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Stabiliser addition

Water

Immobilised yeast bioreactors

Holding tank Filter Heat exchanger

Immobilised Yeast Brewing

Sterile de-aerated dilution liquor

Fig. 5.43 Schematic of a plant for alcohol-free beer production using immobilised yeast bioreactors (redrawn from van Dieren, 1995).

Sterile de-aerated dilution liquor

Fig. 5.43 Schematic of a plant for alcohol-free beer production using immobilised yeast bioreactors (redrawn from van Dieren, 1995).

Inlet

Carrier and biomass bed

Inlet

Carrier and biomass bed

Gas outlet / pressure relief

Sample point

Outlet

Fig. 5.44 Immocon immobilised yeast bioreactor (re-drawn from Mieth, 1995).

Gas outlet / pressure relief

Sample point

Outlet

Fig. 5.44 Immocon immobilised yeast bioreactor (re-drawn from Mieth, 1995).

this case containing immobilised bacteria, Lactobacillus amylovorus. The acidified wort is stored in a holding tank. Before delivery to the immobilised yeast bioreactors, the wort is filtered to remove solids (which could cause clogging) and it is cooled by passage through a heat exchanger. The process is described by Back and Pittner (1993) and Pittner et al. (1993).

The reactors have a capacity of 1.5 m3 and each contain 1 m3 (400 kg) of carrier. The plant is of a modular design with series of reactors. This arrangement provides operational flexibility to allow for fluctuations in demand and permits regeneration and start-up of individual reactors without disrupting production. Regeneration is performed by treating the used carrier with 2% w/v sodium hydroxide at a temperature of 80°C, followed by neutralisation with sterile carbonated water. Regenerated carrier is returned to the reactor and this is then inoculated with a pure yeast culture. Aerated wort at a temperature of 12°C is then circulated for 24 hours through the through the reactor. This promotes yeast growth and allows attachment to occur. At this stage, the reactors contain approximately 50 x 106 yeast cells per gram carrier. Wort flow commences at a rate of approximately 5 hi h 1 and the temperature is held at 4°C. To minimise wort oxidation, strictly anaerobic conditions are maintained. Gradually the flow-rate is increased to 20 hi h 1 and the temperature reduced to between 0 and 1°C. During this stabilisation phase, the yeast concentration doubles to 100 x 106 cells per gram carrier. Wort flow is from top to bottom and because of the lack of mixing is virtually of an ideal plug type. When working at full capacity, individual reactors have productivity of the order of 20 hi h l. Steady state conditions are never achieved since yeast growth continues even at the low operating temperature. Reduction in flow rates due to the high biomass loading eventually becomes rate limiting, thereby providing the requirement for periodic regeneration.

The low temperature and anaerobiosis ensures very limited fermentation and yeast growth but apparently has little, if any, inhibitory effect on the ability of the yeast to reduce the carbonyls which contribute to undesirable 'worty' character. Thus, reduction of 2-methyl propanal, 2-methyl butanal and 3-methyl butanal were reduced by 100, 61 and 48%, respectively. Van Dieren (1995) compared the sugar spectrum of the wort and issuing beer and observed small increases in both the concentrations of glucose and fructose. This was at the expense of a 64% reduction in sucrose levels. Maltose and maltotriose remained virtually unchanged. The ethanol concentration never rose above 100 (ig 1 1. From these data it was concluded that the combination of plug flow, low temperature, low contact time and anaerobiosis ensured that only 20% (7 mM) glucose was utilised by the yeast and that this mainly derived from sucrose hydrolysis. The continued presence of glucose repressed uptake of maltose and maltotriose. Some esters and higher alcohols were formed although at lower levels than would be expected in a standard, full strength lager.

Despite a less than convincing flavour match with standard beer, this plant has been used extensively throughout the world. Thus, Mieth (1995) reported production of 700 000 hi per annum using this system. Operating costs reportedly compare very favourably with other methods for alcohol-free beer production.

5.7.2.2 Continuous maturation. Production scale plant using immobilised yeast reactors for continuous beer maturation has been in operation at the Sinebrychoff Brewery in Helsinki, Finland, since 1990. A new installation capable of producing 1 million hi of beer per annum came on-stream in 1993 (Pajunen & Gronqvist, 1994). The decision to use this approach was taken at a time when an entirely new brewery was constructed on a green-field site (Pajunen & Jaaskelainen, 1993). This is worthy of note, as there will always be a natural inertia which will tend to resist replacing existing plant and process with a substitute that is novel and relatively unproven. Undoubtedly this factor will continue to be the biggest barrier to the more widespread adoption of techniques such as continuous maturation.

The plant is similar, with some modifications, to that used for the production of alcohol-free beer, as described previously (Section 5.7.2.1) and it also uses the same DEAE-cellulose carrier (Spezyme GDC®). Layout of the plant is illustrated in Fig. 5.45. Green beer is delivered from conventional fermenters via a high-performance continuous centrifuge. This serves two purposes. It reduces the yeast count in the green beer stream to a minimum level (< 105 cells ml *), which is essential for avoiding the development of yeast autolytic off-flavours in the subsequent heat treatment. In addition, centrifugation reduces the solids loading of the process flow entering the immobilised yeast reactors, thereby reducing the likelihood of clogging. The authors also noted that the clarification step resulted in an absence of tank bottoms down-stream of the centrifuge and that this contributed to overall process efficiency by minimising beer losses.

After centrifugation the beer is subjected to a heat treatment (90°C for 7 min.) during which a-acetolactate is converted to diacetyl. It is essential to maintain

Green beer feed from primary fermenters

Centrifuge

Heating

Centrifuge

Heating

Fig. 5.45 Continuous maturation using immobilised yeast.

anaerobic conditions during this treatment in order to avoid undesirable oxidation reactions. In addition, in the absence of oxygen, a proportion of the a-acetolactate is converted directly to acetoin, thereby reducing the requirement for diacetyl reduction in the subsequent bioreactor step. The heat treatment has other benefits. It reduces the microbiological loading of the beer which in turn lowers the risk of contamination of the bioreactors. Further, it denatures proteases, which if allowed to persist, can reduce the concentration of foam positive proteins in the finished beer (Muldbjerg et al., 1993). No changes in flavour or colour were observed during the heat treatment apart from increases in the concentrations of the carbonyls, furfural and phenyl ethanal.

Following the heat treatment the beer is cooled to 15°C and then passed through the immobilised yeast bioreactors. The production scale plant has four bioreactors each with a capacity of 7 m3. Contact time within the reactors is 2 hours. Diacetyl is converted to acetoin and 2,3-butanediol, carbonyls are reduced to the same levels, or less, than present in the beer prior to the heat treatment and residual fermentable sugars are assimilated (Gronqvist et al., 1993). A further benefit is that the concentration of some short-chain fatty acids declines during passage through the bioreactors. This was ascribed to non-specific binding to the carrier as opposed to assimilation by the yeast. No changes were observed in the levels of esters and higher alcohols.

The beer issuing from the bioreactor has a yeast count of less than 105 cells ml 1 and is reportedly relatively clear. At this stage, it is cooled to — 1.5°C, prior to further stabilisation treatment. As with the alcohol-free beer bioreactor described in Section 5.7.1.1, the modular design provides operational flexibility. Each reactor may be operated continuously for periods of two to four months, after which time they must be regenerated. This is achieved by treating the carrier with sodium hydroxide (2% w/ v) at a temperature of 80°C. This may be performed in the bioreactor vessels or the carrier may be pumped in the form of slurry to a separate treatment tank. The plant may be operated in a continuous or semi-continuous mode. In the latter case, the process flow may be discontinued for periods of up to a few days as dictated by production requirements, with no apparent detriment to subsequent performance or product quality.

Other large-volume continuous maturation plants exist that are essentially the same as the one described, although different carriers may be employed. For example, Hyttinen et al. (1995) described a production scale facility currently in use at the Hartwall Brewery, also in Finland. In this case, a porous glass carrier (Schott Engineering, Germany) is employed contained in two reactors, each with a volume of 2.5 m3. The direction of flow is upward, which is claimed to give less channelling and plugging compared to the more conventional downward flow. Separation of yeast prior to heat treatment is performed in a hermetically sealed centrifuge with a throughput of 30 hi h l. The heat treatment is 80°C for 10 minutes at a pressure of 3 bar. Residence time within the bioreactors is 0.5 volumes per hour, which gives a volumetric productivity of 2 x 106 hi per annum.

Yet again, the authors stress the need for strictly anaerobic conditions to be maintained throughout the process to maintain product quality. Provided this was done, the total VDK (a-acetolactate + diacetyl) decreased to 30% of the initial concentration after the heat treatment and to 17% after passage through the bioreactor. Presumably, the large change after the heat treatment represents the proportion of a-acetolactate converted directly to acetoin. This is in accord with Inoue et al. (1991) who contended that the bulk of a-acetolactate is converted directly to acetoin by heat treatment (70°C for 30 minutes) under strictly anaerobic conditions and in the absence of yeast. These authors suggested that this approach could be used to replace the immobilised yeast step, or at least reduce the requirement for diacetyl reduction by yeast. Nonetheless, unless very careful control could be guaranteed it would be suspected that the majority of brewers would view such a drastic heat treatment with some trepidation.

The glass carrier would seem to have some operational advantages compared to DEAE-cellulose. Thus, Hyttinen et al. (1995) claimed that with careful handling the same column could be kept in constant operation for periods of up to one year, before regeneration became necessary. However, this did include some downtime for periodic re-pitching. Regeneration is accomplished in situ by treatment with hydrogen peroxide, acid washing and rinsing.

Another facility, albeit only at pilot scale, is described by Groneick et al. (1997). This has been installed at the Iserlohn Brewery in Germany and also uses sintered glass as a carrier. The plant is of the same design as described already; however, the authors make the valuable point that the totally inert nature of the carrier means that the process does not contravene the German beer purity laws.

5.7.2.3 Primary fermentation with immobilised yeast. There is no production scale immobilised yeast bioreactor system currently in use that is capable of carrying out primary fermentation. The immediate likelihood of success remains a remote possibility; however, a few systems have progressed to pilot scale. These share the common feature of having two or more vessels to allow the separation of the stages of primary fermentation necessary for the development of a balanced spectrum of flavour components (see Section 5.7.1). Two systems are described here which appear to show encouraging results.

Yamauchi et al. (1994) reported results obtained with a pilot scale three-stage system shown in diagrammatic form in Fig. 5.46. The first vessel consists of an aerobic stirred reactor of 200 hi capacity (100 hi working volume) maintained at 13°C by cooling through external jackets. A mechanical agitator is provided from the base of which sterile air is introduced. This arrangement ensures good mixing and oxygen solution. Wort is fed in at the top of the vessel close to the wall to avoid undue foaming. The rate of wort addition is controlled automatically, in response to continuous in-tank measurement of the specific gravity within the vessel. The aerobic and highly agitated conditions promote yeast growth at the expense of wort amino nitrogen. Consequently, there is a marked fall in pH and production of considerable higher alcohols.

Wort feed

Wort feed

1 r Continuous centrifuge Yeast

Heating Cooling

Fig. 5.46 System for continuous primary fermentation and maturation using immobilised yeast (adapted from Yamauchi et al., 1994).

1 r Continuous centrifuge Yeast

Heating Cooling

Fig. 5.46 System for continuous primary fermentation and maturation using immobilised yeast (adapted from Yamauchi et al., 1994).

The process stream issuing from the stirred reactor is fed through a continuous centrifuge which reduces the yeast count to less than 1 x 106 cells ml \ prior to infeed into two linked immobilised yeast reactors. These vessels each have bed volumes of 100 hi and use a ceramic bead carrier. The high rates of exothermy associated with primary fermentation and the lack of mixing necessitate high cooling capacity to maintain the set-point temperature of 8°C. To accommodate this, both external wall jackets and internal heat exchanging tubes are provided. In the second stage bio-reactor the apparent extract falls from 8°P to 2.5°Plato. It is during this treatment the bulk of the ethanol and esters are formed.

The third stage vessel is of similar design to the second and is used for continuous maturation. Prior to entry, the green beer is passed through an in-line heat exchanger where it is heated (60 to 80°C for 23-60 min) to convert a-acetolactate to diacetyl and acetoin, then cooled to approximately 0°C.

The total residence time for the process is given as 3-4 days. Times for the individual steps are 20-24 hours in the stirred vessel, 24^8 hours in the second stage immobilised yeast reactors and 24 hours in the maturation vessel. This gives a total process time of 72-96 hours. Comparative data for conventionally and continuously produced beers indicated significant differences, apparently related to altered patterns of yeast growth. Thus, the trial beer contained more ethanol, had a higher pH, increased sulphur dioxide, elevated bitterness and lower levels of higher alcohols. There were also differences in the contents of organic acids, notably reduced pyruvate and acetate but increased citrate, succinate and lactate. Apart from the differences in beer analysis, the process was shown to be unstable such that although six months' operation was achieved, it was not possible to attain a steady state and this resulted in continuously changing performance.

Andries et al. (1997b) reported experiences with a two-stage continuous primary fermentation system which utilises an immobilised yeast reactor of the type shown in Fig. 5.44(d). In this reactor, described in Section 5.7.1.3, the yeast is immobilised in silicon carbide. The in-flowing wort is circulated through the silicon carbide matrix in the first reactor, prior to being transferred to the second vessel. This is of cylin-droconical design and contains free suspended yeast. Good mixing is promoted by an external circulation loop. Attemperation of both reactors is achieved by in-line heat exchangers fitted to the recirculation loops (Fig. 5.47).

In the trials reported by Andries et al. (1997b) using a top-fermenting ale yeast, the residence time in the immobilised reactor was 8 hours at a temperature of 24°C. The

Immobilised reactor

Stirred reactor

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