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The physiology of beer-spoiling yeast

Vegetative growth, cell structure and sexual division

When a yeast culture encounters favourable conditions, it is advantageous for the population to divide vegetatively, or asexually. This allows new individuals to be generated quickly through mitosis, producing cells that are theoretically identical barring random mutation events. The majority of yeasts found within brewery locations reproduce predominantly via this mode of replication. In most instances, vegetative growth occurs through budding, whereby a new cell is produced via localized expansion and extrusion of the mother cell wall. The precise mechanism can vary, with different yeasts employing multilateral or bipolar divisional patterns. It should be noted that other types of yeasts (including those not typically found in breweries) may divide vegetatively via a number of mechanisms, including binary fission (notably Schizosaccharomyces pombe), bud fission, and via stalks or outgrowths. In addition to asexual reproduction, some beer-spoiling yeasts have the ability to reproduce sexually via karyogamy (fusion of cells to form a zygote) and subsequently meio- sis. These events result in the formation of spores contained within an ascus (essentially a sac derived from the original fusion of the two parental cells). Sexual reproduction is relatively common in many beer-spoiling yeasts and can be induced by sudden changes in environmental conditions or nutrient deficiency (starvation).

All yeasts exhibit characteristic cellular structures: a rigid outer wall and a fluid cell membrane envelope the cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, vacuoles, and a variety of vesicles and microbodies. However, despite being virtually identical in terms of cellular constituents, yeasts as a group of organisms are morphologically diverse. A culture of brewing yeast typically comprises a population of uniform cells that are spherical or slightly ellipsoidal in shape, and between 6 and 10 pm in diameter (Fig. 11.1). In contrast, beer-spoiling yeast (as well as other non-domesticated species isolated from the wild) can show a wide variety of cell shapes and sizes (Fig. 11.2). Broadly speaking, vegetative cells belonging to different yeast species can be described as being spherical, ellipsoidal, ovoid (egg-shaped), apiculate (lemon-shaped), pointed, rectangle-like, bottle (or flask)-shaped, or elongated. Yeast cell morphology is influenced by a number of factors, often linked closely to budding or budding patterns. In general, cells that are oval usually exhibit either an axial (bud production occurs adjacent to previous site of division) or a bipolar (cells bud at either polar end of the cell) budding pattern, while cells that are elongated tend to produce buds in an almost exclusively bipolar fashion (Chant and Pringle, 1995). This relationship is true for most strains belonging to the species Hanseniaspora (Kloeckera), for example, which produce lemonshaped cells and display a polar divisional pattern. In contract to brewing yeast strains, which show a high degree of morphological homogeneity, non- domesticated yeasts can be polymorphic; cells within a population are often visually diverse such that they may at first appear to belong to different species. For example, Brettanomyces populations often comprise individuals that are ellipsoidal, elongated, or even rectangular. Similarly, some Hanseniaspora species can show extensive polymorphisms with notable variations in size as well as shape. Differences in morphology within populations are further exacerbated by the pleomorphic nature of yeasts (Fig. 11.3). As described above, under certain conditions yeasts have the ability to form mating aggregates, which lead to the production of sexual spores. Much of our understanding of the process comes from analysis of Saccharomyces strains, where mating is triggered by specific pheromones that bind to receptor sites in two opposite ‘mating types'. In S. cerevisiae, these are known as ‘a' and ‘a', while in Sch. pombe they are referred to as h+ and h- In other types of yeasts, these nomenclature may be used, although often they are referred to as simply plus (P) or minus (M). In many yeast species, cells may also switch mating types, allowing

Types of cell morphology associated with Saccharomyces yeast species at x400 magnification

Figure 11.1 Types of cell morphology associated with Saccharomyces yeast species at x400 magnification. All species typically show a similar uniform ovoid/spherical morphology. Note that the phenolic (A) and diastatic (B) wild S. cerevisiae strains exhibit a smaller cell size when compared to the lager and ale brewing yeast strains shown in C and D, respectively. Non-brewing strains and some ale-type strains may form chains (A), where individual cells are cytoplasmically discrete, but remain connected due to incomplete separation of mother and daughter at the cell wall.

colonies containing both mating types to develop and ensuring that there are always ‘partners' available. Irrespective, the pheromones produced induce mating cells to develop a bottleneck-like projection that extends in the direction of a cell of the opposite mating type. This structure is known as a shmoo and allows for the eventual exchange of genetic material, culminating in spore formation. Typically 1-4 spores are formed within the ascus, the shape of which is highly variable between species. Saccharomyces yeasts tend to produce spores that are held in a tetrahedral formation, while other species have been described as having ellipsoidal, spherical, elongated, hat-shaped, Saturn-shaped, or kidneyshaped spores (Fig. 11.4). It should be noted that, in contrast to bacterial spores, yeast spores are not particularly stress tolerant; ascospores are only slightly more resistant to environmental challenges than vegetative cells.

Many yeast species are also able to adopt a variety of non-sexual structures, including chain formation and pseudohyphal (filamentous) growth. Although budding yeasts do not undergo true hyphal growth, they can exhibit a phenomenon in which cells fully separate by cytokinesis during division, but remain attached to each other due to the presence of specific proteins located in the cell wall. This is termed pseudohyphal growth and is closely linked to nutritional limitation, especially nitrogen deficiency (Gimeno et al., 1992; Kron et al., 1994). The majority of information on how this occurs is based on studies of S. cerevisiae, Sch. pombe, and Candida strains. In S. cerevisiae, it is known that immediately prior to the initiation of filamentous growth, a protein known as Ras2p (localized within the cell membrane) is activated, which in turn stimulates the synthesis of cAMP, an intracellular signalling molecule. This results in the activation

Types of cell morphology associated with non-Saccharomycesyeast species at x400 magnification

Figure 11.2 Types of cell morphology associated with non-Saccharomycesyeast species at x400 magnification. Apiculate cells typical of Hanseniaspora valbyensis (A), rod-shaped cells of Pichia membranifaciens (B), elongated cells such as those associated with Brettanomyces anomalus (C) and ellipsoidal cells as seen in Zygosaccharomyces bailii (D). It should be noted that these images serve as examples and are not representative of all strains within each species. There is considerable variation within species and often individual strains will show several different morphological types, as seen in Pichia anomola (E). This can be compounded by the presence of sexual spores, formation of mating aggregates (shmoos), and the formation of pseudohyphae (F). For the latter, staining was conducted using calcofluor white to reveal the location of chitin deposits within the cell wall, clearing indicating two discrete cells.

of protein kinase A (PKA), which triggers a range of key transcription factors, including products of the STE gene family and Flo8p. These regulate the expression of a huge number of genes contributing to pseudohyphal growth (Jin et al., 2008), the most

well characterized being FLO11. The production of pseudohyphae is distinct from invasive hyphal growth observed in other fungi, but the appearance can be similar with an increase in cell length and enhanced cell-cell adhesion. Pseudohyphal

Forms of yeast growth

Figure 11.3 Forms of yeast growth. Yeast typically divide asexually to produce discrete cells, but failure to separate through budding can lead to chain formation. Cells can also undergo filamentous (pseudohyphal) growth, form biofilms through mat formation, and undergo ‘shmoo’ formation as a means of exchanging DNA during sexual division, which culminates in spore formation.

Spore formation in yeasts as observed under a light microscope at approximately x400 magnification

Figure 11.4 Spore formation in yeasts as observed under a light microscope at approximately x400 magnification. Spore morphology is highly variable and dependent on genus and species. Typically between 1 and 4 globose spores will be formed, enclosed within an ellipsoidal ascus (A and B). Saccharomyces spores can be tetrahedral shaped (C) due to the tight rigidity of the ascus, although some may contain just 2 or 3 spores. Some yeasts such as the fission yeast Schizosaccharomyces pombe form linear asci (D). Pichia species are diverse, showing elongated asci (E), as well as producing spores that can be ‘hat-shaped’ (helmet-shaped) (F) or Saturn-shaped (G). The latter can also be observed in Lindnera saturnus, the genus originally designated for yeast forming such spores, while Hanseniaspora (Kloeckera) and Dekkera (Brettanomyces) form hat-shaped spores. In Zygosaccharomyces cells, a conjugation tube links mating cells together, forming a characteristic dumbbell-shaped zygote (H), while Kluyveromyces marxianus forms kidney- or bean-shaped spores (I). It is common for some species, such as Debaryomyces, to produce lipid bodies as a component of the spore (J), which can be clearly seen under a light microscope. Finally, it should be noted that ascospores can be smooth, warty, or ridged, although this level of detail is not always obvious without the use of a high-powered microscope.

cells are usually elongated (sometimes 20-50pm in length) or ellipsoidal and have constrictions at the septal junctions that connect adjacent cells (Fig. 11.2F). In some instances, complex aggregates of filamentous cells may be observed, including individuals in the process of developing pseudohyphae, which show a variety of intermediate morphologies. The formation of such structures is important from a beer spoilage perspective since elongated or pseudohyphal cells are often those that are able to form a pellicle and float on the surface of liquid media.

Pseudohyphae should not be confused with chain formation (Brown, 1970), which occurs in many yeasts, including some ale strains. In both instances, individual cells are cytoplasmically discrete, but remain connected due to incomplete separation of mother and daughter at the cell wall. However, in chain formation this typically occurs due to a deficiency in CTS1 activity, which codes for chitinase (Kuranda and Robbins, 1991), responsible for digesting chitin scar tissue and allowing separation of a daughter cell from its mother. The impact of chain formation can be similar to pseu- dohyphae in that large clumps of cells can form; however, the significance is arguably of greater impact during fermentation, since chains can act as nucleation points for floc formation, resulting in changes to the flocculation properties of the culture yeast.

Some yeast species will also undergo mat formation in response to nutrient limitation, characterized by an increased ability to ‘stick' to surfaces (Wood et al., 1992; Reynolds et al., 2008). This is also believed to be due to activation of the FLO11 gene, which causes changes in the cell wall that enhance cell-cell adhesion. Interestingly, the mat itself is subject to nutrient and pH gradients, which is believed to impact on the activity of Flo11p rather than gene expression. Cells located at the ‘rim' of the mat structure show decreased adherence properties as a result of higher pH, which may enable the colony to spread more easily (Reynolds, 2008). The ability of cells to stick together in this fashion can be desirable in production yeast strains that are used for bottle conditioning, but can be problematic in beer-spoiling yeasts since this can encourage the production and development of biofilms. Biofilms are aggregates of cells of one or more species (including in some circumstances both yeasts and bacteria together) that adhere to one another and form a community separated from the external environment by the development of a glycoprotein-polysaccharide layer known as a glycocalyx. This serves to protect organisms located within the biofilm structure, with the result that they will often display enhanced resistance to removal through conventional cleaning mechanisms. Biofilm formation in Saccharomyces yeast is known to be an intricate process with the activation of at least 71 genes (Andersen et al., 2014), a phenomenon likely to be equally complex in other yeast species. Once formed, the biofilm is a relatively stable unit and will continue to grow in size until it becomes physically too large to remain attached, or is disrupted in some way, leading to a portion of the biofilm breaking away and cells being released. During biofilm formation, certain yeasts can act as primary colonisers, which then provide a mechanism for the attachment and inclusion of other species, which may not have the capacity to stick to surfaces on their own. Within the brewery environment, this can lead to the development of complex and potentially serious biofilms. For example, it has been shown that non-Saccharomyces yeasts such as Pichia anomola can provide an initial surface attachment for the subsequent colonization by Saccharomyces strains, the presence of which then increases the spoilage potential of the biofilm (Timke et al., 2008).

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