Requirement for oxygen

Failure to provide oxygen at the start of fermentation results in slow fermentation rate, incomplete attenuation and poor yeast growth. Oxygen is required in brewery fermentation to allow yeast to synthesise sterols and unsaturated fatty acids. These lipids are essential components of membranes (Parks, 1978; Brenner, 1984; Weete, 1989; Nes et al., 1993). Thus, S. cerevisiae is capable of growth under strictly anaerobic conditions only when there is an exogenous supply of sterols and unsaturated fatty acids (Andreason & Stier, 1953, 1954). Under aerobic conditions sterols and unsaturated fatty acids may be synthesised de novo from carbohydrates. The ability to grow under strictly anaerobic conditions is relatively rare among yeasts. Indeed, Visser et al. (1990) concluded that S. cerevisiae was a positive exception in this respect. These authors studied the oxygen requirements of type species from 75 genera of yeasts. In oxygen-limited shake flasks using a complex medium supplemented with ergosterol and Tween 80 (a source of unsaturated fatty acids), all stains tested were capable of fermenting glucose to ethanol. However, only 23% actually grew under anaerobic conditions. S. cerevisiae alone was capable of rapid growth at low oxygen tensions. It was suggested that these differences reflected the importance of some mitochondrial functions in growth during anaerobiosis and that S. cerevisiae was less reliant on these than other facultative anaerobes.

The quantity of oxygen required for fermentation is yeast strain-dependent. Kirsop (1974) classified ale strains into four groups based on the oxygen concentration, which produced satisfactory fermentation performance. The required initial oxygen concentrations were half-air saturation, air saturation, oxygen saturation and more than oxygen saturation (see Section 6.1.2). Jacobsen and Thorne (1980) observed similar strain-specific oxygen requirements for lager yeasts. Notwithstanding these strain-specific differences the proportion of oxygen used for lipid synthesis during fermentation is comparatively small. It has been estimated that in an air saturated wort, pitched with lipid depleted yeast, that approximately 10% of the dissolved oxygen is used for sterol synthesis and up to 15% for unsaturated fatty acid synthesis (Aires et al., 1977; Kirsop, 1982). Although a proportion of the oxygen is consumed in oxidation of wort components, it is apparent that the bulk is utilised in reactions other than for synthesis of wort lipids.

Pitching yeast derived from a previous fermentation has an anaerobic (repressed) physiology. Sudden exposure to oxygenated wort provides an opportunity for synthesis of unsaturated lipids and possibly other advantageous aerobic reactions but it also presents a potentially lethal stress in the form of reactive oxygen radicals such as superoxide, hydroxyl and peroxide. In this sense sudden exposure to oxygen presents both threat and opportunity.

Provided the requirement for sterol and unsaturated fatty acid synthesis has been met, alcoholic fermentation and growth proceed under anaerobic conditions. As discussed in the previous section, under aerobic conditions and in the presence of repressing concentrations of sugars, metabolism is also fermentative. Other than lipid synthesis, therefore, there is no apparent role for oxygen in fermentation. However, it has long been recognised that respiratory deficient yeast strains - 'petites' - (see Section produce poor fermentation performance (Ernandes et al., 1993). In addition, fermentation efficiency, as judged by rates of ethanol formation, has been reported to increase under microaerophilic conditions (Grosz & Stephanopoulis, 1990).

As discussed already, Saccharomyces yeasts cannot grow under anaerobic conditions for an indefinite period but can tolerate anaerobiosis. This suggests that the natural environment of these organisms would be microaerophilic/fully aerobic with occasional periods of anaerobiosis. The additional implication is that yeast cells must be capable of responding rapidly to changes in oxygen tension both to ensure survival and to gain selective advantage over those organisms, which are either obligate aerobes or anaerobes.

Yeast cells have several mechanisms for nullifying the damaging effects of oxygen radicals (Krems et al., 1995). The metalloenzymes superoxide dismutases convert the superoxide radical to hydrogen peroxide (Fridovich, 1986). The latter may then be dissimilated by catalase. Yeast cells have two superoxide dismutases (SOD), a Cu,Zn-SOD which is cytosolic and a Mn-SOD which is located in mitochondria. The cytosolic enzyme is constitutive whereas the mitochondrial activity is inducible. Two catalases are present, catalase T, which is cytosolic and catalase A, which is associated with peroxisomes. It has been suggested that catalase is not significant in protection against oxidative stress since the peroxisomal enzyme is associated with growth on fatty acids and is repressed by the presence of glucose (Lee & Hassan, 1987). See Section 4.1.2 for further information on the location of these enzymes.

Reduced glutathione reacts with superoxide and hydrogen peroxide and may represent another protective mechanism, as may sequestration of radicals by transition metals such as copper and iron. Finally, in humans it has been reported that squalene may serve as a scavenger of free radicals and prevent lipid peroxidation in the skin (Kohno et al., 1995). Since yeast cells accumulate squalene in the absence of oxygen a similar mechanism would be plausible.

The effects of free radicals are far-reaching and are associated with degenerative processes such as mutagenesis, transformation of cell lines to malignancy and ageing (see Sections, and for implications in yeast). In respect of these effects, no eukaryotic cells are immune, regardless of the presence of the multiplicity of protective mechanisms. Brewing yeast is no exception. It was demonstrated that during an abrupt transition from anaerobiosis to aerobiosis there was a rapid increase in the specific activity of the CuZn-superoxide dismutase. Increase in the specific activity of the mitochondrial Mn superoxide dismutase occurred only after a lag of several hours. During the transition and before induction of the Mn-superoxide dismutase there was an immediate (5-7%) drop in the viability of the culture. Similar losses of viability were demonstrated when anaerobically-grown yeast was exposed to 0.25 mM potassium superoxide. Aerobically grown cells were unaffected by similar exposure. It was concluded, therefore, that the CuZn-superoxide dismutase was responsible for protection against oxygen radicals in anaerobic cells suddenly exposed to oxygen. The Mn-superoxide dismutase was protective only in aerobic cells (Clarkson et al., 1991).

Zitomer and Lowry (1992) discussed regulation of gene expression by oxygen in S. cerevisiae. These authors described three classes of genes responsive to oxygen tension. First, respiratory growth under aerobic conditions which requires the expression of more than 200 genes. A list of these may be found in Tzagoloff and Dieckmann (1990). Second, there is a class of genes which are expressed only under anaerobic conditions, the functions of which are as yet unknown. Third, the so-called 'hypoxic class of genes' which respond to decreases in oxygen tension and are required for efficient utilisation of low concentrations of oxygen.

The signalling pathway by which molecular oxygen exerts its effects on metabolism remains to be fully elucidated. There is added complexity in that the effects due to oxygen may be modified by other external stimuli such as the overriding of induction of respiration by the presence of a repressing sugar (Section 3.4.1). Haem would appear to be a key intermediate in the oxygen sensing pathway. This molecule is a prosthetic group in cytochromes and also in oxygen-binding enzymes such as catalase. In addition, it serves as an effector metabolite in many of the pathways that involve the utilisation of molecular oxygen (Padmanaban et al., 1989). Oxygen is required for haem synthesis and anaerobically grown yeast contains all the necessary enzymes for haem synthesis. Therefore, the cellular concentration of haem is directly related to oxygen tension. Genes which are induced by haem include those which are involved in respiratory function and a second class which are protective against oxygen radicals. Hypoxic genes are repressed by haem and their products include enzymes involved in the synthesis of sterols, unsaturated fatty acids, haem itself and parts of the electron transport chain (Zitomer & Lowry, 1992). With some exceptions, repression by haem involves activities that are redundant under anaerobic conditions.

In the context of brewing yeast and fermentation, the evidence suggests that apart from lipid synthesis oxygen is required for certain activities which may be induced by haem and are not repressed by glucose and other sugars. These activities are implicated in the partial development of mitochondrial function, which occurs in the repressing but aerobic phase of fermentation. Anaerobic yeast cells contain promitochondria (see Section which develop into fully functional organelles on exposure to oxygen and during derepression (Plattner et al., 1971). Since the enzymes for some essential anabolic reactions are located in the mitochondria it is assumed that in the absence of oxidative phosphorylation another mechanism must exist to generate energy for these reactions and to power transport of precursors and products between mitochondria and the cytosol.

The evidence suggests that during fermentative growth, mitochondrial ATP is derived from substrate level phosphorylation which occurs in the cytosol. Passage of adenine nucleotides between cytosol and mitochondrion is catalysed by an ADP/ATP translocase. Three genes have been identified which encode for this enzyme. One enzyme is constitutive, another is expressed under aerobic conditions and the third is induced by anaerobiosis (Kolarov et al., 1990). Zitomer & Lowry (1992) suggest that the anaerobic enzyme catalyses the reverse of the normal translocase reaction and actually imports ATP from the cytosol into mitochondria. Evidence for this has been provided by the observation that bongkrekic acid, a specific inhibitor of the ATP/ ADP translocase, arrested growth under anaerobic conditions (Subik et al., 1972; Gbelska et al., 1983). O'Connor-Cox et al. (1993), investigating the roles of oxygen in brewery fermentation, also concluded that the ATP/ADP translocase was essential for mitochondrial energy generation. These authors noted that when the translocase was inhibited, also using bongkrekic acid, that yeast growth was reduced, assimilation of wort nitrogen was low and there was over-production of VDK, acetaldehyde, S02 and dimethylsulphide.

3.5.1 Synthesis of sterols and unsaturated fatty acids

In brewery fermentations, a proportion of the requirement for unsaturated fatty acids is obtained from wort. This may be vanishingly small particularly in the case of high-gravity bright worts made with a high level of non-malt adjunct. A small quantity of sterol may be present in wort, although under the conditions of brewery fermentation brewing yeast may not be able to assimilate it, as discussed later. In consequence, both sterol and unsaturated fatty acids must be synthesised during the initial phase of fermentation when oxygen is made available. In the case of sterols, the quantity of oxygen supplied regulates the quantity synthesised. Growth of yeast during the anaerobic phase of fermentation dilutes the pre-formed sterol pool between mother cells and their progeny. Cells divide until sterol depletion limits growth and therefore is responsible for the requirement for more oxygen when the yeast is re-pitched.

The aerobic synthesis of sterol during fermentation is accompanied by dissimilation of glycogen. A linear relationship may be demonstrated between the quantities of sterol formed and glycogen utilised. It is suggested that because of lack of membrane competence in pitching yeast, mobilisation of glycogen provides the metabolic fuel for sterol synthesis (Quain et al., 1981; Quain & Tubb, 1982). The profiles of changes in the concentrations of glycogen and total sterol synthesis during fermentation are shown in Fig. 3.6. The total quantities of sterol synthesised during fermentation are modest, typically increasing from 0.1% of the dry weight in yeast cropped at the end of fermentation up to approximately 1 % at the end of the aerobic phase of fermentation. Greater concentrations of sterol are accumulated in derepressed cells, typically up to 5% of the yeast dry weight (Quain & Haslam, 1979).

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