Film Organisms: Acetic Acid
Bacteria and Yeasts
Michael S. Ramsey
Teaching Laboratory Manager
UCD
mramsey@ucdavis.edu
Outline
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Discussion: VA (volatile acidity) vs. acetic acid
Yeast films
Bacterial films
Symbiosis/Synchronicities?
Microbiological creation of spoilage
compounds
– And some chemical reaction spoilage
• Nutrient additions
• What’s this VA thing all about anyway?
• Vinegar was known early in civilization as the
natural result of exposure of beer and wine to air
– acetic acid-producing bacteria are present globally.
• The use of acetic acid in alchemy extends into the
3rd century BC, when the Greek philosopher
Theophrastus described how vinegar acted on
metals to produce pigments useful in art,
including white lead (lead carbonate) and
verdigris, a green mixture of copper salts
including copper(II) acetate.
• Ancient Romans boiled soured wine, reducing it to a highly
sweet syrup called sapa. Sapa that was produced in lead
pots was rich in lead acetate, a sweet substance also called
sugar of lead or sugar of Saturn, which is believed to have
contributed to lead poisoning among the Roman
aristocracy.
– Lead acetate was a common sweetener even into the
Renaissance with notables such as Pope Clement II and Ludwig
von Beethoven suspected as having died from consumption.
(Carbonate of lead – white, or Venetian, lead - would be used
into the 20th Century)
– Even after the substance’s use in food products was outlawed
it’s use was difficult to trace until fairly modern times.
• In the Renaissance, glacial acetic acid was
prepared through the dry distillation of certain
metal acetates (the most noticeable one being
copper(II) acetate).
• Today, most of the acetic acid used industrially
is produced chemically
VA vs. acetic acid: chemical analysis
• “Volatile acidity” is often wrongly assumed to be the
total acetic acid content of a wine
• Although generally interpreted as acetic acid content, a
“traditional” VA analysis includes any acid, that can be
steam-distilled (or more precisely, steam-stripped),
that is present in the wine
– CO2 (as carbonic acid), SO2 (as sulfurous acid), sorbate,
and lactic, formic, butyric, and propionic acids
• If acetic acid is specifically measured, as by enzymatic –
spectrophotometric methods, results are strictly acetic
acid
I’m Not A Cash Still!
• Most common
apparatus – RD80
Volatile Acid Still
• Not really a “still”
VA vs. acetic acid: sensory analysis
• Acetic acid not as volatile as ethyl acetate
• Acetaldehyde is also often present
• Acetate is often called “acetic nose”
– Wine concentrations range from 10 mg/L to 1200
mg/L
– No legal limit
VA vs. acetic acid
• Some winemakers (including the late Emil
Peynaud) believe the ethyl acetate component
should be the legal indicator of wine spoilage
– Previously, more difficult to measure analytically
– Not as difficult with kits and spectrophotometry
• VA produced by lactic acid bacteria is often
missing the ethyl acetate component (HenickKling, 1993)
Acetic acid
• Can be produced by Brett/Dekkera (but we are
going with surface - film formers)
• Normal byproduct of Saccharomyces growth
– Strains of S. cerevisiae have been shown to produce
acetic acid based on increased activity of the enzyme
acetyl-CoA synthetase
– Fugelsang (1993) reported elevated levels when in coculture with spoilage yeasts
• Can increase as a result of extended aging (1yr) in
new barrels
– Hydrolysis of acetyl groups in wood hemicellulose
Acetic acid
• Acetic acid can result from the oxidation of
wine phenolics which produces hydrogen
peroxide
• Which, in turn, oxides ethanol to acetaldehyde
and then to acetic acid
• Legal limits are still based on “distillable”
Volatile Acidity
• 0.98 g/L OIV
• 1.4 g/L in red wine of this type of harvest (our
experiment)
• Aroma threshold of acetic acid at around 1 g/L
Acetic acid from anaerobic bacteria
• Heterofermentative Lactic Acid Bacteria
ferment glucose with lactic acid,
ethanol/acetic acid and carbon dioxide
(CO2)as by-products
• Important to always remember the other
contributors, other than surface organisms, to
acetic acid
Film Yeasts
• Under oxidative conditions, ethanol, glycerol,
organic acids (esp. malic) can serve as growth
media
• Can synthesize negative aroma compounds
– Ethyl acetate
– Acetoin (buttery cheese)
Film Yeasts
• Candida vini (formerly Candida mycoderma and
often incorrectly identified by Kombucha makers
as Saccharomyces mycoderma)
• Pichia species
• Saccharomyces cereviseae
• Growth may rapidly become pellicle
• Yeasts can initially appear as floating flowers
– “Flowers of wine”
– dusty
Saccharomyces cereviseae
Candida vini
Pichia kluyveri
Acetic Acid Bacteria
• Present on the grapes and in the winery environment
• Several genera and many species, most decline as
ethanol is produced
• Acetobacter species survive through to aging, storage,
and bottling
– Can survive periods of anaerobic conditions
– Begin again with O2 added during fining, racking, stirring,
filtering, etc.
– A. pasteurianus requires less O2 than A. aceti
– Can survive in the bottle and regrow in days in an opened
bottle
Acetobacter aceti
Acetobacter pasteurianus
A Fall Quarter Experiment
• Given oxidative
conditions and
headspace, would a late
addition (post alcohol
fermentation) of a
commercial ML nutrient
increase surface
organism growth?
A Fall Quarter Experiment
• Barrel – aged 2011 California Malbec
• Controls
• One group received commercial ML nutrient
at recommended dose
• Organisms added:
– Flor – forming S. cereviseae
– Pichia kluyveri
– Candida parapsolosis
– Acetobacter aceti
A note on Candida parapsilosis
• Most common yeast
isolated from human
hands and the most
common cause of nail
infections
Nutrients added
• A malolactic fermentation nutrient at
recommended addition
– These are generally blends of “inactive” yeasts to
add amino acids, mineral cofactors, vitamins, cell
wall polysaccharides, and cellulose
A Fall Quarter Experiment
• Visible surface film began
to form within one week
in both control and “plus
nutrients”
• Added organisms could
be seen under the
microscope
• Added organisms were
quickly overwhelmed by
our indigenous
Acetobacter pasteurianus
A Fall Experiment
• By Week 5, our
indigenous Acetobacter
pasteurianus covered
the surface of all
containers
• No cells of any addition
could be seen under the
microscope
Wine data
TA
Etoh VA
RS
pH (g/L) %
(g/L) (g/L)
Base Wine
3.65 6.0 13.0 0.55 0.2
Control
3.43 23.0 11.2 3.92 0.2
Control PLUS 3.54 18.0 11.7 3.43 0.2
Sacch
Sacch PLUS
Acetobacter
aceti
Acetobacter
PLUS
Candida
3.44 21.4 11.3 3.84
3.48 22.1 11.2 3.19
0.2
0.2
3.55 15.4 11.9 3.13
0.1
3.51 20.3 11.3 3.69
3.45 21.8 11.2 3.77
0.1
0.2
Candida PLUS 3.42 27.0 10.6 3.77
Pichia
3.49 18.8 11.6 3.44
Pichia PLUS
3.45 24.2 11.0 4.02
0.3
0.1
0.1
• Although there appear
to be trends…. If we
exclude the Base wine,
and we should, t – tests
indicate there is no
difference in the data
sets
• Drysdale and Fleet (1989) noted that the
presence of Acetobacter resulted in stuck
fermentations
• Doores(1993) found acetic acid to be inhibitory of
Saccharomyces
• Acetobacter has been shown to be inhibitory of
surface forming yeasts (Gilliland and Lacey, 1964)
– Authors proposed some kind of antifungal was
produced
• Is there symbiosis?
• Remember, ethyl acetate and acetaldehyde
were not measured and are not measured by
Cash still or enzyme acetic
• Could this spoilage have been avoided by
gassing the surface?
Three Laws and the Real World
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Henry’s Law
Law of Partial Pressures
Ideal Gas Law
Leaking connections and fittings
Some efficacy in small containers (kegs)
• Could this spoilage have been prevented
strictly through sanitation?
• No. We can not hope to eliminate 100% of all
organisms through normal sanitation
• All it takes is the headspace to favor them
SO2?
• Largely ineffective if headspace
• Pichia and Candida species shown to be
resistant to as much as 3 mg/L molecular SO2
– Pichia membranaefaciens is resistant to benzoate
up to 1.5 grams/Liter
• Generally ineffective once the film is formed
(Thomas and Davenport, 1985)
• Organisms create acetaldehyde, which binds
SO2
The Best Preventative Measure
• Maintain topped tanks and barrels
– Depriving organisms of oxygen and space to grow
• Avoid moldy and damaged grapes
• Good sanitation helps
• Use of slow cellar temperatures can slow
growth
– Less than 60F (15C) can slow growth
– Temperatures of 47F or less may inhibit growth
Conclusions
• Symbiosis?
– Possible chemical differences early. Aerobic
bacteria will win the day
• Did nutrient additions do anything?
• Number one component of creation of acetic
acid was oxygen/headspace
What to do when you forgot about the
headspace
• All methods carry some risk
• Blending
• Refermentation
– Oxidatively growing yeasts can utilize acetic acid
as a carbon source
• Reverse osmosis
– Flavor and aroma modification and stripping
• Ion exchange
– Flavor and aroma modification and stripping
• Smell the glass at the side table
• DO NOT TASTE!
Acknowledgements
• FPM Group 5 – Friday afternoon – VEN 124
Wine Production class