Bacterial Spoilage During Aging:
Acetic Acid Bacteria
Mike Ramsey
Teaching Laboratory Manager
Department of Viticulture and Enology
University of California, Davis
Acetic Acid Bacteria (AABs)
•
•
•
•
•
•
•
Acetobacter
Acidomonas
Gluconobacter
Gluconacetobacter
Kozakia
Asaia
Big reclassification in 1998
Acetic Acid Bacteria (AABs)
• Identification on Genus level is easy; species
level is difficult
• Rod –shaped obligate aerobes
– metabolism is strictly respiratory
– May or may not have flagella
• Very widespread
– Occur mainly in sugary, acidic, or alcoholic
habitats
– Peynaud in 1961 found acetic acid bacteria
on about 50% of all ripe grape clusters
Timeline
Acetobacter aceti
Impact: Acetic Acid
• Acetobacter, Acidomas, Gluconobacter,
Gluconoacetobacter, and Kozakia)
convert ethanol to acetic acid.
– Glucose can be converted to acetic acid
or converted to gluconic acid (main acid
in honey; “umami”)
• Acetobacter, Acidomonas, and
Gloconacetobacter can continue to oxidize
acetic acid to water and CO2 after all the
ethanol has been converted.
Impact: Acetic Acid
• Acetobacter aceti is the most common of
these organisms found in wine and highly
ethanol tolerant, although other species
are common and may dominate your
winery
• The creation of VA is an impact shared by
all of the organisms in this Family, except
Asaia
Impact: Volatile Acidity
• The main component of VA (Volatile
Aciditiy) is acetic acid
– the portion of the organic acids that are
distillable away from the total
Impact: Volatile Acidity
• Important to remember that acetic acid is
also created by lactic acid bacteria
(Lactobacillus, Pediococcus, Oenococcus)
which can create acetic acid during
malolactic fermentation (MLF) and can
covert glucose to acetic acid
– Will not produce ethyl acetate
Impact: Volatile Acidicty
• Yeast genera such as Kloeckera,
Hanseniaspora, and Metchnikowia - which
are found on most grapes - and winery
yeast such as Brettanomyces and
Dekkera produce high amounts of acetic
acid
• Other acetic acid producing wine – related
yeast include Zygosaccharomyces
Impact: Volatile Acidity
• Saccharomyces can produce acetic acid in the
range of 100 to 200 mg/L (ppm) during a normal
fermentation
– Amerine and Ough describe ‘normal’
fermentations as not exceeding 300 mg/L of
acetic acid
• Influenced by yeast strain, fermentation
temperature, and juice nutrition and happens
regardless of exposure to oxygen.
– However, the mechanism in yeast is poorly
understood
Why is acetic acid important?
• Legal and Sensory
• High levels of acetic acid will raise
Titratable Acidity
• Both acetic acid and acetaldehyde are
toxic to Saccharomyces cerevisiae and
may lead to stuck fermentations.
Why is acetic acid important?
• “There are different opinions as to what
level of volatile acidity is appropriate for
higher quality wine.” wine faults –
wikipedia (italics are mine)
– some winemakers seek a low or barely
detectible level of acetic acid to add to the
perceived complexity of a wine.
• Amerine and Ough found it less
objectionable in older wines than in
younger wines
VA Restrictions
Legal Limits:
US
White: 1.2 g/L
Red:
1.4 g/L
CA
OIV
1.1g/L 0.98g/L
1.2g/L 0.98g/L
Threshold of Detection:
 1 g/L
• Can be masked by higher alcohol and sugar
content in wine
• Important to remember that the acetic acid
threshold is lower than the legal limit
Acetic acid measurement
• The most accurate way to measure is by
enzymatic test/spectrophotometer or HPLC/GC
• Wine lab methods
– Sequential Analyzer; Segmented Flow
Analyzer
• The most common method of measurement of
acidic acid is by distillation using a “Cash” still
– SO2, CO2, and other organic acids
(especially lactic) interfere as well as operator
error and cleanliness of equipment
Control of acetic acid production
• As the control of all the compounds
created by AABs are related, we will
review control at end of presentation
VA: Treatment
• Difficult to correct
• Can not neutralize in wine without destroying
the wine
• Refermentation is not an option
– Saccharomyces can not utilize acetic acid
• VA Filtration (Reverse osmosis coupled with ion
exchange)
• Blending
• Bulk/Vinegar
VA Filtration (Reverse osmosis
coupled with ion exchange)
• Permeate = acetic acid, ethanol, water
– Oxidative character may become more
pronounced
– Winemakers report tannin and wine structure
problems
– About 30% removed in one pass
– The filtration companies say it doesn’t change
the wine’s character but it is detrimental to
‘wine quality’ to reduce the VA level below
normal levels
Impact: Ethyl Acetate
• The most common ester in wines
• Chemical image from wikipedia
(http://en.wikipedia.org/wiki/Ethyl_acetate)
Impact: Ethyl Acetate
• More commonly found in red wines
• There is generally a relationship between acetic
acid and ethyl acetate production
• Does not diminish over time, as do some
fermentation esters
• Threshold values reported: 150 – 170 ppm
(Amerine and Ough, 1980)
• Uncited threshold on wikipedia:120 ppm
• Amerine and Ough reported values typical in US
wine: 23 – 135 ppm but it would seem the high
has increased in recent years
Impact: Ethyl Acetate
• Can be formed in excess by indigenous
yeasts, i.e Pichia spp., and Kloeckera and
Hanseniaspora spp.
• Can be formed in excess by Acetobacter
spp.
Impact: Ethyl Acetate
• Can contribute to a general “fruitiness”
character in wine at about 50 ppm
• Excess amounts are considered a “fault”
– Becomes more sharp and acrid as ethanol is
oxidized to acetaldehyde
Impact: Ethyl Acetate
• Aroma of nail polish remover
– Is often used as a solvent
• No easy way to quantify
– There is no set legal limit
• Can impart complexity
• Is not part of Volatile Acidity, so is not measured
by Cash Still/VA Still
• Can be measured by GC – FID (Gas
Chromatography – Flame Ionization Detector)
Control of Ethyl Acetate
• As the control of all the compounds
created by AABs are related, we will
review control at end of presentation
Treatment
• Removal with reverse osmosis and
absorbent media
– Can not be fully removed
• SO2 does not cover the aroma
• Can not referment out
• Blending is difficult as other reds will also
have EA
• Bulk/Vinegar
Impact: Acetaldehyde
• Boiling point = 70 degrees Fahrenheit
• Called “sherry – like” character or green
apple
• Sensory threshold 100 -125 ppm (mg/L)
Impact: Acetaldehyde
• Can be created through the oxidation of ethanol
– during “micro ox” as well as other oxygen
exposure
• Increased production in the presence of acetic
acid bacteria and other wild organisms
– During glycolysis
– Released when conversion to ethanol is
blocked
Impact: Acetaldehyde
• (Fino and Manzanillo sherries are made by
micro organisms creating acetaldehyde)
• Possible carcinogen; definite mutagen
Treatment for Acetaldehyde
• No commercial filtration available
• SO2 binds – but not permanently
• Might be able to reduce by adding fresh
Oenococcus after malolactic fermentation
• These compounds have much lower
sensory threshold than acetic acid—both
acetaldehyde and ethyl acetate are
detectable at less than 200 mg/L in wine.
Impact: Mousiness
• Has been detected in wines all over the
world
• Ability to perceive in tasters seems to have
a genetic predisposition
• The perception is not usually in the aroma,
but is a delayed perception, generally after
swallowing or spitting
– The compounds responsible are not volatile at
wine pH
Impact: Mousiness
• A wine that has been kept away from
oxygen can have mousy taint and not
exhibit it.
– Aeration during filtration and/or bottling
can set it off
• All three compounds can be created by
lactic acid and acetic acid bacteria.
• Two of the compounds can be created by
Brettanomyces
Impact: Mousiness
• Ability to create the compounds varies
within strains of the same organisms
• Generally can not be blended away and
currently there is no technology to remove
it
– Threshold is in parts per trillion
Impact: Mousiness
• Oxygen and iron appear to play a role
• Romano, et al. Journal of Applied
Microbiology Vol 104 issue 6 2007 found if
there were multigram amounts of fructose
or glucose, mousiness was more likely to
be caused by Brett
– Can occur in the bottle
Control of AABs
• Use of undamaged, non – moldy grapes
• Hand harvest in cool temperatures
• Indicators of possible spoilage
– Condition of grapes on arrival
– Presence of Acetobacter species
– Uncontrolled malolactic fermentation
• Reject unsuitable grapes
– Corison (1979) recommended rejection of grapes
– White grapes: 60 mg/L ethyl acetate
– Red grapes: 115 mg/L ethyl acetate
Control of AABs
• SO2 addition and monitoring
– However, some AABs are very resistant to SO2
– Remember your SO2 effectiveness is a function of
your wine pH
• Cold settling, clarification of infected musts
• Quick fermentation with commercial strains of
yeast
• Control O2 exposure
– Cap management, headspace management
– Frequent topping/tight bunging
• Get rid of old cooperage
• Sanitation
– Caustic cleaners
– Sanitizers
Conclusion: AABs
• Can oxidize ethanol to acetic acid
• Threshold of EA and acetaldehyde much
lower than acetic acid
• May cause stuck fermentations
• Sugars to organic acids
• Mousiness
• Turbidity