1. Linda F. Bisson Department of Viticulture and Enology Issues in Fermentation Management, 2011 Yeast Nutrition and Fermentation Progression

2. Outline of Presentation General introduction to nutritional needs during fermentation General introduction to nutritional needs during fermentation Special considerations Special considerations –Native fermentations –Vineyard factors impacting nutrition –Winery production factors impacting nutrition

3. GENERAL INTRODUCTION

4. Yeast Need Nutrients To: Build new cells Build new cells Make and modify metabolic pathways Make and modify metabolic pathways Repair and prevent cell damage Repair and prevent cell damage Adapt to changing environments Adapt to changing environments

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

6. BUILDING NEW CELLS

7. Yeast Nutritional Phases: Building New Cells Cell # Time lag log stationary death Brix

8. Yeast Nutrition: Building New Cells Energy source: Ability to capture and reuse bond energy Energy source: Ability to capture and reuse bond energy Macronutrients: Building blocks needed for new cell material Macronutrients: Building blocks needed for new cell material Micronutrients: Catalysts needed to facilitate biochemical reactions related to growth Micronutrients: Catalysts needed to facilitate biochemical reactions related to growth

9. Yeast Nutrition: Building New Cells Energy source: labor Energy source: labor Macronutrients: bricks and mortar Macronutrients: bricks and mortar Micronutrients: equipment Micronutrients: equipment

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

11. Growth Macronutrients in Juice Carbon: Excess Carbon: Excess Nitrogen: Excess to Deficiency Nitrogen: Excess to Deficiency Phosphate: Sufficient to Deficiency Phosphate: Sufficient to Deficiency Sulfate: Sufficient Sulfate: Sufficient

12. Carbon Sources For carbon component: role in new cell material (bricks and mortar) For carbon component: role in new cell material (bricks and mortar) To be degraded to recapture bond energy: role as energy source to perform needed metabolic reactions (labor) To be degraded to recapture bond energy: role as energy source to perform needed metabolic reactions (labor)

13. Yeast Carbon/Energy Sources in Juice: Monosaccharides: glucose, fructose, galactose, mannose Monosaccharides: glucose, fructose, galactose, mannose Disaccharides: sucrose, maltose, melibiose Disaccharides: sucrose, maltose, melibiose Trisaccharides: raffinose Trisaccharides: raffinose Pentoses: Not used Pentoses: Not used

14. Yeast Carbon (NOT Energy) Sources in Juice: Metabolic Intermediates: TCA cycle acids: malate, fumarate, succinate, oxalacetic acid, citrate Metabolic Intermediates: TCA cycle acids: malate, fumarate, succinate, oxalacetic acid, citrate Oxidative substrates: pyruvate, acetate, lactate, glycerol, ethanol Oxidative substrates: pyruvate, acetate, lactate, glycerol, ethanol See dynamic changes in concentrations in juice during fermentation, role in metabolism and growth not clear See dynamic changes in concentrations in juice during fermentation, role in metabolism and growth not clear

15. Nitrogen Sources For Saccharomyces, function exclusively in building new cells and new pathways For Saccharomyces, function exclusively in building new cells and new pathways Bacteria and some non-Saccharomyces yeasts can use nitrogen compounds as energy sources Bacteria and some non-Saccharomyces yeasts can use nitrogen compounds as energy sources Nitrogen most often the limiting factor in the Saccharomyces environment Nitrogen most often the limiting factor in the Saccharomyces environment

16. Yeast Nitrogen Sources Ammonia Ammonia Most amino acids Most amino acids Degradation may depend upon availability of other components: vitamins and oxygen Degradation may depend upon availability of other components: vitamins and oxygen Utilization impacted by other environmental factors such as pH and ethanol Utilization impacted by other environmental factors such as pH and ethanol

17. Categories of Yeast Amino Acid Nitrogen Sources Compound may be used as that amino acid for biosynthesis Compound may be used as that amino acid for biosynthesis Compound may be converted to related amino acids for biosynthesis Compound may be converted to related amino acids for biosynthesis Compound may be degraded with release of nitrogen (be mindful of potential end products!) Compound may be degraded with release of nitrogen (be mindful of potential end products!)

18. Phosphate Sources Inorganic phosphate Inorganic phosphate Organic phosphate-containing molecules under starvation conditions only Organic phosphate-containing molecules under starvation conditions only Prefer to make their own Prefer to make their own –Internally generated phosphate-containing molecules perform important regulatory roles –If taken in from outside might send false signal Roles as building blocks and in energy movements Roles as building blocks and in energy movements

19. Sulfate Sources Sulfate Sulfate S-containing amino acids S-containing amino acids Used for building new cell material Used for building new cell material Used in formation of catalysts Used in formation of catalysts

20. Micronutrients for Building New Cells Minerals and Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu Minerals and Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu Vitamins: biotin is the only required vitamin, but others are stimulatory Vitamins: biotin is the only required vitamin, but others are stimulatory Generally depending upon how the cells are grown previously micronutrients are not limiting Generally depending upon how the cells are grown previously micronutrients are not limiting

21. MAKING AND MODIFYING METABOLIC PATHWAYS

22. Yeast Nutritional Phases: Modifying Pathways Cell # Time lag log stationary death Brix

23. Making and Modifying Pathways Needs energy for degradation and reconstruction Needs energy for degradation and reconstruction May need influx of net new nutrients if requirements not readily met by degradation (mostly a nitrogen issue) May need influx of net new nutrients if requirements not readily met by degradation (mostly a nitrogen issue) Requires micronutrients for generating the new pathway and for operating the enzymes of that pathway Requires micronutrients for generating the new pathway and for operating the enzymes of that pathway

24. Making and Modifying Pathways Happens during active growth Happens during active growth Happens under non-proliferative but metabolically active growth conditions: sustaining fermentative capacity Happens under non-proliferative but metabolically active growth conditions: sustaining fermentative capacity

25. Most of the fermentation is conducted by “stationary” phase cells Stationary phase means no net increase in viable cells: 1. rate of growth = rate of death 2. quiescent = no growth, no death

26. Making and Modifying Pathways How uniform is the culture? How uniform is the culture? Are there distinct metabolic sub-populations of cells? Are there distinct metabolic sub-populations of cells?

27. Making and Modifying Pathways How uniform is the culture? How uniform is the culture? ANSWER: It depends, but highly likely to not be uniform ANSWER: It depends, but highly likely to not be uniform Are there distinct metabolic sub-populations of cells? Are there distinct metabolic sub-populations of cells? ANSWER: YES Goal of fermentation management: keeping cells optimally active Goal of fermentation management: keeping cells optimally active

28. Keeping Cells Metabolically Active Feeding enough at the appropriate time so they have what they need Feeding enough at the appropriate time so they have what they need Being mindful of factors that alter nutritional needs: Being mindful of factors that alter nutritional needs: –Demand new pathways (have to do something different) –Impose metabolic stress (have to do it in a different way) Understanding the needs of the strains being used (commercial or native) Understanding the needs of the strains being used (commercial or native)

29. REPAIRING AND PREVENTING CELL DAMAGE

30. Yeast Nutritional Phases: Repair and Prevention of Damage Cell # Time lag log stationary death Brix

31. Repairing and Preventing Damage Adaptation to ethanol Adaptation to ethanol Addressing presence of stressors in fermentation Addressing presence of stressors in fermentation Requires “survival factors” Requires “survival factors” Mother Nature selects for surviving rather than completing your fermentation Mother Nature selects for surviving rather than completing your fermentation

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

33. Survival Factors Oxygen Oxygen Fatty Acids Fatty Acids Sterols Sterols Nutritional Factors Nutritional Factors

34. 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

35. ADAPTATION TO CHANGING ENVIRONMENTS

36. Yeast Nutritional Phases: Adaptation Cell # Time lag log stationary death Brix

37. The Need to Adapt Can happen at any time Can happen at any time Temperature shift Temperature shift Introduction or bloom of other organisms Introduction or bloom of other organisms Change in chemical environment (pH, sugar content, ethanol content) Change in chemical environment (pH, sugar content, ethanol content)

38. The Need to Adapt May exceed internal capacity of nutrient recycling May exceed internal capacity of nutrient recycling If need new nutrients their choice is continue to be active or use those nutrients to enter a stable dormant phase If need new nutrients their choice is continue to be active or use those nutrients to enter a stable dormant phase If stress is too severe they might not be able to readjust and adapt If stress is too severe they might not be able to readjust and adapt Again, Mother Nature selects for survival not completion of your fermentation Again, Mother Nature selects for survival not completion of your fermentation

39. Outline of Presentation General introduction to nutritional needs during fermentation General introduction to nutritional needs during fermentation Special considerations Special considerations –Native fermentations –Vineyard factors impacting nutrition –Winery production factors impacting nutrition

40. Native Fermentations Nutritional requirements of cells present not known Nutritional requirements of cells present not known More typically than not, a mixed population of Saccharomyces and non- Saccharomyces More typically than not, a mixed population of Saccharomyces and non- Saccharomyces Saccharomyces population is not going to be uniform Saccharomyces population is not going to be uniform

41. Mixed Populations More competition for nutrients More competition for nutrients Accumulation of inhibitory end products Accumulation of inhibitory end products Non-homogeneity of Saccharomyces Non-homogeneity of Saccharomyces –In some regions this leads to domination by less fermentatively robust strains = strong initiators are not a priori strong finishers

42. Heterogeneity The Good News: The Good News: –Greater complexity –Selection for survivalists –Slower fermentations The Bad News: The Bad News: –Higher risk of something going wrong –Need to pay closer attention than with commercial inoculants

43. Viticultural Factors Impacting Nutrition First source of nutrients First source of nutrients Can be a source of inhibition Can be a source of inhibition Rot changes fermentation dynamics Rot changes fermentation dynamics Sometimes need to get yeast nutrition right in the vineyard, winery additions do not seem to compensate Sometimes need to get yeast nutrition right in the vineyard, winery additions do not seem to compensate

44. Winery Production Factors Impacting Nutrition Manipulations of microbial populations Manipulations of microbial populations  Cold soaks  Heat treatments  Holding of fruit Manipulation of fermentation environment Manipulation of fermentation environment  Temperature  pH adjustments  Aeration and mixing practices  Tank dimensions