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Lecture 10: Problem Fermentations. Reading Assignment: Text, Chapter 4, pages 168- 181. This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and off-character production. Problem Fermentations.

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slide1

Lecture 10:

Problem Fermentations

slide2

Reading Assignment:

Text, Chapter 4, pages 168- 181

slide3

This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and off-character production

problem fermentations
Problem Fermentations
  • Slow (sluggish) fermentation
  • Stuck (incomplete, arrested) fermentation
  • Off-character production
    • Hydrogen sulfide
    • Sulfur volatiles
    • Acetic acid
    • Undesired Esters
stuck and sluggish fermentations6
Stuck and Sluggish Fermentations
  • Characterized by failure of yeast to consume sugar
  • Multiple causes
  • Difficult to treat
  • Leads to reduced wine quality
slide7

The main challenge of slow and arrested fermentations is that they are not recognizable until after fermentation rate has changed. At this point it may be too late to change the adaptive response of the yeast.

slide9

Fermentation Profile

5

1

2

Brix

3

4

Time

1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness

fermentation profile
Fermentation Profile
  • Lag time
    • Duration?
  • Maximum fermentation rate
    • Rate value?
    • Duration?
  • Transition point
    • At what Brix level?
    • How sharp?
  • Post-transition fermentation rate
    • Value relative to max fermentation rate?
    • Length of time?
    • Brix/ethanol/nitrogen level at which it occurs?
  • Overall time to dryness
fermentation capacity is a function of
Fermentation Capacity Is a Function of:
  • Yeast Biomass Concentration
  • Fermentative Ability of Individual Cells
nutrient limitation nitrogen
Nutrient Limitation: Nitrogen
  • Nitrogen: most often limiting
  • Amino acids
    • Can be degraded as N source via transamination
    • Can be interconverted with related amino acids
    • Can be used as that amino acid
  • Ammonia
    • Mobilized by direct amination
transamination
Transamination

Glutamate + X -ketoglutarate + N-X

Glutamine + X’ glutamate + N-X’

Alanine + X’’ pyruvate + N-X’’

Where “X” is an intermediate in amino acid/ nucleotide biosynthesis, and “N-X” is an amino acid or nucleotide base.

amination
Amination

NH4 + -ketoglutarate glutamate

NH4 + glutamate glutamine

preference for nitrogen sources
Preference for Nitrogen Sources
  • How readily can it be converted to NH4, glutamate or glutamine?
  • Expense of utilization (ATP, cofactor, oxygen requirement)
  • Toxicity of C-skeleton
  • What else is available?
amino acid transport
Amino Acid Transport

H+

H+

Amino acid

Transporter

ATPase Pump

ATP ADP

Amino acid

H+

factors affecting nitrogen compound utilization and preference
Factors Affecting Nitrogen Compound Utilization and Preference
  • pH
    • Transport is coupled to H+ ion movements
  • Ethanol
    • Inhibits amino acid transporter function (80% at 5% ethanol for the general amino acid permease)
    • Increases passive proton flux
  • Other N compounds
    • Competition for uptake
    • Nitrogen repression
    • Induction
  • Yeast strain differences
sources of nutrients
Sources of Nutrients
  • Grape
  • Nutrient additions (winemaker)
    • Diammonium phosphate
    • Yeast extracts
    • Yeast “ghosts”
    • Proprietary yeast nutrient mix
  • Yeast autolysis
causes of stuck sluggish fermentations20
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Ionic imbalance
ionic imbalance
Ionic Imbalance

Ratio of K+:H+

Must be at least 25:1

Needs to be adjusted early in fermentation

Probably important in building an ethanol tolerant membrane

causes of stuck sluggish fermentations22
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
substrate inhibition
Substrate Inhibition

Transporters with a high substrate affinity can get “jammed” at high substrate concentrations

G

G

F

F

F

G

causes of stuck sluggish fermentations24
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
  • Ethanol toxicity
ethanol toxicity
Ethanol Toxicity

Plasma membrane is the most ethanol-sensitive cell structure:

Composition: Protein 50%

Lipid 40%

Other 10%

Functions:

Permeability barrier

Regulation of uptake

Mediates response to environment

Maintains electrochemical gradients

Mediates cell-cell interactions

ethanol toxicity26
Ethanol Toxicity

Impact of ethanol

Perturbs membrane structure at protein:lipid interface

Leads to increased “passive proton flux” and acidification of cytoplasm

Inhibits protein activity

Affects membrane “fluidity”

ethanol toxicity28
Ethanol Toxicity

Adaptation of membrane requires:

Increasing content of sterols

Increasing relative content of proteins

Increasing level of desaturation (number of double bonds) in fatty acid side chains

Modification of phospholipid head groups?

fatty acid saturation
Fatty Acid Saturation

Saturated Unsaturated

phospholipid head groups
Phospholipid Head Groups

FA

PO4

FA

PO4

CH2

CH

FA

PO4

CH2

CH2

NH2

FA

PO4

CH2

CH2

N -CH3

HO

OH

HO

OH

H3C-

OOC NH2

+

CH3

OH

Phoshpatidyl-

Inositol Serine Ethanolamine Choline

ethanol toxicity32
Ethanol Toxicity

Sterol and fatty acid desaturation are Oxygen-requiring processes

New protein synthesis requires nitrogen be available

Phospholipid head group synthesis requires cofactors (S-adenosyl- methionine) be available

causes of stuck sluggish fermentations33
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
  • Ethanol toxicity
  • Presence of toxic substances
presence of toxic substances
Presence of Toxic Substances
  • Toxins may arise from the metabolic activity of other microbes
  • Toxins may arise from metabolic activity of Saccharomyces
  • Toxins may have arisen in vineyard, but are not inhibitory until ethanol has accumulated
the most common toxins
The Most Common Toxins
  • Acetic acid
  • Higher organic acids (C2 – C4)
  • Medium chain fatty acids/fatty acid esters
  • Acetaldehyde
  • Fungicide/Pesticide residues
  • Higher alcohols
  • Higher aldehydes
  • Killer factors
  • Sulfur dioxide
causes of stuck sluggish fermentations36
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
  • Ethanol toxicity
  • Presence of toxic substances
  • Poor adaptation of strain
poor adaptation of strain
Poor Adaptation of Strain
  • Strain may not display ethanol tolerance
  • Strain may have high nitrogen/vitamin requirements
  • Strain may be a poor fermentor, but capable of dominating the fermentation
  • Temperature effects
causes of stuck sluggish fermentations38
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
  • Ethanol toxicity
  • Presence of toxic substances
  • Poor adaptation of strain
  • Low pH
slide39
pH
  • pH is reduced by metabolism of Saccharomyces
  • Low pH musts (below pH 3.0) may drop to an inhibitory level (pH 2.7)
  • Dependent upon K+ concentration
causes of stuck sluggish fermentations40
Causes of Stuck/Sluggish Fermentations
  • Nutrient limitation
  • Nutrient imbalance
  • Substrate inhibition
  • Ethanol toxicity
  • Presence of toxic substances
  • Poor adaptation of strain
  • Low pH
  • Temperature shock
temperature shock
Temperature Shock
  • Super-cooling/heating of tank due to equipment failure
  • High temperature fermentations becoming too warm due to yeast metabolism
slide42

The factors leading to arrest of fermentation are interacting. Limitation for nutrients enhances the toxicity of ethanol as does high temperature and the presence of other toxic substances.

the saccharomyces off characters
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
volatile sulfur compounds
Volatile Sulfur Compounds
  • Hydrogen Sulfide: H2S
  • Methanethiol: CH3-SH
  • Ethanethiol: C2H5-SH
  • Dimethyl sulfide: CH3-S-CH3
  • Dimethyl disulfide: CH3-S-S-CH3
  • Diethyl sulfide: C2H5-S-C2H5
  • Diethyl disulfide: C2H5-S-S-C2H5
sources of sulfur compounds
Sources of Sulfur Compounds
  • Sulfate reduction pathway
  • Degradation of sulfur containing amino acids
  • Inorganic sulfur
    • Non-enzymatic
    • Requires reducing conditions established by yeast
  • Degradation of S-containing pesticides/fungicides
hydrogen sulfide formation
Hydrogen Sulfide Formation
  • Due to nitrogen limitation
  • Sulfate reduction regulated by nitrogen availability
  • Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H2S
  • Strain variation
higher sulfides
Higher Sulfides
  • Come from degradation of sulfur containing amino acids
  • From reaction of reduced sulfur intermediates with other cellular metabolites?
  • Formed chemically due to reduced conditions?
current understanding of h 2 s formation
Current Understanding of H2S Formation
  • Nitrogen levels not well-correlated with H2S formation, but generally see increased H2S at lower nitrogen
  • Under complex genetic control
  • Tremendous strain variation in H2S production
factors impacting h 2 s formation
Factors Impacting H2S Formation
  • Level of total nitrogen
  • Level of methionine relative to total nitrogen
  • Fermentation rate
  • Use of SO2
  • Vitamin deficiency
  • Presence of metal ions
  • Inorganic sulfur in vineyard
  • Use of pesticides/fungicides
  • Strain genetic background
timing of formation of h 2 s52
Timing of Formation of H2S

Early (first 2-4 days): due to N imbalance

Late (end of fermentation): due to autolysis, degradation of S-containing compounds

H2S produced early can be driven off by carbon dioxide during active phase of fermentation

slide53

Sulfate Reduction Pathway

SO4

SUL1, SUL2

SO4

MET3

Adenylylsulfate

MET14

Phosphoadenylylsulfate

MET16 (1,8,20,22)

Sulfite

MET10 (1,5?,8,20)

Sulfide

MET17/25/15

Cysteine Cystathionine Homocysteine Methionine

CYS3

CYS4

MET6

regulation of the sulfate reduction pathway
Regulation of the Sulfate Reduction Pathway
  • Methionine (SAM) Repression
  • Cysteine Inhibition of Inducer Production (O-acetyl serine)
  • General Amino Acid Control
  • Sub-PathwayControls
slide55

Homocysteine

Methionine

S-Adenosylhomocysteine

Met-tRNA

S-Adenosylmethionine

slide56

Cysteine

Cys-tRNA

γ-Glutamylcysteine

GlutathioneRDGlutathioneOX

methionine repression antagonized by
Methionine Repression Antagonized by:
  • Threonine
  • Serine
  • Aspartate
  • Glycine
  • Glutamate
  • Histidine
  • Lysine
the amino acids of general amino acid control
The Amino Acids of General Amino Acid Control
  • Lysine
  • Histidine
  • Arginine
  • Leucine
  • Valine
  • Serine
  • Phenylalanine
  • Tryptophan
  • Proline
  • Methionine
the saccharomyces off characters60
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
  • Acetic Acid
acetic acid production by saccharomyces
Acetic Acid Production by Saccharomyces
  • Levels made by Saccharomyces are generally low, below threshold of detection
  • Strain differences in amount formed
  • Derived from:
    • Fatty acid biosynthesis/degradation
    • Amino acid degradation
the saccharomyces off characters62
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
  • Acetic Acid
  • Higher Alcohols
    • Fusel oils
    • Phenethyl alcohol
higher alcohols c2
Higher Alcohols ( C2)
  • Also called “fusel oils”
  • Formed during amino acid degradation

R R R

HCNH C=O HC=O

COOH COOH

RCHOH RCOOH

Deamination

Decarboxylation

Amino Acid Alcohol Acid

Reduction Oxidation

phenethyl alcohol
Phenethyl Alcohol

OH

CH2

H2C

Generic “floral”

May be too intense for some wines

the saccharomyces off characters65
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
  • Acetic Acid
  • Higher Alcohols
  • Acetaldehyde/Higher Aldehydes
aldehyde production
Aldehyde Production
  • Acetaldehyde from glycolysis
    • Released when conversion to ethanol is blocked
    • Released as SO2 adjunct
  • Higher aldehydes from amino acid degradation
    • Released when formation of higher alcohols is blocked
the saccharomyces off characters67
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
  • Acetic Acid
  • Higher Alcohols
  • Acetaldehyde/Higher Aldehydes
  • Unwanted Esters
    • Fatty acid metabolism
    • Amino acid metabolism
      • Phenethyl Acetate
unwanted esters
Unwanted Esters

Esters form from the reaction of an alcohol and an acyl-CoA molecule

O O

R1-OH + R2-CSCoA R1-O-C-R2

source of esters
Source of Esters
  • Most common ester is ethyl acetate made from the reaction of ethanol with acetyl-CoA
  • Esters can derive from amino acid degradation and reaction of acids with ethanol or of alcohols with acetyl-CoA
  • Esters can derive from fatty acid metabolism
phenethyl acetate
Phenethyl Acetate
  • Degradation product of phenyalanine
  • Characteristic “rose oil” odor
  • May be too pungent

O

CH2-CH2O-C-COOH

the saccharomyces off characters71
The Saccharomyces Off-Characters
  • Volatile sulfur compounds
  • Acetic Acid
  • Higher Alcohols
  • Acetaldehyde/Higher Aldehydes
  • Unwanted Esters
  • Vinyl Phenols
vinyl phenols
Vinyl Phenols

OH

H

OH

OH

H

H

CH

CH

COOH

CH

CH2

CH2

CH3

Decarboxylase Vinyl Phenol

Reductase

vinyl phenols73
Vinyl Phenols
  • Responsible for sweaty, horsy, stable,pharmaceutical off aromas
  • Usually formed by Brettanomyces
  • Saccharomyces possesses the enzymes needed to make vinyl phenols and there are reports that it will make them under certain conditions
slide74

Moral:

Yeast needs are simple, but it can be challenging to keep them happy.