a semi-continuous basis; when acetification is nearly complete and acetic
acid levels are typically around 10–14% w/v, a proportion of the
fermenter’s contents is removed and replaced with an equal volume of
fresh alcoholic vinegar stock. Since a substantial amount of finished
vinegar is retained in the fermenter, this conserves the culture and means
that a relatively high level of acidity is maintained throughout the
fermentation, protecting against contamination. It also protects against
over-oxidation as it has been found thatAcetobacter europaeus, a species
commonly found in commercial vinegar fermenters, will not over-oxidize
when the acetic acid concentration is more than 6%.
Many of the acetic acid bacteria associated with commercial acetifica-
tion are difficult to culture on conventional solidified media, although
some success has been enjoyed using a double-layer medium which
provides colonies growing on the surface with a constant supply of
ethanol and moisture from a lower, semi-solid layer. As a result, vinegar
fermentations are usually initiated with seed or mother vinegar, an
undefined culture obtained from previous fermentations. Depending
on the type of acetification, the culture can be quite heterogeneous
andA. europaeus,A. hansenii, A. acidophilum,A. polyoxogenes, and
A. pasteurianushave all been isolated from high-acidity fermentations.
Oxidation of ethanol to acetic acid is the relatively simple pathway by
which acetic acid bacteria derive their energy. It occurs in two steps
mediated by an alcohol dehydrogenase and an aldehyde dehydrogenase
(Figure 9.14). Both enzymes are associated with the cytoplasmic mem-
brane and have pyrroloquinoline quinone (PQQ) as a coenzyme. PQQ
acts as a hydrogen acceptor which then reduces a cytochromeviaan
intermediate quinone. The consequent electron transport establishes a
proton motive force across the membrane which can be used to synthe-
size ATP.
Overall, acetification can be represented chemically by:
C 2 H 5 OHþO 2 ¼CH 3 COOHþH 2 O ð 9 : 2 ÞFrom the stoichiometry of the equation it can be calculated that 1 litre of
ethanol should yield 1.036 kg of acetic acid and 0.313 kg of water. This
leads to the approximate relationship that 1% v/v ethanol will give 1%
w/v acetic acid, and this is used to predict the eventual acidity of a
vinegar and to calculate fermentation efficiency. It implies that, in the
absence of over-oxidation, evaporative losses and conversion to biomass,
the sum of the concentration of ethanol (%v/v) and the concentration of
acetic acid (%w/v), known as the total concentration or GK (German:
Gesammte Konzentration) should remain constant throughout acetifi-
cation. The GK yield is the GK of the final vinegar expressed as a
percentage of the GK at the start of acetification.
Chapter 9 357