Yeast

• Acetate esters and higher alcohols production in S. cerevisiae are temperature-dependent
• Most commercial S. cerevisiae strains are mesophilic (20-30°C), with Tmax 35 – 43°C
• What S. cerevisiae needs for survival and growth: Temperature, Water, Media pH & pO2, Other Stresses (physical & chemical)
• Regulatory mechanisms

  1. Catabolite repression - glucose or an initial product of glucose metabolism represses the synthesis of various respiratory and gluconeogenic enzymes

  2. Glucose repression - an adaptation that degrades glucose exclusively to ethanol and CO2

  3. Catabolite inactivation - glucose-induced deactivation of some respiratory and gluconeogenic enzymes
• Heat-shock response is brought about by Hsps (Heat-shock proteins)
• Glycolysis


  General pathway for conversion of glucose to pyruvate, whereby production of energy in the form of ATP is coupled to the generation of intermediates and reducing power in the form of NADH for biosynthetic pathways
• Low-temperature stress (cold-shock) protection by intracellular trehalose and a cold-shock protein
• Chemoorganoheterotrophic organism

  Requires organic substrates to get its carbon for growth and development, and produces energy from oxidoreduction of an organic compound
• Most commercial S. cerevisiae strains are regarded as “non-osmotolerant”
• Carbohydrates in the form of sugar

  Used to generate energy. In the absence of air, these sugars are converted to alcohol and carbon dioxide
• Thermotolerance in S. cerevisiae is related to pH: it is at a maximum when external pH declines to 4
• Media acidified with organic acids are more inhibitory to S. cerevisiae growth than mineral acids: they lower intracellular pH more
• Overall cell volume of S. cerevisiae is observed to change when osmotic potential of medium changes: cell walls are relatively elastic
• The growth phase of the yeast is dependent on how much air/oxygen has been added to the fermentation substrate. Requires 8 to 20 ppm of oxygen
• The Pasteur effect is the inhibiting effect of oxygen on fermentation. When the fermentation process is aerated, this could result in an increase of yeast cell growth which would conversely result in a decrease in fermentation rates. The Pasteur effect occurs only if glucose concentration is low and under limited concentrations of nitrogen and other nutrients
• What S. cerevisiae needs for survival and growth

  • Water
  • Media pH & pO2
  • Other Stresses
• The influence of oxygen and sugar availability lead to various metabolic phenomena: Pasteur effect, Crabtree effect, Facultative anaerobe characteristics
• S. cerevisiae characteristics: Grow aerobically or anaerobically, Anaerobic growth only for a few generations, Preferentially use oxygen, Fermentation is preferred catabolic route, Lower glycolytic rate in presence of oxygen (Pasteur effect), Glycolytic rate is similar in presence and absence of oxygen (no noticeable Pasteur effect)
• Cold shock

  1. Sub-lethal growth arrest during cell division following a cold shock

  2. Cold shock induces specific protein biosynthesis, known as cold-shock proteins
• Acid washing of brewer’s yeasts: hold at pH 2.2 – 2.5 at 4°C. for “a few hours”, killing most contaminating bacteria
• Trehalose is also an osmoprotectant in the early stages of fermentation of “high-” and “very high gravity” worts
• S. cerevisiae is not a facultative anaerobe: it is auxotrophic for oleic acid and ergosterol under anaerobic conditions. The biochemical pathways leading to these essential biomolecules require the input of O2
• Physical stresses on S. cerevisiae include high hydrostatic pressure, high CO2 gas pressure, mechanical shear stress, temperature shock stress, and osmotic shock stress
• Most commercial S. cerevisiae strains grow well between pH 4.5 – 6.5
• Some protection is afforded by coordinating intracellular Na+, K+ and glycerol levels
• Crabtree effect is the phenomenon in which yeast produces ethanol in aerobic conditions at high external glucose concentrations
• Adaptive physiological responses to ethanol stress

  1. Decreased membrane saturated fatty acids (e.g. palmitate)

  2. Increase in membrane unsaturated fatty acids (e.g. oleate)

  3. Greater ergosterol synthesis

  4. Elevated intracellular trehalose

  5. Increased mitochondrial superoxide dismutase activity

  6. Increased stress protein synthesis

  7. Increased Cytochrome P450

  8. Increased phosphatidylinositol biosynthesis
• Sulphite uptake in S. cerevisiae

  • Sulphite in medium liberates SO2 which enters freely
• Nutrition sources for S. cerevisiae

  • Carbon
  • Nitrogen
  • Sulphur
  • Phosphate
  • Mineral elements
  • Vitamins
• Monosaccharide uptake in S. cerevisiae

  • 20 hexose transporters identified; rate of alcohol production linked to sugar uptake
• Assessing viability of S. cerevisiae growth

  Plate count; Slide count; Vital stains (e.g. methylene blue); Metabolic activity (e.g. ATP bioluminescence); Cellular compounds (e.g. glycogen, trehalose); Intracellular pH (spectrofluorophotometry)
• Cell death: The Hayflick limit for S. cerevisiae is around 25
• Vmax of the main glycolytic enzymes


  • Information about the maximum rate of the main glycolytic enzymes
• Effects of ethanol on cell physiology

  • Membrane structure and function
  • Alteration in fatty acid and sterol composition
  • Induced lipolysis of cellular phospholipids
  • Increased ionic permeability
  • Inhibition of nutrient uptake
  • Affects membrane polarization and proton-motive force
• Using viability estimates in S. cerevisiae growth

  To determine "pitching" rate of inoculum into production fermenters; generally try for 10^7 viable cells/ml and inoculum volume of 1% of substrate feedstock
• S. cerevisiae Growth

  Multilateral, asymmetric budding initiated when mother reaches a critical size; daughter always smaller at separation; symmetric budding during pseudohyphal growth
• Periplasm is a thin (35-45 A), cell wall-associated region external to the plasma membrane and internal to the cell wall. It contains secreted proteins (mannoproteins) that are unable to permeate the cell wall but fulfill essential functions in hydrolyzing substrates that do not cross the plasma membrane
• Cell envelope consists of the plasma membrane, the periplasmic space, and the cell wall and other extracellular features. The cell envelope takes ca. 15% of the total cell volume and has a major role in controlling the osmotic and permeability properties of the cell
• Cytological methods for yeasts

  1. Light microscopy: phase-contrast + appropriate staining techniques

  2. Fluorescence microscopy with fluorescent dyes

  3. Reporter molecules such as green fluorescent protein (GFP)

  4. Monospecific antibodies coupled with fluorescent dyes

  5. Flow cytometry

  6. Electron microscopy (transmission and scanning)

  7. Atomic force microscopy
• Yeast cytoplasm is an acidic (pH 5.25) colloidal fluid, mainly containing ions and low or intermediate molecular weight organic compounds, and soluble macromolecules (e.g., enzymes, factors, glycogen). Enzymes include those in the glycolytic pathway, acid syntha