Vinegar Production By

Microorganisms
 What is vinegar?
Vinegar is acetic acid which is produced
by acetic acid bacteria (Gluconobacter
and Acetobacter) oxidizing an alcohol-
containing fruit juice. Distilled vinegar
comes from use of pure ethanol and
refers to its prior distillation Acetobacter
aceti, which is normally used to produce
vinegar, can make vinegar up to 14 per
cent acetic acid. When cider, wine or
malt is used as starting material about 5
per cent acetic acid will be produced
 Traditional production
• vinegar was produced in casks filled with wood
shavings on which wine was sprayed. When
vinegar is distilled it will have no color
• When cider, wine or malt is used as starting
material about 5 per cent acetic acid will be
produced. The color and taste of the vinegar will
depend on the source (cider, wine, beer, barley
malt) and production method
 Vinegar Production
• Sour (spoiled ) wine
• Vin and aigre (sour) production in the US about
160 million gallons per year (2/3 used in
commercial products such as sauces and
dressings, production of pickles and tomato
products) the acetic acid bacteria divided into
two genera: Acetobacter aceti and
Gluconobacter oxydans obligate aerobes that
oxidize sugar, sugar alcohols and ethanol with
the production of acetic acid as the major end
product ethanol oxidation occurs via two
membrane-associated
• Dehydrogenase and acetaldehyde
dehydrogenase
 Process
• Open vat (Orleans) – shallow vats /slime/ lots of
air
• Trickle Generator: beech wood shavings
covered by a
• Trickle of alcoholic substrate, with air entry at the
base. Recycle the product until 4% acetic
acid is obtained
• Bubble method: Large scale ferment or with
massive air. Input (and heat removal). Efficiency
90-98% conversion
 Acetic acid
• Introduction
– Represented organic chemical and an
important feedstock for various chemicals
such as vinyl acetate polymer, cellulose
acetate, terephthalic acid, dimethyl
terephthalate, acetic acid ester, acetic
anhydride, and calcium magnesium acetate
 Acetic acid fermentation
• Acetic acid is produced by fermenting various
substrates (starchy solution, sugar solutions or
alcoholic foodstuffs such as wine or cider) with
Acetobacter bacteria
• Gram-negative, aerobic, rod-shaped bacteria
• Acid tolerant, growing well below pH 5.0
 Industrial Production of Acetic
Acid

• Trickling filter

• Submerged, batch process (Frings

acetator)
 Trickling filter
• Trickling filter, wooden bioreactor (volume up to
60 m3) filled with beech wood
• Shavings, acetic acid bacteria grow as biofilm
• The ethanolic solution is sprayed over the
surface and trickles through the
• Shavings into a collection basin, and
recirculated
• Temperature maintained at 29-35°C
• About 12% acetic acid produced in 3 days
• The life of a well-packed and maintained
generator is about 20 years
 Submerged, batch process
(Frings acetator)
• Stainless steel tank with a high-speed mixer
• Microbes, air, ethanol and nutrients are mixed to
provide a favorable environment
• For microbial growth
• 30°C maintained by circulation of cooling water
• 12% acetic acid in about 35 h
• Production rate per m3 over 10 times higher
than with surface “fermentation” and
• Over 5% higher than with trickling filter
 Material And Methods
• Microorganism
– Acetobacter aceti
– Gluconobacter sp.
– Medium And Culture Condition

– For inoculum preparation, the cells from stock

cultures were transferred
– to 50 mL of MRS broth in a 250 mL
Erlenmeyer flask, and then this was
– incubated at 30°C for 48 h in a shaking
incubator
• 30 g ethanol, 10 g glycerol, 10 g corn
steep liquor (CSL), 2.0 g K2HPO 4, 0.5 g
MgSO4.7H20, and 10 g CaCO 3. The
initial pH of the fermentation medium was
adjusted to 6.5. The main cultivation for
acetic acid production was conducted in a
shaking incubator set to 30°C and 200 rpm
for 48 h
 Analytical Method

• Acetic acid concentration was quantified

by a Waters high-performance liquid
chromatography (HPLC) system (Millipore,
Milford, MA, USA) equipped with a Waters
486 tunable absorbance detector set to
210 nm. ion-exclusion column temperature
was maintained at 40° Analytical Method
 Effect Of Carbon Source

• After 48 h of incubation at 30°C and 200

rpm, Acetobacter sp. RKY4 showed
different characteristics on the utilization of
each carbon source
 Effect Of Ethanol

• The bacterial tolerance to acetic acid can

be increased by increase in ethanol
concentration in the cultivation medium
• The maximum concentration of acetic acid
was obtained at 45.5 g/L from the medium
containing 40 g/L ethanol The maximum
concentration of acetic acid was obtained
at 45.5 g/L from the medium containing 40
g/L ethanol
 Effect Of Nitrogen Sources

• The complex nitrogen sources such as

yeast extract, peptone, CSL, and beef
extract were superior to the inorganic
nitrogen sources such as NH4C1,
(NH4)2SO4, and urea
• Acetobacter sp. increased linearly with
increases in those concentrations up to 10
g/L, but then remained constant or
decreased beyond this value
 Effect of MgSO4°7H20
concentration
• Acetic acid concentration increased with
increases in MgSO4°7H20 concentration
up to 0.5 g/L, in which the molar yield of
acetic acid based on the consumed
ethanol was 87%, but then decreased
beyond this value. Therefore, Acetobacter
spmay require a mineral source such as
magnesium for efficient production of
acetic acid
 Effect of Phosphate Sources
• The monobasic phosphate sources such as
showed better results in terms of acetic acid
formation than dibasic phosphate sources such
as K2HPO 4, Na2HPO
• Acetobacter sp. increased with increases in
(NH4)H2PO 4 concentration up to I g/L, but then
decreased beyond this value Consequently, the
optimum medium for acetic acid formation by
batch culture of Acetobacter sp.
 Uses of acetic acid
• In adhesives, textiles, paints and paper in the
form of vinyl acetate monomer (VAM)
• Its derivative purified terephthalic acid (PTA)
is used in polyethylene terephthalate (PET)
bottles
• Acetic anhydride is a reagent for the production
of aspirin, heroin, and other compounds
• In the form of vinegar
• As a solvent for re-crystallization to purify
organic compounds
• Use in de-scaling agents to remove lime-scale
from taps and kettles
 Future uses
• Low cost
• Utilize the waste material
• Addition of sugar
• Commercial production
conclusion
THANKS