Fig. 6.11 Wort collection of 2000 hi cylindroconical ale fermentation filled with 5 x 400 hi batches of wort. Yeast slurry was pitched with the first and fourth batches of wort at the times indicated. The wort volume is shown together with the measured yeast count and predicted yeast count, assuming no growth (Boulton & Clutterbuck, unpublished data).

during wort collection and taking into account the dilution effect of the increasing wort volume.

As may be seen during the first 5 hours there was no increase in cell count and the actual and predicted yeast counts could be superimposed. Some 6 hours into collection the actual and predicted cell counts began to gradually diverge indicating that cell proliferation had commenced. At the end of addition of the third batch of wort, immediately prior to injection of the second tranche of pitching yeast, the measured cell count was 5.7 x 106ml 1. This compared to a predicted count of 1.7 x 106 ml \ assuming no growth had occurred. After 20 hours, when collection was complete, the actual cell count was 10.3 x 106, compared with a predicted count of 2.5 x 106ml 1. At this time, the yeast pitched with the fourth batch of wort would have been exposed to oxygenated wort for 8 hours. The first pitched yeast, after 8 hours, had increased in number by a factor of 1.5. Assuming the same behaviour for the later pitched yeast and subtracting this figure from the total count at 20 hours, this indicated that at this time the first pitched yeast had increased 8-fold, or 3 budding cycles. In fact, microscopic examination of the yeast cells at 20 hours revealed a more complex picture. Thus, prior to the addition of the second batch of yeast the majority of the cells were in the form of short chains and clearly in the process of active proliferation. At 20 hours, the majority of cells had a similar appearance; however, there were also significant numbers of larger single cells showing no obvious signs of budding. This suggested that the late-pitched yeast (which at the time of addition would still have been in the stationary phase) was unable to compete for wort nutrients and oxygen with the actively growing yeast cells derived from the first pitch.

When stationary phase yeast is exposed to oxygen, whether suspended in wort or not, it has a relatively low affinity for assimilation of oxygen. With prolonged exposure to oxygen, there is a marked increase in the ability of the yeast to take up this nutrient (see Section At a temperature of 20°C, the specific oxygen uptake rate (% decrease in oxygen saturation per minute per gram dry weight of yeast) increases roughly five-fold during 3-5 hours' exposure to oxygen (Boulton et al., 1991). Under carbon catabolite repressed conditions, such as would be the case in aerated wort, this relatively higher affinity for uptake of oxygen is maintained. It is likely, therefore, that in the case of this fermentation, the later pitched yeast would have a much lower affinity for oxygen than that pitched earlier and possibly would be at a competitive disadvantage. If yeast is pitched continuously throughout prolonged wort collection, the situation is even more complex. In this case, it would be predicted that when collection was complete there would be a mixed population of yeast with varying affinity for oxygen, and therefore inconsistent physiological condition. Clearly, this cannot be conducive to good fermentation control.

This example highlights the fact that yeast pitching and wort oxygenation must be co-ordinated processes. Thus, the length of time that yeast is exposed to oxygen is as significant to subsequent fermentation performance as the total mass of oxygen supplied. The data in Fig. 6.12 shows a recording of the output from a dissolved oxygen probe suspended in fermenter during wort collection of the fermentation described in Fig. 6.11. It may be seen that as each new batch of wort was added to the vessel it was accompanied by an additional charge of oxygen. This dissolved oxygen decreased due to consumption by the yeast during the periods when wort pumping stopped. Therefore, the rate of decline of dissolved oxygen concentration during these periods provided an indication of the oxygen uptake rate due to the yeast. These rates increased with each consecutive addition of batch of wort. The increased affinity of the yeast for oxygen was further demonstrated by the fact that rates of increase and peak values of DOT associated with fresh wort additions decreased with each consecutive batch of wort. In part, this must have been due to the gradual increase in cell count. However, this would not explain the increase in oxygen uptake rates between

Fig. 6.12 Trace of dissolved oxygen concentration of wort in fermenter during collection of a 2000 hi ale fermentation filled with 5 x 400 hi batches of air saturated wort (Boulton & Clutterbuck, unpublished data).

the first and second batches of wort since during this same time period there was little change in yeast concentration (Fig. 6.11). In fact, during the first 8 hours of collection, the yeast count declined slightly due to dilution by the in-flowing wort. The oxygen uptake rate did not increase markedly with collection of the fourth batch of wort. This coincided with addition of the remainder of the pitching yeast, perhaps also indicating that the later pitched yeast made only a small contribution to total oxygen consumption and by inference to the subsequent fermentation.

O'Connor-Cox and Ingledew (1990) studied the effects on fermentation performance of the timing of oxygen addition. This has been discussed in Section 6.1.2. However, these authors concluded that it might be advantageous to pitch yeast with the first brew-length under anaerobic conditions. Oxygen should only be added with subsequent brew-lengths. The utility of this approach has as yet been explored at laboratory scale only. Undoubtedly it merits further investigation.

Several general lessons may be drawn from these examples. In the first instance, the introduction of large capacity vessels without concomitant improvements in the capabilities of the brewhouse should perhaps be discouraged. If this is not possible, and fermenters require several hours to fill with multiple batches of wort, the manner of oxygenation and pitching should be given careful consideration. In order to establish a rapid and consistent start to fermentation it is preferable to pitch all the yeast over as short a time period as possible and at the beginning of collection. The oxygenation regime should be chosen on the basis of that which provides the desired fermenter residence time, extent of yeast growth relative to the pitching rate and beer analysis. Whatever regime this happens to be, it should be adhered to with the greatest possible rigour in order to maximise the chances of obtaining consistent fermentation performance.

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