Whilst the largest industrial fermentations utilizing yeasts are the brewing of beer and the production of biomass, recent processes have also been established for the production of recombinant products.
It is common practice in the British brewing industry to use the yeast from the previous fermentation to inoculate a fresh batch of wort. The brewing terms used to describe this process are 'crop', referring to the harvested yeast from the previous fermentation, and 'pitch', meaning to inoculate. One of the major factors contributing to the continuation of this practice is the wort-based excise laws in the United Kingdom where duty is charged on the sugar consumed rather than the alcohol produced. Thus, dedicated yeast propagation systems are expensive to operate because duty is charged on the sugar consumed by the yeast during growth. It can then be appreciated that the reduced cost of using yeast from a previous fermentation is an attractive proposition (Boulton, 1991). The dangers inherent in this practice are the introduction of contaminants and the degeneration of the strain, the most common degenerations being a change in the degree of flocculence and attenuating abilities of the yeast. In breweries employing top fermentations in open fermenters these dangers are minimized by collecting yeast to be used for future pitching from 'middle skimmings'. During the fermentation the yeast cells flocculate and float to the surface, the first cells to do this being the most flocculent and the last cells the least flocculent. As the head of yeast develops, the surface layer (the most flocculent and highly contaminated yeasts) is removed and discarded and the underlying cells (the 'middle skimmings') are harvested and used for subsequent pitching. Therefore, the 'middle skimmings' contain cells which have the desired floccu-lence and which have been protected from contamination by the surface layer of the yeast head. The pitching yeast may be treated to reduce the level of contaminating bacteria and remove protein and dead yeast cells by such treatments as reducing the pH of the slurry to 2.5 to 3, washing with water, washing with ammonium persulphate and treatment with antibiotics such as polymixin, penicillin and neomycin (Mandl et al., 1964; Strandskov, 1964; Roessler, 1968; Reed and Nagodawithana, 1991a).
However, traditional open vessels are becoming increasingly rare and the bulk of beer is brewed using cylindro-conical fermenters (see Chapter 7). In these systems the yeast flocculates and collects in the cone at the bottom of the fermenter where it is subject to the stresses of nutrient starvation, high ethanol concentration, low water activity, high carbon dioxide concentration and high pressure (Boulton, 1991). Thus, the viability and physiological state of the yeast crop would not be ideal for an inoculum. The viability of the crop may be assessed using a biomass probe of the type described earlier, thus ensuring that at least the correct amount of viable biomass is used to start the next fermentation. However, the physiological state of the biomass will not have been influenced by such monitoring procedures. The situation is further complicated by the fact that the harvested yeast is stored before it is used as inoculum. Metabolic activity is minimized during this time by chilling rapidly to about 1°, suspending in beer and storing in the absence of oxygen. If oxygen is present during the storage period then the yeast cells consume their stored glycogen which renders them very much less active at the start of the fermentation (Pickerell et al., 1991).
One of the key physiological features of yeast inoculum is the level of sterol in the cells. Sterols are required for membrane synthesis but they are only produced in the presence of oxygen. Thus, we have the anomaly of oxygen being required for sterol synthesis, yet anaerobic conditions are required for ethanol production. This anomaly is resolved traditionally by aerating the wort before inoculation. This oxygen allows sufficient sterol synthesis early in the fermentation to support growth of the cells throughout the process, that is after the oxygen is exhausted and the process is anaerobic. Boulton et al. (1991) developed an alternative approach where the pitching yeast was vigorously aerated prior to inoculation. The yeast was then sterol rich and had no requirement for oxygen during the alcohol fermentation.
The difficulties outlined above and the likelihood of strain degeneration and contamination mean that yeasts are rarely used for more than five to ten consecutive fermentations (Thorne, 1970; Reed and Nagodawithana, 1991a) which necessitates the periodical production of a pure inoculum. This would involve developing sufficient biomass from a single colony to pitch a fermentation at a level of approximately 2 grams of pressed yeast per litre. Hansen (1896) pioneered the use of pure inocula and devised a yeast propagation scheme utilizing a 10% inoculum volume at each stage in the programme and employing conditions similar to those used during brewing. However, modern propagation schemes use inoculum volumes of 1% or even lower and may use conditions different from those used during brewing. Therefore, continuous aeration may be used during the propagation stage which seems to have little effect on the beer produced in the subsequent fermentation (Curtis and Clark, 1957). Yeast inoculum produced in this way would also be sterol rich, obviating the need for aerated wort.
A number of yeast propagators (which are basically closed, aerated vessels) have been described in the literature. The simplest type of progagator is a singlestage system resembling an unstirred, aerated fermenter which is inoculated with a shake-flask culture developed from a single colony (Gilliland, 1971). Curtis and Clark (1957) and Thorne (1970) described two-stage systems which could be operated semi-continuously. Thome's propagator consisted of two linked vessels, 1.5 and 150 dm3 respectively. The smaller vessel was filled with wort, sterilized, cooled, aerated and inoculated with a flask-grown culture. After growth for 3 to 4 days the culture was forced by air pressure into the second vessel which had been filled with sterilized, cooled wort and aerated. An aliquot of 1.5 dm3 was forced back into the first vessel after mixing. In a further 3 to 4 days the larger vessel contained sufficient biomass to pitch a 1000 dm3 fermenter and the first vessel contained sufficient inoculum for another second stage. However, although this procedure should produce a pure inoculum there is a danger of strain degeneration occurring in such a semi-continuous system.
Boulton (1991) speculated that when the UK moves to an alcohol-based tax then the economics of propagating inoculum for each brew would be considerably more attractive. He suggested that the bakers' yeast aerobic fed-batch inoculum programme could be adopted to produce sterol-rich catabolite-derepressed cells. Such an inoculum would remove the necessity for aerating the wort prior to inoculation because sterol synthesis would not be necessary due to the high sterol content of the cells.
The commercial production of bakers' yeast involves the development of an inoculum through a large number of aerobic stages. Although the production stages of the process may not be operated under strictly aseptic conditions a pure culture is used for the initial inoculum, thereby keeping contamination to a minimum in the early stages of growth. Reed and Nagodaw-ithana (1991b) discussed the development of inoculum for the production of bakers' yeast and quoted a process involving eight stages, the first three being aseptic while the remaining stages were carried out in open vessels. The yeast may be pumped from one stage to the next or the seed cultures may be centrifuged and washed before transfer, which reduces the level of contamination. The yields obtained in the first five stages are relatively low because they are not fed-batch systems, whereas the last three stages are fed-batch (the fed-batch bakers' yeast fermentation is considered in more detail in Chapter 2). A summary of a typical inoculum development programme for the production of bakers' yeast is given in Fig. 6.2.
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