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Fig. 4.26 Events during the first 12 hours of a stirred laboratory fermentation (redrawn from Quain, 1998). Units are biomass (0. mg.ml-1 - for real figure -h by 10): cell number (•, 106.ml_1): budded cells (A. %). cell volume (□. |im3 - for real value x 2) and present gravity (■).

stationary phase ('GO') cells presumably 'sense' the presence of nutrients and shift into the G1 phase and START. During these first two hours cell volume increases perceptibly but PG and cell number remain static whereas total biomass declines by almost 20%. Arguably, glycogen breakdown during this time fuels the GO to G1 transition and, specifically, new lipid synthesis. As pitching yeast is 'lipid depleted', the formation of sterols and unsaturated fatty acids is a critical requirement for cell division to occur in brewery fermentations. Entry into S phase is apparent with the onset of budding at 2 hours, which peaks at almost 90% of the population within 6 hours of pitching. By now the population is asynchronous as, against a budding index of 80%, new daughter cells begin to accumulate and the cell number increases from 7 hours onwards. At this point changes are detectable in biomass and sugar uptake (PG). As would be predicted, these early events in fermentation are accompanied by an increase in cell size, which peaks 20% higher at 6 hours. Presumably, this reflects the need for individual cells to achieve a critical cell size before entering the cell cycle.

This frenetic early period of activity prepares the cell for its future role in fermentation. During active fermentation (Fig. 4.27) there are about 3.5 rounds of cell division as cell numbers increase from 10 to 100 x 106 cells ml 1. Other indicators of cell cycle activity (budding index and cell volume) decline.

Of course, such measures are very much an average of what is a changing and heterogeneous cell population with, typically, 50% virgin daughter cells, 25% single budded mothers and 25% multi-budded mother cells (Deans el al., 1997). Interestingly (Fig. 4.28), cell division does not continue throughout fermentation but 'arrests' at about the 'mid-point'. This is reciprocated by the budding index declining to basal level. It is generally accepted that division is not arrested by nutrient deficiency in the fermenting wort but by dilution by cell division of essential lipids. As noted in more detail elsewhere the sterols and, to a lesser extent the unsaturated fatty acids, determine yeast growth and the rate (and extent) of fermentation. Consequently, brewery

Fig. 4.27 Events during a stirred laboratory fermentation (see Fig. 4.26) (unpublished observations of Fintan Walton and David Quain). Units are biomass (♦. mg.ml-1 - for real figure -h by 10); cell number (▲. 106.ml_1>; budded cells (•. %). cell volume (■. |im3) and present gravity ( x. as a % of the original gravity. 1060).

Fig. 4.27 Events during a stirred laboratory fermentation (see Fig. 4.26) (unpublished observations of Fintan Walton and David Quain). Units are biomass (♦. mg.ml-1 - for real figure -h by 10); cell number (▲. 106.ml_1>; budded cells (•. %). cell volume (■. |im3) and present gravity ( x. as a % of the original gravity. 1060).

Fig. 4.28 Relationship between cell division and fermentation (same experiment as Fig. 4.26) (unpublished observations of Fintan Walton and David Quain). Units are budded cells (♦. % of total cells) and cell number (■. 106.ml_1).

fermentations are in a sense nutrient limited! The point of difference being that the nutrient is supplied and consumed some days before 'deficiency' takes effect.

4.3.3.3 Stationary phase. In the 'real world', nutrient starvation is often the 'norm' for micro-organisms. As noted by Werner-Washburne et al. (1996), 'yeast cells can be translocated from relatively nutrient-rich environments, e.g. ripe grapes and plant leaves (especially wilted plant leaves), to nutrient-poor environments as rapidly as a

Fig. 4.28 Relationship between cell division and fermentation (same experiment as Fig. 4.26) (unpublished observations of Fintan Walton and David Quain). Units are budded cells (♦. % of total cells) and cell number (■. 106.ml_1).

cell is blown from its birth place or becomes lodged on the leg of a fly'. Accordingly, under conditions of starvation, yeast stops growing and enters a quiescent 'stationary phase' or, in cell cycle terminology, GO.

Shutting down metabolism and entry into stationary phase is the cell's strategy for long-term survival for months, or perhaps even years (Werner-Washburne et al., 1996). So substantial is the cell's response to starvation that wholesale changes in physiology, genetics and morphology are seen. Stationary phase cells (i) are more heat resistant (Walton et al., 1979), (ii) accumulate trehalose and glycogen (Lillie & Pringle, 1980), (iii) are more resistant to the deleterious effects of oxygen radicals (Werner-Washburne et al. (1996) and (iv) have a low adenylate energy charge (Ball & Atkinson, 1975). Stationary phase also triggers what are occasionally dramatic changes in cell morphology. GO cells show (i) thicker cell walls which are more resistant to enzymic removal (Deutch & Parry, 1974), (ii) intracellular vacuoles increased in size and number (Schwencke, 1991), (iii) cell shape which can become distorted ('schmoo's') (Wheals, 1987) and (iv) some strains form pseudohyphae (Kuriyama & Slaughter, 1995).

Most of the contemporary studies on the stationary phase in yeast focus on cell biochemistry and genetics. A notable publication by Fuge et al. (1994) overcame some of the criticism of some of the work in the area by monitoring cells that were genuinely in stationary phase. This work sought to clarify the role of protein synthesis over a protracted 23-day stationary phase cells and to establish whether certain proteins were formed uniquely during this time. As perhaps would be anticipated, the rate of protein synthesis during stationary phase is about 300-fold lower than in exponentially growing cells. However, what is perhaps surprising is that the portfolio of proteins synthesised during active growth are also formed in stationary phase cells. This suggests that the majority of proteins provide 'housekeeping functions' in exponentially and stationary phase cells. Seemingly, only a few proteins are unique to stationary phase function or growth.

As ever, the genetics of the stationary phase are detailed and complex. Although a number of genes have been reported to be induced prior to or during entry into stationary phase, one, SNZ1, stands out as being unusual (Werner-Washburne et al., 1996). Expression of this 'snooze' gene occurs two days after glucose exhaustion and increases 14-fold between exponential and stationary phase cells. Although the function of the encoded protein (SNZlp) is currently unknown, it is found in phy-logenetically diverse organisms and is one the most evolutionary conserved proteins, which suggests that it has a bigger role in biology (Braun et al., 1996).

Although subject to a formidable amount of work, our understanding of stationary phase in yeast is based almost exclusively on aerobic cultures of haploid strains growing on glucose in defined laboratory media at 30° C. Little work has been published on stationary phase in brewery fermentations. Consequently, as conditions in brewery fermentations are almost diametrically opposed to the laboratory model, it is something of an act of faith that stationary phase is similar in brewing yeast. A further rider is that the trigger for entry into stationary phase is not solely exhaustion of a (usually fermentable) carbon source but includes nitrogen and sulphur limitation. The onset of stationary phase in (anaerobic) brewery fermentations has not been explicitly studied. Whether entry into stationary phase at the end of fermentation is triggered by exhaustion of fermentable carbohydrate or some other nutrient is not entirely clear. Indeed, the presence of residual fermentables (notably maltotriose) in green beer possibly suggests a more complex interpretation than simply the apparent lack of external nutrients. In this case, it may be more appropriate to consider that sugars become 'unavailable' because of changes (brought about by the effects of lipid depletion on membrane function) in the affinity of transport proteins or through cell flocculation. Certainly, brewery fermentations can 'arrest' or 'stick', in the presence of large amounts of fermentable carbohydrate, when lipid synthesis is limited by the addition of insufficient oxygen at the beginning of fermentation. Of course, with so little hard evidence, the simple view that the unavailability of fermentable carbohydrate triggers entry into stationary phase does not preclude the possibility that the exhaustion of some other nutrient may initiate this transition.

Much of what is known about stationary phase in brewery fermentations relates to the consequences of yeast storage and its subsequent 'fitness for purpose'. Physiologically, although apparently quiescent, brewing yeast in stationary phase continues to 'tick over' - the rate of which is dependent on temperature. In gross terms, stationary phase cells are under threat and metabolism is focused on survival in the hope that the cells' environment will eventually improve. In common with laboratory observations under aerobic conditions (Lillie & Pringle, 1980), stored (stationary phase) brewing yeast dissimulates accumulated storage carbohydrates such as glycogen and, to a lesser extent, trehalose (Quain et al., 1981). Quantitatively, the contribution of glycogen to survival in stationary phase is substantial. As if in recognition of this, glycogen accumulation during active brewery fermentation is substantial, accounting for up to 40% or more of the cell biomass. The extent of glycogen turnover during stationary phase in fermenter or in storage vessel can be extensive depending on duration and temperature. Presumably, glycogen breakdown provides energy for ongoing cell maintenance during stationary phase. Although the significance is not clear, the rate of glycogen breakdown during stationary phase in brewing strains is found to vary by up to three-fold (Quain, 1988).

The advent of the concept of 'vitality' and 'vitality testing' (see Section 7.4.2) has resulted, indirectly, in an improved understanding of the physiology of cells in stationary phase. Numerous measures have been advocated to assess the vitality of stored yeast. Most focus on metabolic response post-feeding with glucose, such as uptake of oxygen, evolution of carbon dioxide, intra- or extracellular changes in pH. Whatever their utility in establishing a population's 'fitness' for pitching-on, these tests provide a useful benchmark for the varying physiological status of batches of yeast in store. However, interpretation of 'cause and effect' is difficult, as these measures are unable to differentiate between the impact of yeast storage and physiological changes brought about by stationary phase.

In passing, although stationary phase cells are physiologically better equipped to withstand the rigours of yeast storage and handling, such cells will be more resistant to killing through pasteurisation.

4.3.3.4 Ageing. Understanding the phenomena of cellular ageing has a universal and popular appeal. Together with the nematode C. elegans (whose genome is being sequenced, Section 4.3.2.1) and the fruit fly, Drosophila, S. cerevisiae is at the fore front of high-profile studies that seek to model and unravel the mechanisms of cell ageing. The profile is such that national newspapers and press agencies report developments in this field. For example, work at the Massachusetts Institute of Technology (MIT) created a furore with 'Yeast gene yields clues to the ageing process' (Reuters, 28/8/97) and 'Yeast cells may hold key to human ageing' (The Times, 26/12/ 97). Simplistically the media see the prize of such work as being understanding and even control of human ageing. Inevitably, this ever-changing area is continually subject to review. Recent general reviews include those by Sinclair et al. (1998) and Jazwinski (1999) together with more applied reviews by Smart (1999) and Powell et al. (2000).

The yeast cell has a finite life span, measured by the number of divisions and not by chronological age. On stopping division, the cell becomes senescent and eventually dies. In addition to the usual arguments about being a model eukaryote, S. cerevisiae lends itself to experimental studies on ageing. The reasons for this are numerous but includes: (i) the cell and organism are one and the same, (ii) the cell is thought not to be affected by extracellular factors (hormones, other cells), (iii) cells can be isolated singly or in bulk and (iv) bud scars act as a 'biomarker' for cell age (Jazwinski, 1990). Although currently fashionable, the study of the life span of yeast is by no means new, dating back to work by Mortimer and Johnston (1959). This important paper concluded that the life span of a diploid strain of S. cerevisiae was an average of 24 generations, with a maximum of about 40. Some decades later and using the same methodology as Mortimer and Johnston (1986), Egilmez and Jazwinski (1989) reported remarkably similar data for a haploid strain, which had a mean life span of 24 + 9 (standard deviation) generations. Other strains have a shorter life span. Work with a polyploid lager strain (Barker & Smart, 1996) reported a mean life span of 17 generations and a maximum of 29 generations. Similar data was reported for a haploid strain (mean = 17, maximum = 31) (Austriaco, 1996). In passing, the approach first described by Mortimer and Johnston (1959) requires long hours of tiring microscopic examination. Given this, it is not surprising that yeast cells under study require to be refrigerated overnight so as to slow division and to 'provide relief for the investigator' (D'mello et al., 1994)! Such treatment has no bearing on determinations of life span (Egilmez & Jazwinski, 1989).

The physiology of ageing in yeast is fascinating. Morphologically, the cell takes on an aged appearance, becoming granular, crenellated and wrinkled (Mortimer & Johnston, 1959; Jazwinski, 1990; Barker & Smart, 1996) and accumulates lipid granules (Austriaco, 1996). Further, as first noted by Mortimer and Johnston (1959), yeast cell size and, to an extent, generation time, increase with successive generations. The increase in cell size is linear with age (Jazwinski, 1990). With lager yeast NCYC 1166 there is a six-fold difference in cell volume (Fig. 4.29 ) between young new mothers (163 |im3) and senescent cells (950 |^m3) at the end of their life span (Barker & Smart, 1996). The relationship between generation time and cell age is less pleasing to the eye! Egilmez and Jazwinski (1989) noted that a 'pattern of moderate increase in generation time between the ages of 10 and 20 generations was always followed by a sharper increase after generation 20'. Barker and Smart (1996) (Fig. 4.30) reached broadly similar conclusions although the generation time was stable for almost 70% of the cell's life span before increasing dramatically during the last two or three divisions.

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