It has been said that yeast is known more for what it can do than what it is (Robinow & Johnson, 1991). Until comparatively recently this was certainly the case and, thus, early brewers had by and large to be content with gauging yeast condition on the basis of observed relationships between behaviour and macro-morphological appearance. For example, assessing fermentation progress based on the appearance of the yeast head in an open square fermenter. In this sense yeast was (and perhaps still is by some practitioners of a less sensitive nature) treated as a bulk ingredient rather than a population of individuals.
Nevertheless, brewers were also relatively early converts to recognising the value of direct observation of the appearance of individual yeast cells as evidenced by the fact that as early as the late nineteenth century a microscope was considered an essential piece of laboratory apparatus. For example, Lindner (1895) discussed the application of microscopy to the study of pure yeast culture, yeast morphology and the theory of infection. In a later publication (Hatch, 1936) stressed the value of microscopic examination of pitching yeast under bright field. He provided the description 'healthy, vigorous yeast will show cells uniform in size and shape with walls clearly defined but not thickened. Few or no granular cells should be present and the number of bacteria should not exceed 2 or 3% of the total number of yeast cells'.
Direct examination of yeast cells using a light microscope continues to provide a method for detecting morphological abnormalities in the population of yeast used in the brewery. For example, an abrupt shift in microscopic appearance which might suggest that a process failure or change in practice was having a deleterious effect on the yeast. Similarly it provides a means of detecting gross contamination with bacteria and possibly other yeast strains, providing the latter have sufficiently different morphology to render them distinguishable from the native population.
A detailed elucidation of cellular ultrastucture requires microscopic tools with a precision and discrimination greater than that afforded by simple optical methods. Several such methods have been developed and yeast has been used frequently in these studies as a convenient model eukaryotic cell. Similarly, the brewing industry has been at the forefront in adopting new approaches for examining yeast cell morphology and cytology. Thus, Mitchison et al. (1956) using interference microscopy measured changes in cell dry mass, volume and number in growing and dividing Schizosaccharomyces pombe. Royan and Subramaniam (1956) used phase contrast and dark field microscopy to investigate the appearance of the nucleus and vacuole in yeast cells growing in wort. In the same period reports of the use of the electron microscope begin to appear, for example Bradley (1956) described the use of a carbon replica shadowing method for visualising the surface of yeast cells.
Further refinements in light microscopy continue to provide ever more powerful tools for studying cell ultrastructure. For example, the use of fluorescence microscopy (Robinow, 1975; Pringle et al., 1989) and confocal microscopy (Bacalfao & Stelzer, 1989). The latter method allows the production of three-dimensional images of yeast cells using a laser scanning device in which the resultant fluorescence light intensity is detected via a photomultiplier. Coincident with advances in light microscopy have been the development of ever more elegant incarnations of the electron microscope. Some of these as applied to yeast cells are described by Kopp (1975) and Wright and Rine (1989).
The development of the methods outlined in the previous discussion has allowed a detailed picture of yeast ultrastructure to be drawn up. Although this has been a rewarding exercise in itself, it is, of course, all the more valuable to be able to link structure with function. In this regard the development of a battery of additional techniques for the isolation and fractionation of individual yeast cell organelles has been of great importance. Thus, disruption of yeast cells under carefully controlled conditions and the subsequent collection of individual organelles followed by appropriate analysis has allowed the intracellular location of a multitude of biochemical pathways to be identified. Such studies have demonstrated that compart -mentalisation plays a key role in the regulation of metabolism of individual cells. Furthermore, changes in physiological condition of yeast cells may be accompanied by visible changes in cytology. In this respect the effects on yeast of catabolite repression, exposure to oxygen, starvation and cellular ageing are all pertinent to brewing.
Apart from the localisation of groups of pathways into individual cellular compartments there is strong evidence that metabolism is ordered both biochemically and spatially in a co-ordinated fashion within cellular compartments. Several studies suggest that glycolytic enzymes are physically associated with the cytoskeleton in an ordered fashion such that products and substrates of individual enzymes are channelled in a controlled manner through the pathway (Shearwin & Masters, 1990; Masters, 1992). Recently, Götz et al. (1999) provided evidence that changes at the enzyme level associated with shifts from glycolytic to gluconeogenic modes of metabolism, in yeast, could be mediated by the cytoskeleton.
Several review articles describe procedures for sub-cellular fractionation and collection of yeast organelles, for example, Walworth et al. (1989) and Lloyd and Cartledge (1991). More specific methods are described for isolating protoplasts (Kuo & Yamamoto, 1975); nuclei (Duffus, 1976); vacuoles (Wiemken, 1976); plasma membranes (Henschke & Rose, 1991) and mitochondria (Linnane & Lukins, 1976; Deters et al., 1978; Guerin, 1991).
As alluded to already, a considerable body of work is now available in the literature providing great detail of the relationships between yeast cell structure and function. Much of this work has been performed on brewing or closely related yeast strains. However, there is much less literature describing how the conditions to which yeast is exposed during brewing result in modifications to cellular morphology and ultra-stucture. In particular the effects of serial fermentation with associated transient aerobiosis and growth followed by intermittent periods of storage under starvation conditions. Some of the effects of brewing on yeast morphology are summarised in subsequent sections of this chapter. In some cases a lack of published data has necessitated a degree of speculation.
Lodder (1970) describes the cells of S. cerevisiae as being spheroidal, subglobose, ovoid, ellipsoidal or cylindrical to elongate, occurring singly, in pairs, occasionally in short chains or clusters. Cells may be grouped into three classes on the basis of size. A large type, 4.5-10.5 x 7.0-21.0 (im (microns); a small-cell type falling within the range 2.5-7.0 x 4.5-11 (im and an intermediate group with cells measuring 3.5-8.0 x 5.0-11.0 (im. Some yeasts may form filaments which may be up to 30 (m in length. Brewing yeast cells fit into any of these categories; however, they tend to be quite large cells, a consequence of polyploidy. Vagvolgyi et al. (1988) demonstrated the effect of ploidy on yeast cell size, thus, the mean diameters of hapoid, diploid and triploid S. cerevisiae cells being 4.2, 5.2 and 5.9 (im, respectively (see also Section 126.96.36.199). The relationship between volume and surface area for an ellipsoidal yeast cell (e.g. S. cerevisiae) has been given as 45 and 71 (im2 for haploid and diploid cells with volumes of 29 and 55 (im3, respectively (Hennaut et al., 1970). See Section 188.8.131.52 for a fuller discussion on ploidy and cell size.
The mean cell size of a particular yeast strain is not a constant but varies according to the stage in the growth cycle, the growth conditions and the age of the individual cell. Changes in cell size associated with age (see Section 184.108.40.206 for a full discussion on ageing.) were described by Woldringh et al. (1995) using a centrifugal elutriation system which allowed the collection of new-born daughter cells. These authors reported that the volume of these increased from a mean value of 17 (im3 to 34 (im3 after five generations and 81 (im3 at 15 generations (see also Section 220.127.116.11). Hartwell and Unger (1977) concluded that the increase in cell volume occurred during the phase in the cell cycle when budding has finished. In fact, the mean cell volume decreases by approximately a third during the budding phase. Apart from volume changes, predictably, there are also cell cycle-associated oscillations in cell mass. Baldwin and Kubitschek (1984) demonstrated that the cellular density reached a peak during the mid-growth cycle and this was attributed to a loss of water with a concomitant increase in dry mass. Such observations serve to illustrate the fact that caution should be exercised in interpreting experimental data, expressed as percentage dry mass, regarding fluctuation in the concentrations of individual cellular components during growth.
The growth conditions also influence cell size. Robinow and Johnson (1991) reviewed the effects of incubation temperature on cell size and reported a variable response depending on the yeast. Thus, most yeast types, probably including Sac-charomyces, showed a temperature dependent increase in cell size. However, Schizosaccharomyces pombe cells increased in size at temperatures higher and lower than the optimum. Brewing yeast cells growing fermentatively on maltose are significantly bigger than the same cells growing oxidatively on ethanol (C. A. Boulton, unpublished data).
Short-term perturbations in cell size may also occur, presumably as a result of osmotic effects, when yeast is transferred between different media. For example, the effects of pitching yeast suspended in barm ale into wort. Thus, Quain (1988) observed that in the first 4 hours after pitching yeast, at laboratory scale, into wort there was a transient increase in cell size. The mean cell volume increased from approximately 170 (im3 to 200 (im3. The volume changes occurred before the onset of budding and cell proliferation and were independent of cell dry weight. These results suggest that the degree of turgor of the plasma membrane exerts an influence on cell size and that the cell wall is sufficiently flexible to accommodate such short-term fluctuations.
Changes in cell size over longer periods have also been recorded. Cahill et al. (1999b) reported the application of image analytical techniques as a method for improving the control of pitching rate. The apparatus was used to monitor cell size of stored pitching yeast. It was observed that for both ale and lager yeast the mean cell volume reduced by 19% (302 to 244 |im3) and 7 % (208 to 194 (im3), respectively during storage over a period of 14 days at 4°C. The changes in mean cell volume were correlated with glycogen content, the latter being utilised for maintenance energy during storage.
A diagrammatic representation of a section through an idealised yeast cell indicating the major organelles is shown in Fig. 4.1. Not all of these features are visible within the cell at all times; indeed some may not be present in yeast at all, as discussed later. A brief discussion of the structure and function of some of these organelles follows. The description is not comprehensive; however, an attempt has been made to draw out those features which might be expected to be characteristic of brewing yeast.
18.104.22.168 Cell composition. Most published data for yeast composition relates to bakers' yeast which predictably will have significant differences in some cellular
Fig. 4.1 A diagrammatic cross section of a yeast cell.
Fig. 4.1 A diagrammatic cross section of a yeast cell.
components compared to brewing yeast. In particular, levels of trehalose are engineered to be high in bakers' yeast to ensure good resistance to drying (see 22.214.171.124). In addition, sterol levels are high in bakers' yeast, a consequence of catabolite derepression (see Section 3.5.1).
The molecular composition of dried wine yeast is given in Table 4.1. From these data Rosen (1989) gave a rough molecular formula for this yeast as:
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