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