brewing industry and restored enthusiasm. Actually only the term was new; the technique had been used quite traditionally for example in production of acetic acid in which wood chips are used as carriers. Anyway, some groups started again to study continuous fermentation in brewing, this time with immobilized yeast. Main fermentation is biochemically rather complex. Most of the avour compounds are formed at this stage. Moreover, a number of technical problems in a continuously operated bioreactor must be solved. These include removal of excess yeast, removal of carbon dioxide, sustaining of yeast viability, optimization of oxygen (air) feed, prevention of microbial contaminations, prevention of clogging or channelling of the reactors, and regeneration of large amounts of carrier. The most severe economical limitation is the price of the carrier. In main fermentation the residence time in the bioreactors would be at least one day, which is 10-fold longer than with the system developed to replace lagering. This means that the total bioreactor volume needed is also 10-fold greater. Consequently, the carrier cost is quite decisive in terms of the economic feasibility. In general, the following properties, at least, should be considered when choosing the carrier (Linko et al., 1997): price; convenience of immobilization; type of immobilization; cell loading; mass transfer limitations; channelling or blocking of the reactor; stability; rigidity; regeneration; sterilization; binding of contaminating microorganisms; possibility to use various reactor designs; possibility of uidization (also for regeneration); approval for food use. Many of these points may alone prevent the use of a carrier, for example a high price. Some of them are interdependent: if the price is very low, there is not necessarily any need for regeneration. At least the following types of carriers are available at present: cellulose derivatives, porous glass, silicon rubber, diatomaceous earth, silicon carbide and wood chips (Linko et al., 1997). The price of wood chips is a fraction of the price of other available carriers. Several species of wood, e.g. beech, aspen or birch, may be useful in the

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M. Linko et al. / Journal of Biotechnology 65 (1998) 8598 Table 1 Advances in the malting and brewing industry Subject Transgenic barleys Lactic starters in malting High gravity brewing Liquid CO2 hop extracts Genetically modied yeasts h-Acetolactate decarboxylase Amyloglucosidase i-Glucanase Super-occulants Maturation with immobilized yeast Main fermentation with immobilized yeast Status Rapid development Full-scale trials Common practice Industrial plants Technically ready Technically ready Technically ready Technically ready Industrial plants Pilot plants

form of small relatively uniform chips: length and width a few millimeters. The wood chips used in traditional brewing to facilitate secondary fermentation and clarication of beer are much bigger, length 400500 mm, width 40 60 mm, thickness 1 3 mm. Regeneration is, of course, necessary if the carrier is expensive. The possible regeneration procedure should be simple, preferably in the reactor, without discharge of the carrier. A more attractive alternative would be a carrier cheap enough to make the regeneration unnecessary. Wood chips could meet this requirement. Anyway, wood chips could also be treated with hot water or steam. A large amount of yeast per reactor volume is a clear target when aiming at an efcient process. It is generally agreed that also continuous fermentation should proceed through changing stages, resembling the conventional batch fermentation, to ensure proper avour formation. This can be achieved with plug-ow through the reactor. An alternative would be a series of CSTRs (continuously stirred tank reactors). A small feed of air into the bioreactor is necessary. This allows some growth of the yeast and leads to balanced avour formation. Aeration can be arranged in a prefermenter, in which the rst part, 2050%, of the main fermentation occurs (Kronlof, 1994; Kronlof et al., 1995) In the Japanese version the rst stage is a CSTR (Yamauchi and Kashihara, 1995). In principle any brewers yeast strain can be immobilized, including genetically modied yeasts (Kronlof and Linko, 1992). Strongly occulent yeasts are especially suitable, because a high yeast concentration can be easily achieved and maintained. This is quite pronounced when comparing a genetically modied super-occulent yeast with its parent strain, but it can also be seen in a comparison of weakly and strongly occulent yeasts taken from industrial beer production (Watari et al., 1994; Linko et al., 1997). In all bioreactors with immobilized yeast a signicant amount of cells are actually free, not bound to the carrier. It is difcult to measure the proportion of free yeast and even more difcult to elucidate the participation of the free cells in the fermentation, especially when using porous carriers.

The occulation characteristics of yeast affect the performance of immobilized yeast bioreactors in many ways. The extreme is a genetically modied super-occulent strain, which is not useful in conventional batch fermentation because of too early occulation and sedimentation. The carrier used for this type of yeast should obviously be coarse enough to allow smooth ow through the bioreactor. It seems possible that instead of typical immobilization on a carrier simply some kind of supporting structure could be designed to prevent the yeast from forming too tight blocks in the reactor (Linko et al., 1997). This way of thinking approaches reinventing the once abandoned tower fermenters (Klopper et al., 1965). However, with a super-occulent yeast the size of the fermenters could be less gigantic because of the extremely high yeast concentration.

6. Conclusions Some recent achievements and their present status in the malting and brewing industry have been presented in Table 1.

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Big breweries produce typically 500 million litres of beer in a year, some of them even more. These breweries must be efcient to survive in the rude world of competition. They also have to be sure that the quality of beer is always good and uniform. Big breweries are obviously interested in all technical novelties making the processes cheaper, easier and safer. At the same time they must guard the image of their products carefully. This may cause some delay in adopting new techniques, even when it can be conrmed that there is no change in the quality of the nal beer. The approach of small breweries is quite different. Limited quantities of interesting special beers can be produced with somewhat higher costs and each batch may also be slightly different in avour. In fact, small breweries and their products are very popular in many countries.

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