Linda F. BissonLinda F. Bisson

Department of Viticulture and Department of Viticulture and EnologyEnology

Issues in Fermentation Issues in Fermentation Management, 2011Management, 2011

Yeast Nutrition and Yeast Nutrition and Fermentation Fermentation ProgressionProgression

Outline of PresentationOutline of Presentation

General introduction to nutritional General introduction to nutritional needs during fermentationneeds during fermentation

Special considerationsSpecial considerations– Native fermentationsNative fermentations– Vineyard factors impacting nutritionVineyard factors impacting nutrition– Winery production factors impacting Winery production factors impacting

nutritionnutrition

GENERAL INTRODUCTIONGENERAL INTRODUCTION

Yeast Need Nutrients To:Yeast Need Nutrients To:

Build new cellsBuild new cells Make and modify metabolic Make and modify metabolic

pathwayspathways Repair and prevent cell damageRepair and prevent cell damage Adapt to changing environmentsAdapt to changing environments

Yeast Nutritional Phases

Cell #

Time

lag

log

stationary

death

Brix

BUILDING NEW CELLSBUILDING NEW CELLS

Yeast Nutritional Phases: Building New Cells

Cell #

Time

lag

log

stationary

death

Brix

Yeast Nutrition: Building New CellsYeast Nutrition: Building New Cells

Energy source: Energy source: Ability to capture and Ability to capture and reuse bond energyreuse bond energy

Macronutrients:Macronutrients: Building blocks needed Building blocks needed for new cell materialfor new cell material

Micronutrients:Micronutrients: Catalysts needed to Catalysts needed to facilitate biochemical reactions related to facilitate biochemical reactions related to growthgrowth

Yeast Nutrition: Building New CellsYeast Nutrition: Building New Cells

Energy source: Energy source: laborlabor Macronutrients:Macronutrients: bricks and mortar bricks and mortar Micronutrients:Micronutrients: equipment equipment

MacronutrientsMacronutrients Carbon/Energy Sources:Carbon/Energy Sources: glucose, glucose,

fructose, sucrose fructose, sucrose Nitrogen Sources:Nitrogen Sources: amino acids, amino acids,

ammonia, nucleotide bases, peptidesammonia, nucleotide bases, peptides Phosphate Sources:Phosphate Sources: inorganic inorganic

phosphate, organic phosphate phosphate, organic phosphate compoundscompounds

Sulfur Sources:Sulfur Sources: inorganic sulfate, inorganic sulfate, organic sulfur compoundsorganic sulfur compounds

Growth Macronutrients in Growth Macronutrients in JuiceJuice

Carbon: Carbon: ExcessExcess Nitrogen: Nitrogen: Excess to DeficiencyExcess to Deficiency Phosphate: Phosphate: Sufficient to DeficiencySufficient to Deficiency Sulfate: Sulfate: SufficientSufficient

Carbon SourcesCarbon Sources

For carbon component: role in new For carbon component: role in new cell material (bricks and mortar)cell material (bricks and mortar)

To be degraded to recapture bond To be degraded to recapture bond energy: role as energy source to energy: role as energy source to perform needed metabolic reactions perform needed metabolic reactions (labor)(labor)

Yeast Carbon/Energy Sources Yeast Carbon/Energy Sources in Juice:in Juice:

Monosaccharides:Monosaccharides: glucose, fructose, glucose, fructose, galactose, mannosegalactose, mannose

Disaccharides:Disaccharides: sucrose, maltose, sucrose, maltose, melibiosemelibiose

Trisaccharides:Trisaccharides: raffinose raffinose Pentoses:Pentoses: Not used Not used

Yeast Carbon (NOT Energy) Yeast Carbon (NOT Energy) Sources in Juice:Sources in Juice:

Metabolic Intermediates: Metabolic Intermediates: TCA cycle acids: TCA cycle acids: malate, fumarate, succinate, oxalacetic malate, fumarate, succinate, oxalacetic acid, citrateacid, citrate

Oxidative substrates: Oxidative substrates: pyruvate, acetate, pyruvate, acetate, lactate, glycerol, ethanollactate, glycerol, ethanol

See dynamic changes in concentrations in See dynamic changes in concentrations in juice during fermentation, role in juice during fermentation, role in metabolism and growth not clearmetabolism and growth not clear

Nitrogen SourcesNitrogen Sources

For For SaccharomycesSaccharomyces, function exclusively , function exclusively in building new cells and new pathwaysin building new cells and new pathways

Bacteria and some non-Bacteria and some non-SaccharomycesSaccharomyces yeasts can use nitrogen compounds as yeasts can use nitrogen compounds as energy sourcesenergy sources

Nitrogen most often the limiting factor Nitrogen most often the limiting factor in the in the SaccharomycesSaccharomyces environment environment

Yeast Nitrogen SourcesYeast Nitrogen Sources

AmmoniaAmmonia Most amino acidsMost amino acids Degradation may depend upon availability Degradation may depend upon availability

of other components: vitamins and oxygenof other components: vitamins and oxygen Utilization impacted by other Utilization impacted by other

environmental factors such as pH and environmental factors such as pH and ethanol ethanol

Categories of Yeast Amino Acid Categories of Yeast Amino Acid Nitrogen SourcesNitrogen Sources

Compound may be used as that amino Compound may be used as that amino acid for biosynthesisacid for biosynthesis

Compound may be converted to related Compound may be converted to related amino acids for biosynthesisamino acids for biosynthesis

Compound may be degraded with release Compound may be degraded with release of nitrogen (be mindful of potential end of nitrogen (be mindful of potential end products!)products!)

Phosphate SourcesPhosphate Sources Inorganic phosphateInorganic phosphate Organic phosphate-containing molecules Organic phosphate-containing molecules

under starvation conditions onlyunder starvation conditions only Prefer to make their ownPrefer to make their own

– Internally generated phosphate-containing Internally generated phosphate-containing molecules perform important regulatory rolesmolecules perform important regulatory roles

– If taken in from outside might send false If taken in from outside might send false signalsignal

Roles as building blocks and in energy Roles as building blocks and in energy movementsmovements

Sulfate SourcesSulfate Sources

SulfateSulfate S-containing amino acidsS-containing amino acids Used for building new cell materialUsed for building new cell material Used in formation of catalystsUsed in formation of catalysts

Micronutrients for Building New Micronutrients for Building New CellsCells

Minerals and Trace Elements:Minerals and Trace Elements: Mg, Ca, Mg, Ca, Mn, K, Zn, Fe, CuMn, K, Zn, Fe, Cu

Vitamins:Vitamins: biotin is the only required biotin is the only required vitamin, but others are stimulatoryvitamin, but others are stimulatory

Generally depending upon how the cells Generally depending upon how the cells are grown previously micronutrients are are grown previously micronutrients are not limitingnot limiting

MAKING AND MODIFYING MAKING AND MODIFYING METABOLIC PATHWAYSMETABOLIC PATHWAYS

Yeast Nutritional Phases: Modifying Pathways

Cell #

Time

lag

log

stationary

death

Brix

Making and Modifying Making and Modifying PathwaysPathways

Needs energy for degradation and Needs energy for degradation and reconstructionreconstruction

May need influx of net new nutrients if May need influx of net new nutrients if requirements not readily met by requirements not readily met by degradation (mostly a nitrogen issue)degradation (mostly a nitrogen issue)

Requires micronutrients for Requires micronutrients for generating the new pathway and for generating the new pathway and for operating the enzymes of that operating the enzymes of that pathwaypathway

Making and Modifying Making and Modifying PathwaysPathways

Happens during active growthHappens during active growth Happens under non-proliferative but Happens under non-proliferative but

metabolically active growth metabolically active growth conditions: sustaining fermentative conditions: sustaining fermentative capacitycapacity

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 Making and Modifying PathwaysPathways

How uniform is the culture?How uniform is the culture? Are there distinct metabolic sub-Are there distinct metabolic sub-

populations of cells?populations of cells?

Making and Modifying Making and Modifying PathwaysPathways

How uniform is the culture?How uniform is the culture?

ANSWER: It depends, but highly ANSWER: It depends, but highly likely to not be uniformlikely to not be uniform

Are there distinct metabolic sub-Are there distinct metabolic sub-populations of cells?populations of cells?

ANSWER: YESANSWER: YES Goal of fermentation management: Goal of fermentation management:

keeping cells optimally activekeeping cells optimally active

Keeping Cells Metabolically Keeping Cells Metabolically ActiveActive

Feeding enough at the appropriate time Feeding enough at the appropriate time so they have what they needso they have what they need

Being mindful of factors that alter Being mindful of factors that alter nutritional needs: nutritional needs: – Demand new pathways (have to do Demand new pathways (have to do

something different)something different)– Impose metabolic stress (have to do it in a Impose metabolic stress (have to do it in a

different way)different way) Understanding the needs of the strains Understanding the needs of the strains

being used (commercial or native)being used (commercial or native)

REPAIRING AND PREVENTING REPAIRING AND PREVENTING CELL DAMAGECELL DAMAGE

Yeast Nutritional Phases: Repair and Prevention of Damage

Cell #

Time

lag

log

stationary

death

Brix

Repairing and Preventing Repairing and Preventing DamageDamage

Adaptation to ethanolAdaptation to ethanol Addressing presence of stressors in Addressing presence of stressors in

fermentationfermentation Requires “survival factors”Requires “survival factors” Mother Nature selects for surviving Mother Nature selects for surviving

rather than completing your rather than completing your fermentationfermentation

Role of Survival FactorsRole of Survival Factors

Maintain viability of cellsMaintain viability of cells Increase ethanol toleranceIncrease ethanol tolerance Maintain energy generationMaintain energy generation

Survival FactorsSurvival Factors

OxygenOxygen Fatty AcidsFatty Acids SterolsSterols Nutritional FactorsNutritional Factors

Survival FactorsSurvival Factors

Needed to alter composition of the plasma Needed to alter composition of the plasma membrane (sterols, fatty acids and membrane (sterols, fatty acids and proteins) so that it can withstand the proteins) so that it can withstand the perturbing effects of ethanolperturbing effects of ethanol

Both phospholipid and protein content must Both phospholipid and protein content must be adjustedbe adjusted

ADAPTATION TO CHANGING ADAPTATION TO CHANGING ENVIRONMENTSENVIRONMENTS

Yeast Nutritional Phases: Adaptation

Cell #

Time

lag

log

stationary

death

Brix

The Need to AdaptThe Need to Adapt

Can happen at any timeCan happen at any time Temperature shiftTemperature shift Introduction or bloom of other Introduction or bloom of other

organismsorganisms Change in chemical environment Change in chemical environment

(pH, sugar content, ethanol content)(pH, sugar content, ethanol content)

The Need to AdaptThe Need to Adapt May exceed internal capacity of nutrient May exceed internal capacity of nutrient

recyclingrecycling If need new nutrients their choice is If need new nutrients their choice is

continue to be active or use those continue to be active or use those nutrients to enter a stable dormant phasenutrients to enter a stable dormant phase

If stress is too severe they might not be If stress is too severe they might not be able to readjust and adaptable to readjust and adapt

Again, Mother Nature selects for survival Again, Mother Nature selects for survival not completion of your fermentationnot completion of your fermentation

Outline of PresentationOutline of Presentation

General introduction to nutritional General introduction to nutritional needs during fermentationneeds during fermentation

Special considerationsSpecial considerations– Native fermentationsNative fermentations– Vineyard factors impacting nutritionVineyard factors impacting nutrition– Winery production factors impacting Winery production factors impacting

nutritionnutrition

Native FermentationsNative Fermentations

Nutritional requirements of cells Nutritional requirements of cells present not knownpresent not known

More typically than not, a mixed More typically than not, a mixed population of population of SaccharomycesSaccharomyces and and non- non- SaccharomycesSaccharomyces

Saccharomyces Saccharomyces population is not population is not going to be uniformgoing to be uniform

Mixed PopulationsMixed Populations

More competition for nutrientsMore competition for nutrients Accumulation of inhibitory end Accumulation of inhibitory end

productsproducts Non-homogeneity of Non-homogeneity of SaccharomycesSaccharomyces

– In some regions this leads to domination In some regions this leads to domination by less fermentatively robust strains = by less fermentatively robust strains = strong initiators are not a priori strong strong initiators are not a priori strong finishersfinishers

Heterogeneity Heterogeneity

The Good News:The Good News:– Greater complexityGreater complexity– Selection for survivalistsSelection for survivalists– Slower fermentations Slower fermentations

The Bad News:The Bad News:– Higher risk of something going wrongHigher risk of something going wrong– Need to pay closer attention than with Need to pay closer attention than with

commercial inoculantscommercial inoculants

Viticultural Factors Viticultural Factors Impacting NutritionImpacting Nutrition

First source of nutrientsFirst source of nutrients Can be a source of inhibitionCan be a source of inhibition Rot changes fermentation dynamicsRot changes fermentation dynamics Sometimes need to get yeast Sometimes need to get yeast

nutrition right in the vineyard, winery nutrition right in the vineyard, winery additions do not seem to additions do not seem to compensatecompensate

Winery Production Factors Winery Production Factors Impacting NutritionImpacting Nutrition

Manipulations of microbial populationsManipulations of microbial populations Cold soaksCold soaks Heat treatmentsHeat treatments Holding of fruitHolding of fruit

Manipulation of fermentation Manipulation of fermentation environmentenvironment

TemperatureTemperature pH adjustmentspH adjustments Aeration and mixing practicesAeration and mixing practices Tank dimensionsTank dimensions