Yeast Nutrition and Fermentation Progression

Linda F. Bisson Department of Viticulture and Enology Issues in Fermentation Management, 2011

Outline of Presentation

 General introduction to nutritional needs during fermentation  Special considerations – Native fermentations – Vineyard factors impacting nutrition – Winery production factors impacting nutrition

GENERAL INTRODUCTION

Yeast Need Nutrients To:

 Build new cells  Make and modify metabolic pathways  Repair and prevent cell damage  Adapt to changing environments

Yeast Nutritional Phases

stationary

Cell #

log lag death

Brix Time

BUILDING NEW CELLS

Yeast Nutritional Phases: Building New Cells

stationary

Cell #

log lag death

Brix Time

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

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

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

Growth Macronutrients in Juice

 Carbon: Excess  Nitrogen: Excess to Deficiency  Phosphate: Sufficient to Deficiency  Sulfate: Sufficient

Carbon Sources

 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)

Yeast Carbon/Energy Sources in Juice:

 Monosaccharides: glucose, fructose, galactose, mannose  Disaccharides: melibiose sucrose, maltose,  Trisaccharides: raffinose  Pentoses: Not used

Yeast Carbon (NOT Energy) Sources in Juice:

 Metabolic Intermediates: TCA cycle acids: malate, fumarate, succinate, oxalacetic acid, citrate  Oxidative substrates: pyruvate, acetate, lactate, glycerol, ethanol  See dynamic changes in concentrations in juice during fermentation, role in metabolism and growth not clear

Nitrogen Sources

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

Yeast Nitrogen Sources

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

Categories of Yeast Amino Acid Nitrogen Sources

 Compound may be used as that amino acid 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!)

Phosphate Sources

 Inorganic phosphate  Organic phosphate-containing molecules under starvation conditions only  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

Sulfate Sources

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

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

MAKING AND MODIFYING METABOLIC PATHWAYS

Yeast Nutritional Phases: Modifying Pathways

stationary

Cell #

log lag death

Brix Time

Making and Modifying Pathways

 Needs energy for degradation and reconstruction  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

Making and Modifying Pathways

 Happens during active growth  Happens under non-proliferative but metabolically active growth conditions: sustaining fermentative capacity

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

Making and Modifying Pathways

 How uniform is the culture?

 Are there distinct metabolic sub-populations of cells?

Making and Modifying Pathways

 How uniform is the culture?

 ANSWER: It depends, but highly likely to not be uniform Are there distinct metabolic sub-populations of cells?

 ANSWER: YES Goal of fermentation management: keeping cells optimally active



Keeping Cells Metabolically Active

Feeding enough at the appropriate time so they have what they need   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)

REPAIRING AND PREVENTING CELL DAMAGE

Yeast Nutritional Phases: Repair and Prevention of Damage

stationary

Cell #

log lag death

Brix Time

Repairing and Preventing Damage

 Adaptation to ethanol  Addressing presence of stressors in fermentation  Requires “survival factors”  Mother Nature selects for surviving rather than completing your fermentation

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

Survival Factors  Oxygen  Fatty Acids  Sterols  Nutritional Factors

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

ADAPTATION TO CHANGING ENVIRONMENTS

Yeast Nutritional Phases: Adaptation

stationary

Cell #

log lag death

Brix Time

The Need to Adapt

 Can happen at any time  Temperature shift  Introduction or bloom of other organisms  Change in chemical environment (pH, sugar content, ethanol content)

The Need to Adapt

 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 stress is too severe they might not be able to readjust and adapt  Again, Mother Nature selects for survival not completion of your fermentation

Outline of Presentation

 General introduction to nutritional needs during fermentation  Special considerations – – – Native fermentations Vineyard factors impacting nutrition Winery production factors impacting nutrition

Native Fermentations

 Nutritional requirements of cells present not known   More typically than not, a mixed population of Saccharomyces Saccharomyces and non Saccharomyces population is not going to be uniform

Mixed Populations

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

Heterogeneity

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

Viticultural Factors Impacting Nutrition

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

Winery Production Factors Impacting Nutrition

  Manipulations of microbial populations    Cold soaks Heat treatments Holding of fruit Manipulation of fermentation environment  Temperature  pH adjustments  Aeration and mixing practices  Tank dimensions