1. OVERVIEW OF PROBLEM FERMENTATIONS Linda F. Bisson Department of Viticulture and Enology, UCD

2. Types of Problem Fermentations Lack of fermentation progression Off-character production

3. PROBLEMS IN FERMENTATION PROGRESSION

4. Stuck and Sluggish Fermentations Characterized by failure of yeast to consume all available sugar Multiple causes Can be difficult to treat as cells have arrested metabolism Leads to reduced wine quality

5. Typical Fermentation Profile 1 2 3 4 5 1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness Brix Time

6. Stages of Fermentation Stage 1: Lag due to need to adapt then grow to maximize population levels Stage 2: Fastest rate of fermentation: largest population of cells supported by medium, absence of inhibitory ethanol Stage 3: Transition: at this point ethanol has become inhibitory to continued maximal fermentation; cells are adjusting plasma membrane composition and internal membranes and proteins to an ethanol tolerant form Stage 4: Maximal fermentation rate of ethanol-adapted cells Stage 5: Complete metabolic consumption of sugar, cells enter decline or death phase

7. Fermentation Progression Assessment Lag time Duration? Maximum fermentation rate Rate value? Duration? Transition point At what Brix level? How sharp? Post-transition fermentation rate Value relative to max fermentation rate? Length of time? Brix/ethanol/nitrogen level at which it occurs? Overall time to dryness

8. Fermentation Capacity Is a Function of: Yeast Biomass Concentration Fermentative Ability of Individual Cells

9. Fermentation Capacity Is a Function of: Yeast Biomass Concentration Fermentative Ability of Individual Cells Both are dependent upon: Adequate nutrition Absence of inhibitors Permissive growth conditions Permissive fermentation (energy generation) conditions

10. Causes of Stuck/Sluggish Fermentations Nutrient limitation

11. Nutrient Limitation Nitrogen: most often limiting Phosphate can also be limiting Depending upon circumstances, micronutrient limitation may also be a problem Pitching yeast Deficiency in vineyard Microbial blooms prior to fermentation Loss during rehydration

12. Causes of Stuck/Sluggish Fermentations Nutrient limitation Ionic imbalance

13. Ionic Imbalance Ratio of K + :H + Must be at least 25:1 Needs to be adjusted early in fermentation Probably important in building an ethanol tolerant membrane

14. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Ethanol toxicity

15. Ethanol Toxicity Ethanol disrupts membrane component interactions affecting transporter activity and ability to remove toxic substances like free protons from the cell Cells adapt to ethanol by changing the composition of the membrane and by changing the species of the proteins present Adaptation requires the ability to make new proteins and membrane components (lipids, sterols) If conditions do not enable development of ethanol tolerance, fermentation rates will decrease

16. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Ethanol toxicity Presence of toxic substances

17. Presence of Toxic Substances Toxins may arise from the metabolic activity of other microbes Toxins may arise from sub-optimal metabolic activity of Saccharomyces (such as acetic acid production) Toxins may have arisen in vineyard, but are not inhibitory until ethanol has accumulated

18. The Most Common Toxins Acetic acid Higher organic acids (C 2 – C 4 ) Medium chain fatty acids/fatty acid esters Acetaldehyde Fungicide/Pesticide residues Higher alcohols Higher aldehydes Killer factors Sulfur dioxide

19. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Ethanol toxicity Presence of toxic substances Poor adaptation of strain

20. Poor Adaptation of Strain Strain may not display sufficient ethanol tolerance Strain may have high nitrogen/vitamin requirements that are not being met Strain may be a poor fermentor, but capable of dominating the fermentation Rehydration/Implantation issues: Strain not rehydrated properly, stored correctly or is being used past its expiration date Temperature shock effects during rehydration and pitching into the tank

21. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Ethanol toxicity Presence of toxic substances Poor adaptation of strain Low pH

22. pH pH is reduced by metabolism of Saccharomyces Low pH musts (below pH 3.0) may drop to an inhibitory level (pH 2.7) Dependent upon K + concentration

23. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Ethanol toxicity Presence of toxic substances Poor adaptation of strain Low pH Temperature shock

24. Temperature Shock Superheating or supercooling Will impact cell membrane functionality and ability to tolerate ethanol Main issue is a ratcheting down of maximal level of ethanol tolerance

25. Problem Fermentation Profiles

28. Causes of Long Lag Poor health of starter culture Presence of inhibitors Poor grape quality Mold infestation Premature initiation of fermentation

29. Poor Health of Starter Culture Active Dry Yeast: Past expiration date Not hydrated properly Not stored properly Natural Fermentation: Yeast numbers low Inhibitory microbes present (heavy rot of fruit) Poor yeast strain present (high N requirements, lack of ethanol tolerance)

30. Presence of Inhibitors Sulfur dioxide concentration too high Sulfur dioxide added improperly Microbial competition for nutrients Microbial activity generating inhibitory compounds Enzymatic competition for oxygen Pesticide/fungicide residues on grapes at harvest Temperature of must/juice too high/low

31. Poor Grape Quality Infected grapes: loss of micronutrients Infected grapes: high microbial loads Loss of free oxygen

33. Causes of Slow Rate Over Entire Time Course Failure to reach maximum cell density Nutrient (growth factor) limitation Strain a poor choice for conditions Inhibitory fermentation conditions: temperature, pH, ionic imbalances, organic acids

35. Causes of a Decrease in Rate Poor ethanol tolerance Loss of viability Loss of fermentative capacity Nutrient (survival factor) limitation Poor strain

37. Causes of an Abrupt Stop Temperature shock Rapid build up of inhibitors: acetic/organic acids: generally caused by addition of active ML culture pH decreases too much Strain very ethanol sensitive

38. Most Common Causes of Stuck/Sluggish Fermentations Temperature extreme Ethanol intolerance Nutrient deficiency Deficient yeast strain Microbial incompatibility Presence of an inhibitory substance Poor fermentation management decisions

39. Why are stuck fermentations difficult to treat? Cells adapt to adverse conditions by reducing fermentation capacity Biological adaptation difficult to reverse Diagnosis of cause of fermentation problem difficult Conditions that cause stuck fermentations are also conducive to cell death New inocula respond to cell death by arresting activities

40. Best Advice Pay attention to strain selection: match strain to fermentation conditions Provide adequate nutrient levels For active dry yeast: follow packet instructions for rehydration and adaptation to juice For native fermentations: be aware of microbiota issues, heed historical observations for nutrient requirements and ethanol tolerance Temperature Shock: should be avoided but if it happens consider racking and re-inoculation with unshocked yeast Adjust juices of marginal pH (too high or too low) Limit activity of inhibitory microbes (wild lactics)