1. Linda F. Bisson Department of Viticulture and Enology Issues in Fermentation Management, 2011 Temperature and Other Stressors

2. Outline of Presentation Types of fermentation stressors Types of fermentation stressors Fermentation progression and stress Fermentation progression and stress Stress management Stress management

3. TYPES OF FERMENTATION STRESSORS

4. Types of Fermentation Stressors Internal Biological Internal Biological External Biological External Biological Abiological Abiological

5. Internal Biological Stressors Sub-optimal nutrition Sub-optimal nutrition Inability to adapt to environment Inability to adapt to environment Metabolic imbalances: cytoplasmic redox potential, cytoplasmic pH Metabolic imbalances: cytoplasmic redox potential, cytoplasmic pH

6. External Biological Stressors Presence of inhibitory metabolites Presence of inhibitory metabolites Competition for metabolites Competition for metabolites Changes to chemical environment Changes to chemical environment

7. Abiological Stressors Temperature Temperature Reductive conditions of tank/vessel Reductive conditions of tank/vessel Lack of mixing/cell buoyancy: cell settling Lack of mixing/cell buoyancy: cell settling

8. FERMENTATION PROGRESSION AND STRESS

9. Yeast and Stress Grape juice is a relatively harsh environment Grape juice is a relatively harsh environment –Carbon-rich –Nitrogen-limited –Extremes of pH Will be inhibited by accumulation of their own metabolic end products Will be inhibited by accumulation of their own metabolic end products –ethanol –acetaldehyde –CO 2

10. Yeast and Stress Common stress response Common stress response –Countering different stressors involves the same cellular metabolic responses –A graded response Presence of multiple stressors often synergistic (sum is worse than its parts) Presence of multiple stressors often synergistic (sum is worse than its parts) Metabolically assess if it is worth it to keep going: cell growth, cell metabolism Metabolically assess if it is worth it to keep going: cell growth, cell metabolism

11. Fermentation Stress Factors Ethanol Ethanol Acetaldehyde Acetaldehyde

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

13. How Do Membranes Normally Work? Maintain permeability barrier Maintain permeability barrier Gated: let desirable things in get undesirable things out and keep them there Gated: let desirable things in get undesirable things out and keep them there Movements of things are protein dependent Movements of things are protein dependent Must allow movement of proteins Must allow movement of proteins

14. Membrane Fluidity Is Required for Transport G G G

15. What Impacts Membrane Function? Temperature: lipid structure responsive to temperature Temperature: lipid structure responsive to temperature Ethanol: impacts lipid structure and hydration Ethanol: impacts lipid structure and hydration Exceeding capacity of membrane carriers to export toxic substances (equivalent of cellular kidney failure) Exceeding capacity of membrane carriers to export toxic substances (equivalent of cellular kidney failure) Most toxic substance? Protons Most toxic substance? Protons Protons co-transported with desired compounds Protons co-transported with desired compounds

16. Proton Export: the PMA1 pump ATPADP + Pi H+

17. Ethanol Toxicity Perturbs membrane structure at protein:lipid interface Perturbs membrane structure at protein:lipid interface Leads to increased “passive proton flux” and acidification of cytoplasm Leads to increased “passive proton flux” and acidification of cytoplasm Inhibits protein activity Inhibits protein activity Affects membrane “fluidity” Affects membrane “fluidity” Ultimately leads to lipid interdigitation and membrane failure Ultimately leads to lipid interdigitation and membrane failure

18. Ethanol Toxicity Plasma membrane is the most ethanol-sensitive cell structure: Composition: Protein 50% Lipid 40% Lipid 40% Other 10% Other 10%Functions: Permeability barrier Regulation of uptake Mediates response to environment Maintains electrochemical gradients Mediates cell-cell interactions

19. Lipid Bilayers Normal Interdigitation

20. Ethanol Toxicity Adaptation of membrane requires: Adaptation of membrane requires: –Increasing content of sterols –Increasing relative content of proteins –Increasing level of desaturation (number of double bonds) in fatty acid side chains –Modification of phospholipid head groups? Failure to adapt means cell death Failure to adapt means cell death

21. Fatty Acid Saturation Saturated Unsaturated

22. Phospholipid Head Groups FA PO 4 OH HO FA PO 4 CH 2 CH OOC NH 2 FA PO 4 CH 2 NH 2 FA PO 4 CH 2 N -CH 3 CH 3 H 3 C- + Phoshpatidyl- Inositol Serine Ethanolamine Choline

23. Ergosterol HO

24. Membrane Adaptation Desaturation and sterols both function to stabilize bilayer and prevent interdigitation: integrate within membrane; stabilize cytoplasmic face of membrane Desaturation and sterols both function to stabilize bilayer and prevent interdigitation: integrate within membrane; stabilize cytoplasmic face of membrane

25. Lipid Bilayers Normal Stabilized

26. Ethanol Toxicity Sterol and fatty acid desaturation are Oxygen-requiring processes New protein synthesis requires nitrogen be available Phospholipid head group synthesis requires cofactors (S-adenosyl- methionine) be available

27. Acetaldehyde as Stressor Accumulates along with ethanol Accumulates along with ethanol Impacts cytoplasmic redox status Impacts cytoplasmic redox status Reactive molecule: interferes with metabolism and protein structure Reactive molecule: interferes with metabolism and protein structure

28. External Stress Factors Those that impact ethanol tolerance Those that impact ethanol tolerance –Temperature –Organic acids Independent of ethanol tolerance Independent of ethanol tolerance –Peptide killer factors –Redox status stressors

29. Temperature Shock Super-cooling/heating of tank due to equipment failure Super-cooling/heating of tank due to equipment failure High temperature fermentations becoming too warm due to yeast metabolism High temperature fermentations becoming too warm due to yeast metabolism

30. Temperature Also impacts membrane Also impacts membrane –Warmer the temperature the more fluid –Warmer the temperature the more likely interdigitation will occur Affects protein activity Affects protein activity Affects protein conformation and complex formation Affects protein conformation and complex formation Affects metabolic rates Affects metabolic rates

31. Organic Acids C6, C8, C10, C12 C6, C8, C10, C12 From yeast or from bacteria From yeast or from bacteria Enter yeast cell with protons adding to proton stress of cytoplasm Enter yeast cell with protons adding to proton stress of cytoplasm Exceed proton pump to get rid of them Exceed proton pump to get rid of them

32. Fermentation Stressors All converge on membrane All converge on membrane Alter fluidity (temperature) Alter fluidity (temperature) Alter proton accumulation and excretion Alter proton accumulation and excretion Once membrane becomes dysfunctional cell fate is sealed Once membrane becomes dysfunctional cell fate is sealed Viable cells in population respond to this type of stress by shutting down Viable cells in population respond to this type of stress by shutting down