Transcript VEN124 Section III

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
Daughter Cell
Mother 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
Mating Pair
a


a
a/
Budding
Zygote
Haploid Cells
Diploid Cell
Yeast Life Cycles: Sporulation
Ascus
Spore
Vegetative Cell
2N
Tetrad
4 x 1N
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 (phosphomannoprotein)
• Mitochondria: site of oxidative reactions
• Vacuoles: site of storage and hydrolysis
• Secretory pathway
• Nucleus
Saccharomyces
Nucleus
Mitochondrion
Secretory
Pathway
Golgi
Vacuole
Endoplasmic reticulum
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
ATP
fructose
glucose-6-phosphate
fructose-6-phosphate
ATP
fructose 1,6-diphosphate
dihydroxyacetone phosphate
glyceraldehyde 3-phosphate
NAD+
NADH
1,3 -diphosphoglycerate
ATP
3-phosphoglycerate
2-phosphoglycerate
phosphoenol pyruvate
ATP
pyruvate
“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
Pyruvate
CO2 + CH3-CHO
Acetaldehyde
NADH
H+
NAD+
CH3-CH2OH
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
100
Glucose
Fructose
Cell Mass
Viable Cells
120
100
10
80
60
1
40
20
0.1
0
0
2
4
6
-20
8
10
0.01
Time (days)
Absorbanc (580 nm) - Colony
forming units (x10E6)
Sugar (g/L) - Fermentation
Rate (g/L/day)
140
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
stationary
death
Cell #
log
Brix
lag
Time
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