general information about yeast and its importance in alcoholic fermentation

1. Importance of yeast for
alcoholic fermentation
Mustafa. H. Vohra
Research Associate
Department of Alcohol Technology
Vasantdada Sugar Institute

2. Yeast
 Yeast is generally defined as unicellular fungi, which in a stage
of their life cycle occurs as a single cell reproducing commonly
by budding or less frequently by fission.
 Occurrence: Widely distributed in nature, soil, marine, organic
matter, fruits.
 Isolation: Soil of wine yards, fruits, insects, distillery
environment.
 Generally in distilleries Saccharomyces cerevisiae and
Schizosaccharomyces pombe are used.
 Pyscrophilic – Below 15oC
 Mesophilic- 20-35oC
 Thermophilic- above 45oC
 Taxonomy: 50 Genera, 500 Species and 1500 Strains.

3. Morphology- (shapes)
 Vegetative cells are spherical, global & oval –
Depending on age & cultural conditions- Elongated
special shapes
 Some cells form pseudomycellium under stress conditions.
(Size) depends on age/cultural conditions
 Width- 1 to 9µm
 Length- 2- 10 µm (20-50 µm)
 Spherical cells- 4-6 µm much larger than bacteria (µm)

5. Required characteristic of better yeast for
alcoholic fermentation
 High alcohol tolerance (above 12%).
 High ethanol yield and productivity.
 Tolerance to inhibitors.
 Minimum nutrient supplementation
 High sugar tolerance.
 High temperature tolerance (above 400C).
 Flocculating type.
 Less by products formation during the fermentation.
 Broad substrate utilization range.

6. Saccharomyces cerevisiae
 “Sacchoaromyces” is Greek for “sugar mold” and
“cerevisiae” is Latin for “for beer”.
 Used since ancient times in baking and brewing.
 Significant eukaryotic model organism.
 First isolated from the skin of grapes.
 Flocs and settles well, thus it is easy to filter out.
– Flocculation- process of coming out of solution
and clumping together.
One such yeast…….

7. Saccharomyces
Mitochondrion
Vacuole
Nucleus
Endoplasmic reticulum
Secretory
Pathway
Golgi

8. Yeast cell structure
 Cell wall: Surrounded by a rigid envelope, which comprised of
carbohydrate (glucan, mannan) and glycosylated protein
(phosphomanno-protein).
 Determines the shape of the cell. Provides a barrier against
high osmotic pressure in cytoplasm and generally maintains the
integrity of the cell.
 The cell wall is a complex extra cellular organelle, capable of
dynamic changes in response to changes in external conditions
and to different stages of the life cycles.
 Capsular material: Produced extracellularly.
 Cytoplasmic membrane: present between cell wall &
cytoplasm.
 Function: Selective transport of low molecular weight
compounds.

9.  Cytoplasm: Ground matrix- Cell structures are suspended.
Consists of polyphosphates, glycolytic & hydrolytic, pentose
cycle enzymes, glycogen, trehalose, ribosomes.
 Nucleus: Nucleolus- 90% DNA, RNA, Polyphosphate
compounds.
 Number of chromosomes vary.
 Nuclear membrane: pair of unit membrane with pores. (85
nm).
 Cell division: Nucleus divides.
 Function: Genetic and hereditary control.
 Endoplasmic reticulum: Double membrane system.
Ribosomes are present which are responsible for protein
synthesis.

10.  Vacuoles: Spherical, more transparent, vary in size,
site of storage and hydrolysis
 In active cells more vacuoles are present & in old
cells single large vacuoles are present surrounded by
unit membrane.
 Contain ribonucleases, proteases, esterases, free
amino acids, polyphosphates.
 Function: Autolysis.
 Mitochondria: Powerhouse of cell, site of oxidative
reactions.
 Contains DNA, RNA, RNA-Polymerase, Enzymes of
TCA & ETC.
 Lipid globules: Contain lipids, no membrane.

11. Yeast Life Cycle : Asexual
New daughters must grow before initiating
their first cell cycle

12. Yeast Life Cycle: Sexual
Haploid Cells
Mating Pair
Budding Zygote
Diploid Cell
a
a/
a

13. Yeast Nutrition
Macronutrients: Building blocks needed for
new cell material.
Micronutrients: Catalysts needed to facilitate
biochemical reactions.

14. Macronutrients
Carbon/Energy Sources: Glucose, fructose,
sucrose, mannose (No L- sugars).
Nitrogen Sources: Amino acids, ammonia,
nucleotide bases, peptides.
Phosphate Sources: Inorganic phosphate,
organic phosphate compounds.
Sulfur Sources: Inorganic sulfate, organic
sulfur compounds.

15. Macronutrient Energy Sources
 Monosaccharides: Glucose, fructose, galactose,
mannose.
Disaccharides: Sucrose, maltose, melibiose.
Trisaccharides: Raffinose.
Pentoses: None.
Oxidative substrates: Pyruvate, acetate,
lactate, glycerol, ethanol.

16. 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.

17. Yeast Nitrogen Sources
Degradation may depend upon availability of
other components: vitamins and oxygen.
Utilization impacted by other environmental
factors such as pH.
Inorganic nitrogen like most ammonium salt
are used.
Organic nitrogen like Amino acids & urea are
used.

18. Micronutrients
Minerals and Trace Elements: Mg, Ca, Mn, K, Zn,
Fe, Cu.
The requirement of minerals are is very low but they
are essential for the growth.
Vitamins: act as catalytic functions.
Some yeast can synthesize their own vitamins while
some have multiple requirements like biotin,
pantothenic acid, folic acid, niacin, p-amino benzoic
acid, inositol, thiamin, riboflavin, pyridoxine, etc.
All these vitamins are present in molasses.

19. Yeast Nutritional Phases
Cell #
Time
lag
log
stationary
death
Brix

20. 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

21. 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.

22. Cultural conditions for yeast
As the yeast is facultative organism, for growth
aerobic conditions are required.
MGYP medium:
Malt extract- 0.3%
Glucose- 1.0%
Yeast extract- 0.3%
Peptone- 0.5%
D/W (make up volume) 100 ml
Physical parameters:
Temperature- 32.50C
pH- 4.50
RPM- 180

23. Yeast Growth/metabolism
 Yeast can either produce energy by fermentation or
oxidative phosphorylation (ox/phos).
 Yeast greatly prefer fermentation over ox/phos and
will not start ox/phos until all the sugars are
converted to ethanol.
 Ethanol is a good source of energy and in the
presence of oxygen yeast use it up as well.
 Fermentation also produces 2 CO2 molecules per
molecule of sugar-- as a by product. =)

24. Metabolism
Special flavors and aromas of beers arise from minor biochemical reactions
Major Reaction: Glucose to Carbon Dioxide and Ethanol

25. Fundamentals of yeast growth
(facultative veg. Reproduction – td = 2h)
Aerobic Anaerobic
Glycolytic (EMP) pathway
(cytoplasm)
EMP-TCA-oxidative
phosphorylation
EtoH is final e- acceptor O2 is final e- acceptor
Partial degradation Complete degradation
2 ATP/ glucose 36 ATP/ glucose
Weak cell membrane Cell membrane with ufa &
sterols (nicotinic acid)
fermentation EtoH tolerance, respiration

26. Time (h)
Growth kinetics (Batch growth)Logno.ofcells/ml
No.ofcells/ml
µmax= 0.26 hr-1
td = ?
5
6
7
8
108
107
106
105
5 10 15 25 30 35 40 450

27. A- Lag phase dx/dt = 0
 Synthesis & degradation of cellular material
 Adaptation to new environment
 Process design-Avoid lag phase
 Composition of growth medium kept same
B- Accelerating phase dx/dt = µx ----------- (1)
(µ relevant to medium & strain)
µ = 0 to µmax
C- Exponential phase dx/dt = µmax ------------(2)
 Reproduction & multiplication at max. rate
 Necessary nutrient in excess amount
 No hidden limitation by incomplete mixing
 Absence of inhibitors( SO2, Sulphites, org.acids, cupric ion,
ethanol, DCO2

28. On integration, ln x-ln xo= µmax . t ------------------------(3)
Plot of ln x Vs t , slope = µmax (experiment)
When eq (3) is expressed in form
ln (x/xo) = µmax . t ------------------------ (4)
The fundamental eq. For batch growth
x = xo e(µmax . t ) ------------------------(5)
When x=2xo, t = td = doubling time
td = ln 2/ µmax = 0.693/ µmax --------------- (6)
D-Decelarating phase: again dx/dt = µt
 Concentration of one of the nutrient is low.
 Accumulation of inhibitory compounds.

29. E-Stationary phase
 Substrate is exhausted
 No growth
 Beginning of starvation, degradation of cellular material
 Later- cell lysis – nutrients
 For other cell- few cells still divide
 So keep your culture in exponential phase & reduce down time.
Oxygen solubility
 In liquid culture O2 is obtained by yeast from dissolved state
 Aeration – blowers/ compressors
 Solubility of O2 in DW at 20oC = 9.2 mg/L. Decreases with
increasing temp & increasing solute conc.
 In its life cycle- yeast cell divides on an average 24 times (bud scars,
chitin)
 Below 1PPM DO – yeast cannot multiply for move than 4-5
generations.
Therefore, keep low sugar & high aeration rate

30. Importance of Yeast in Brewing
Fermentation Flavors
 What are some of the brewing fermentation
flavor compounds?
 What influences their production?
 Sensory evaluation of some of these flavor
compounds.

31. Control of Fermentation Flavors
Important Factors
 Yeast
– Strain
– Pitching rate and consistency
– Viability (autolytic “yeasty” flavours)
– Contaminants
 Wort
– Gravity, pH, dissolved oxygen, contaminants, yeast foods
 Fermentation
– Design/geometry, temperature, pressure

32. Beer Fermentation Flavors
WORT
(sugars, amino acids, etc.)
Acids (organic & fatty)
HigherAlcohols
(fusel oils)
Glycerol
Esters
Aldehydes
& Ketones
Sulfur compounds
Phenols
Yeast autolytic
compounds
bacteria, wild yeast

33. Ester production by yeast
 Over 90 distinct esters in beer - flowery
and fruity flavors and aromas.
 Desirable at low concentrations, but
undesirable at high concentrations.
 Important esters: Isoamyl acetate, ethyl
acetate, isobutyl acetate, ethyl caproate,
2-phenyl acetate.

34. Glycerol Production by Yeast
Glycerol is quantitatively one of the most
important products of yeast fermentation
and contributes to the viscosity and “body”
of beer (and wine).

35. Higher alcohols
 Increased by
– high fermentation temp
– mixing during
fermentation
– low concentration of
amino-acids in wort
– intensive aeration
– high gravity
 Reduced by
– high pitching rate
– cold pitching temperature
and fermentation
– pressure during
fermentation
– avoid oxygen after pitching
– enough amino-acids
 80 % of higher alcohols are built during fermentation.
They can´t be influenced during maturation.

36. Higher Alcohols
n-propanol
i-butanol
2-methyl-butanol-1
3-methyl-butanol-1
aliphatic-alcohols
2-phenyl-ethanol
tyrosol
tryptophol
aromatic alcohols
2,3-butandiol
glycerol
multivalent alcohols
higher alcohols

37. Yeast Autolysis
Flavors and Aromas
 Gives meat-like flavors and aromas, harsh
bitterness
Yeast viability significantly decreases.
Yeast releases proteases.
pH increases.
 Increased by:
– Length of time beer sits on yeast.
– Temperature of yeast.
 Reduced by:
– Timely remove settled yeast.

38. Sulfur Aromas
 Increased by:
– Low wort oxygenation.
– Restricted yeast growth.
– Slow fermentation.
– Poor venting of fermenter gas.
– Time on yeast.
– Short storage time.

39. Products of Fermentation
• Ethanol and CO2 are the main products of
fermentation.
• Glycerol is sometimes produced and it can
smoothen the taste and impart viscosity.
• Higher-alcohol esters and aldehydes can be
produced as flavor compounds.

40. Unpleasant By-products
• Succinic and acetic acid production causes
wine defects.
• Malic acid occurs naturally in some grapes and
can give wine a very sour taste (it is used to
produce SweetTarts candy).
• Malolactic fermentation using Oenococcus
oeni can decrease malic acid content thus
lowering acidity of the wine.