1. VEN124 Section III The Alcoholic Fermentation

2. Lecture 8: Yeast Biology

3. Reading Assignment: Text, Chapter 4, pages123-168

4. In this lecture we will cover the basic biology of yeast and the topics of yeast nutrition and selection for wine production.

5. The Alcoholic Fermentation The microbiological conversion of the grape sugars, glucose and fructose, to the end product, ethanol, is called the alcoholic fermentation. Fermentation means that an organic compound serves as terminal electron acceptor.

6. The Alcoholic Fermentation Is conducted by the yeast Saccharomyces cerevisiae or Saccharomyces bayanus

7. Characteristics of Saccharomyces Eukaryote: possesses a membrane bound nucleus Nucleus Endoplasmic reticulum Nucleus is surrounded by a double membrane layer with the outer membrane contiguous with the endoplasmic reticulum

8. Characteristics of Saccharomyces Eukaryote: possesses a membrane bound nucleus Reproduces by budding

9. Reproduction by Budding Mother Cell Daughter Cell

10. Characteristics of Saccharomyces Eukaryote: possesses a membrane bound nucleus Reproduces by budding Grows vegetatively as haploid (1N) or diploid (2N)

11. Yeast Life Cycle New daughters must grow before initiating their first cell cycle

12. Characteristics of Saccharomyces Eukaryote: possesses a membrane bound nucleus Reproduces by budding Grows vegetatively as haploid (1N) or diploid (2N) Capable of conjugation (1N ⃗ 2N) and sporulation (2N ⃗ 1N)

13. Yeast Life Cycles: Conjugation Haploid Cells Mating Pair Budding Zygote Diploid Cell  a a/  a 

14. Yeast Life Cycles: Sporulation Vegetative Cell 2N Tetrad 4 x 1N Spore Ascus

15. Characteristics of Saccharomyces Eukaryote: possesses a membrane bound nucleus Reproduces by budding Grows vegetatively as haploid (1N) or diploid (2N) Capable of conjugation (1N ⃗ 2N) and sporulation (2N ⃗ 1N) Non-motile

16. Characteristics of Saccharomyces: Sub-Cellular Organization Plant-like cell wall: comprised of carbohydrate (glucan, mannan) and glycosylated protein (phosphomanno- protein) Mitochondria: site of oxidative reactions Vacuoles: site of storage and hydrolysis Secretory pathway Nucleus

17. Saccharomyces Mitochondrion Vacuole Nucleus Endoplasmic reticulum Secretory Pathway Golgi

18. Glycolysis The set of biochemical reactions converting hexose (6 carbon) sugars to two 3 carbon pyruvate molecules, during which energy is released and recaptured in the form of ATP.

19. Glycolysis Glucose + 2 ATP + 2 NAD + + 2 ADP + 2 Pi 2 Pyruvate + 4 ATP + 2 NADH + heat

20. Glycolysis glucose glucose-6-phosphate fructose-6-phosphate fructose 1,6-diphosphate dihydroxyacetone phosphate glyceraldehyde 3-phosphate 1,3 -diphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate phosphoenol pyruvate pyruvate fructose ATP NAD + NADH ATP

21. “Upper Glycolysis”: consumes two molecules of ATP “Lower Glycolysis”: produces four molecules of ATP NET PRODUCTION: TWO MOLECULES OF ATP

22. Where does ethanol come from? The end products of glycolysis are pyruvate and 2 molecules of the reduced co-factor NADH. Yeast cells regenerate NAD + by transferring the hydrogen molecule (electron) to an organic molecule: acetaldehyde

23. Ethanol Formation CH 3 -CO-COOH CO 2  + CH 3 -CHO CH 3 -CH 2 OH NADH NAD + H+H+ Pyruvate Acetaldehyde Ethanol

24. Other organisms use different strategies to regenerate NAD + Their presence in wine leads to a diversity of end products of sugar catabolism

25. Carbon Distribution at End of Fermentation 95% = ethanol + carbon dioxide 1% = new cells 4% = other end products –Pyruvate –Acetate –Acetaldehyde –Glycerol –Lactate

26. Ethanol Yield 1M glucose (fructose) 2 M CO 2 + 2 M ethanol Theoretical Maximum: 180 g 2(46g) = 92/180 = 51.1% w/w = 63.9% v/w =  0.6 original Brix value

27. Yeast will ferment even in the presence of oxygen. Why?

28. Fermentation vs. Respiration Fermentation: 2 ATP/glucose(fructose) Respiration: 36-38 ATP/ glucose Efficiency of ATP yield is only an issue if sugar is limiting

29. In Saccharomyces, glucose concentration regulates the switch between fermentation and respiration.

30. Regulation of Glycolysis Transport: site of global rate control Allosteric enzymatic steps: localized rate control –Hexokinase –Phosphofructokinase –Pyruvate kinase Effectors of regulation: ATP, ADP, AMP fructose 2,6 bisphosphate, citrate, glucose

31. Yeast Choice and Nutrition

32. Yeast Choice: Desirable Traits Fermentation to dryness Reasonable rate of fermentation Predictable fermentation characteristics Good ethanol tolerance Good temperature tolerance Sulfur dioxide tolerance

33. Yeast Choice: Desirable Traits Little to no off-character production –Sulfur volatiles –Acetic acid –Ethyl carbamate Little to no inhibition of other desirable microbes Killer factor resistant Production of desired aroma characters

35. Yeast Nutrition Macronutrients: Building blocks needed for new cell material Micronutrients: Catalysts needed to facilitate biochemical reactions

36. Macronutrients Carbon/Energy Sources: glucose, fructose, sucrose Nitrogen Sources: amino acids, ammonia, nucleotide bases, peptides Phosphate Sources: inorganic phosphate, organic phosphate compounds Sulfur Sources: inorganic sulfate, organic sulfur compounds

37. Macronutrient Energy Sources Monosaccharides: glucose, fructose, galactose, mannose Disaccharides: sucrose, maltose, melibiose Trisaccharides: raffinose Pentoses: None Oxidative substrates: pyruvate, acetate, lactate, glycerol, ethanol

38. Categories of Yeast Nitrogen Sources Compound may be used as that compound for biosynthesis Compound may be converted to related compounds for biosynthesis Compound may be degraded with release of nitrogen

39. Yeast Nitrogen Sources Degradation may depend upon availability of other components: vitamins and oxygen Utilization impacted by other environmental factors such as pH

40. Micronutrients Minerals and Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu Vitamins: biotin is the only required vitamin, but others are stimulatory

41. Nutritional Requirements of Different Phases of Fermentation Growth Phase: Building blocks and catalysts Stationary Phase: Survival factors

42. Yeast Nutritional Phases Cell # Time lag log stationary death Brix

43. Most of the fermentation is conducted by stationary phase cells Stationary phase: 1. rate of growth = rate of death 2. quiescent, no growth, no death

44. Role of Survival Factors Maintain viability of cells Increase ethanol tolerance Maintain energy generation

45. Survival Factors Oxygen Fatty Acids Sterols Nutritional Factors

46. How Does Ethanol Inhibit Yeast? Displaces water of hydration changing the properties of protein-lipid interactions Denatures proteins Disrupts protein active sites Allows increased passage of protons from the medium into the cell leading to acidification of the cytoplasm Removal of protons requires expenditure of energy

47. Survival Factors Needed to alter composition of the plasma membrane (sterols, fatty acids and proteins) so that it can withstand the perturbing effects of ethanol Both phospholipid and protein content must be adjusted