VEN124 Section III
The Alcoholic Fermentation
Lecture 8:
Yeast Biology
Reading Assignment:
Text, Chapter 4, pages123-168
In this lecture we will cover the basic biology of yeast and the topics of yeast nutrition and selection for wine production.
The Alcoholic Fermentation
The microbiological conversion of the grape sugars, glucose and fructose, to the end product, ethanol, is called the alcoholic fermentation.
Fermentation means that an organic compound serves as terminal electron acceptor.
The Alcoholic Fermentation
Is conducted by the yeast Saccharomyces cerevisiae or Saccharomyces bayanus
Characteristics of Saccharomyces
• Eukaryote: possesses a membrane bound nucleus
Nucleus Endoplasmic reticulum
Nucleus is surrounded by a double membrane layer with the outer membrane contiguous with the endoplasmic reticulum
Characteristics of Saccharomyces
• Eukaryote: possesses a membrane bound nucleus
• Reproduces by budding
Reproduction by Budding
Mother Cell
Daughter Cell
Characteristics of Saccharomyces
• Eukaryote: possesses a membrane bound nucleus
• Reproduces by budding
• Grows vegetatively as haploid (1N) or diploid (2N)
Yeast Life Cycle
New daughters must grow before initiating their first cell cycle
Characteristics of Saccharomyces
• Eukaryote: possesses a membrane bound nucleus
• Reproduces by budding
• Grows vegetatively as haploid (1N) or diploid (2N)
• Capable of conjugation (1N ⃗ 2N) and sporulation (2N ⃗ 1N)
Yeast Life Cycles: Conjugation
Haploid Cells
Mating Pair
Budding Zygote
Diploid Cell
a
a/
a
Yeast Life Cycles: Sporulation
Vegetative Cell
2N
Tetrad
4 x 1N
Spore
Ascus
Characteristics of Saccharomyces
• Eukaryote: possesses a membrane bound nucleus
• Reproduces by budding• Grows vegetatively as haploid (1N) or
diploid (2N)• Capable of conjugation (1N ⃗ 2N) and
sporulation (2N ⃗ 1N) • Non-motile
Characteristics of Saccharomyces: Sub-Cellular Organization• Plant-like cell wall: comprised of
carbohydrate (glucan, mannan) and glycosylated protein (phosphomanno-protein)
• Mitochondria: site of oxidative reactions• Vacuoles: site of storage and hydrolysis• Secretory pathway• Nucleus
Saccharomyces
Mitochondrion
Vacuole
Nucleus
Endoplasmic reticulum
Secretory
Pathway
Golgi
Glycolysis
The set of biochemical reactions converting hexose (6 carbon) sugars to two 3 carbon pyruvate molecules, during which energy is released and recaptured in the form of ATP.
Glycolysis
Glucose + 2 ATP + 2 NAD+ + 2 ADP + 2 Pi
2 Pyruvate + 4 ATP + 2 NADH + heat
Glycolysis glucose
glucose-6-phosphate
fructose-6-phosphate
fructose 1,6-diphosphate
dihydroxyacetone phosphate glyceraldehyde 3-phosphate
1,3 -diphosphoglycerate
3-phosphoglycerate
2-phosphoglycerate
phosphoenol pyruvate
pyruvate
fructoseATP
ATP
NAD+
NADH ATP
ATP
“Upper Glycolysis”: consumes two molecules of ATP
“Lower Glycolysis”: produces four molecules of ATP
NET PRODUCTION: TWO MOLECULES OF ATP
Where does ethanol come from?
The end products of glycolysis are pyruvate and 2 molecules of the reduced co-factor NADH.
Yeast cells regenerate NAD+ by transferring the hydrogen molecule (electron) to an organic molecule: acetaldehyde
Ethanol Formation
CH3-CO-COOH
CO2 + CH3-CHO
CH3-CH2OH
NADH
NAD+H+
Pyruvate
Acetaldehyde
Ethanol
Other organisms use different strategies to regenerate NAD+
Their presence in wine leads to a diversity of end products of sugar catabolism
Carbon Distribution at End of Fermentation
• 95% = ethanol + carbon dioxide• 1% = new cells• 4% = other end products
– Pyruvate– Acetate– Acetaldehyde– Glycerol– Lactate
Ethanol Yield 1M glucose (fructose)
2 M CO2 + 2 M ethanol
Theoretical Maximum:
180 g 2(46g) = 92/180 = 51.1% w/w = 63.9% v/w = 0.6 original Brix value
Yeast will ferment even in the presence of oxygen. Why?
Fermentation vs. Respiration
Fermentation: 2 ATP/glucose(fructose)
Respiration: 36-38 ATP/ glucose
Efficiency of ATP yield is only an issue if sugar is limiting
In Saccharomyces, glucose concentration regulates the switch between fermentation and respiration.
Regulation of Glycolysis
• Transport: site of global rate control• Allosteric enzymatic steps: localized
rate control– Hexokinase– Phosphofructokinase– Pyruvate kinase
• Effectors of regulation: ATP, ADP, AMP fructose 2,6 bisphosphate, citrate, glucose
Yeast Choice and Nutrition
Yeast Choice: Desirable Traits
• Fermentation to dryness
• Reasonable rate of fermentation
• Predictable fermentation characteristics
• Good ethanol tolerance
• Good temperature tolerance
• Sulfur dioxide tolerance
Yeast Choice: Desirable Traits
• Little to no off-character production– Sulfur volatiles– Acetic acid– Ethyl carbamate
• Little to no inhibition of other desirable microbes
• Killer factor resistant• Production of desired aroma characters
Synthetic Grape Juice Fermentation
-20
0
20
40
60
80
100
120
140
0 2 4 6 8 10
Time (days)
Su
gar
(g
/L)
-
Fer
men
tati
on
R
ate
(g/L
/day
)
0.01
0.1
1
10
100
Ab
sorb
anc (580 n
m) - C
olo
ny
form
ing
un
its (x10E6)
Glucose
Fructose
Cell Mass
Viable Cells
Yeast Nutrition
• Macronutrients: Building blocks needed for new cell material
• Micronutrients: Catalysts needed to facilitate biochemical reactions
Macronutrients• Carbon/Energy Sources: glucose,
fructose, sucrose
• Nitrogen Sources: amino acids, ammonia, nucleotide bases, peptides
• Phosphate Sources: inorganic phosphate, organic phosphate compounds
• Sulfur Sources: inorganic sulfate, organic sulfur compounds
Macronutrient Energy Sources
• Monosaccharides: glucose, fructose, galactose, mannose
• Disaccharides: sucrose, maltose, melibiose
• Trisaccharides: raffinose• Pentoses: None• Oxidative substrates: pyruvate, acetate,
lactate, glycerol, ethanol
Categories of Yeast Nitrogen Sources
• Compound may be used as that compound for biosynthesis
• Compound may be converted to related compounds for biosynthesis
• Compound may be degraded with release of nitrogen
Yeast Nitrogen Sources
• Degradation may depend upon availability of other components: vitamins and oxygen
• Utilization impacted by other environmental factors such as pH
Micronutrients
• Minerals and Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu
• Vitamins: biotin is the only required vitamin, but others are stimulatory
Nutritional Requirements of Different Phases of Fermentation
• Growth Phase: Building blocks and catalysts
• Stationary Phase: Survival factors
Yeast Nutritional Phases
Cell #
Time
lag
log
stationary
death
Brix
Most of the fermentation is conducted by stationary phase cells
Stationary phase:
1. rate of growth = rate of death
2. quiescent, no growth, no death
Role of Survival Factors
• Maintain viability of cells
• Increase ethanol tolerance
• Maintain energy generation
Survival Factors
• Oxygen
• Fatty Acids
• Sterols
• Nutritional Factors
How Does Ethanol Inhibit Yeast?• Displaces water of hydration changing the
properties of protein-lipid interactions• Denatures proteins• Disrupts protein active sites• Allows increased passage of protons from the
medium into the cell leading to acidification of the cytoplasm
• Removal of protons requires expenditure of energy
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