Eur Food Res Technol (2004) 219:393–397
DOI 10.1007/s00217-004-0947-9
ORIGINAL PAPER
Hip
lito Rubio-Fernndez ·
Mara Desamparados Salvador · Giuseppe Fregapane
Influence of fermentation oxygen partial pressure
on semicontinuous acetification for wine vinegar production
Received: 19 November 2003 / Revised: 29 April 2004 / Published online: 11 August 2004

Springer-Verlag 2004
Abstract This paper describes semicontinuous acetic acid
fermentations for wine vinegar production carried out with
different aerating gas compositions ranging from 21%
(air) to 63% oxygen content and using low aeration
(3.7 h1, vvm), in order to study the influence of the
oxygen partial pressure on the aerating gas supplied to the
reactor in this industrial biotransformation process. The
acetification process was conducted in 6- to 100-l reac-
tors. The overall acetic acid productivity increased from
0.72 g l1 h1 with air to 1.35 g l1 h1 when oxygen-rich
(36%) air was used. The same behaviour was observed for
the maximum acetification rate, and therefore the total
process time was reduced in proportion to the increase in
productivity, from 65 h using air to 35 h using an aerating
gas mixture containing 36% oxygen. The yield of the
process was high, 96–99%; the final concentration of
acetic acid reached was 116–118 g l1; and the substrate
yield coefficient based on ethanol metabolised was higher
using oxygen-rich air than with air. It was not feasible to
carry out semicontinuous acetification cycles with an
oxygen content higher than 40%, and when the oxygen
content was 63%, the process stopped during the first
cycle with very little acetic acid production. Moreover, an
inverse relationship between the acetic acid formation rate
profile in the course of the acetification process and the
amount of dissolved acetaldehyde in the fermentation
broth formed by the acetic bacteria was observed.
Keywords Wine vinegar · Acetic acid fermentation ·
Aeration · Oxygen · Acetaldehyde
H. Rubio-Fernndez · M. D. Salvador · G. Fregapane ())
Department of Food Science and Technology,
Faculty of Chemistry,
University of Castilla–La Mancha,
Avda Camilo Jos Cela 10, 13071 Ciudad Real, Spain
e-mail: giuseppe.fregapane@uclm.es
Tel.: +34-926-295300
Fax: +34-926-295318
Introduction
Wine vinegar possesses good sensory qualities and is a
highly appreciated component of the diet in Mediter-
ranean countries [1, 2]. This product is particularly im-
portant in the Castilla–La Mancha region of Spain, which
produces more than 60% of all Spanish wine vinegar. In
2001 the figure was about 28 million litres (10% acetic
acid).
Nowadays, industrial wine vinegar is mainly produced
by submerged culture in semicontinuous processes [3].
The concentration of dissolved oxygen in the culture broth
during fermentation has a significant effect on bacterial
growth and on the production rate of acetic acid [4, 5],
since oxygen is a substrate in this process. The most im-
portant factors affecting dissolved oxygen are the oxygen
transfer rate, the air flow rate and the oxygen partial
pressure in the air supply to the bioreactor.
A high aeration flow is undesirable for improvement of
the acetic acid production rate in that it reduces the pro-
cess yield and the organoleptic characteristics of wine
vinegar, because it tends to strip off volatile components,
including ethanol, which is the substrate of the acetifi-
cation reaction [6, 7, 8]. For this reason, some authors [4,
9, 10, 11] have suggested the use of oxygen-rich air at a
lower flow rate; however, none of these assays were
carried out under industrial acetification conditions (11–
12% acetic acid). Moreover, the behaviour of various
fermentation parameters, for example, acetification rate
and cell viability, was not specifically analysed when
oxygen-rich air was used, so the results are contradictory
[12].
To reduce the loss of volatile components, a pilot
fermenter has been developed, equipped with a closed gas
recirculation system [8]. Yields close to 100% have been
attained; however, this type of acetator is expensive and
requires the use of pure oxygen.
The aim of the present work was to investigate the
effect of oxygen partial pressure on acetic acid fermen-
tation for wine vinegar production. To this end, semi-
continuous acetifications were conducted using air and
394
oxygen-rich air (up to 63% oxygen content) in the aer-
ating gas supplied to the fermentation medium in reactors
of up to 100-l capacity.
Materials and methods
Strain
An industrial culture was used, obtained from a local vinegar fac-
tory (Parras, Toledo). It was maintained in a liquid medium (30:70
wine/vinegar mixture) and stored in 20% (v/v) glycerol at 50 C
[6, 13].
Growth media
The growth medium was a commercial white table wine of the
Airen variety, obtained directly from a local producer. The com-
position was as follows: 13 (%, v/v) alcohol, corresponding to
103 g l1 ethanol; 4.7 g l1 total acidity as tartaric acid; 0.8 g l1
volatile acidity as acetic acid; pH 3.4; 0.5 g l1 residual sugars,
glucose and fructose; and 70 ppm SO2, which was removed by
bubbling with air.
Semicontinuous fermentation
A 6-l and a 100-l noncommercial bubble column reactor were
employed [6, 13]. To start the first fermentation, two 250-ml aer-
ated shake flasks with 100 ml of a wine/vinegar/water mixture
(40 g l1 ethanol and 10 g l1 acetic acid) were inoculated with 4 ml
of the stored industrial culture and incubated at 30 C and 200 rpm
until the concentration of acetic acid reached 25 g l1 (4–6 days).
These flasks were then used as an inoculum for the 6-l fermenter
with a wine/vinegar mixture (40 g l1 ethanol and 45 g l1 acetic
acid) as a growth medium at 30 C. When an acetification cycle had
been completed, about 40% of the fermented broth was discharged
as end-product vinegar, at an acetic acid concentration close to
115–120 g l1. The same volume of fresh wine was then fed into the
fermenter and a new production cycle started. Initial fermentation
conditions of the wine/vinegar mixture (40:60) were 47€1 g l1
ethanol and 70€1 g l1 acetic acid. Two litres of the fermenting
medium from the laboratory fermenter at the exponential growing
stage were used as the inoculum in the pilot acetator.
In acetification, the oxygen content of the aerating gas supplied
to the fermenter was varied from 21% (air) up to 63%. The specific
composition of the oxygen-rich air was controlled using two flow
meters: one to control the amount of pure oxygen introduced and
the second to measure the total flow supplied to the reactor. In all
fermentations, the aeration was 3.7 h1, expressed as vvm (air flow
rate/reactor working volume ratio). The temperature of the fer-
menting broth was maintained (€0.5 C) by circulating water
through an internal stainless-steel coil. Two oxygen probes (Syland
Scientific) inserted in the vessel and in the recirculation line were
used to monitor the dissolved oxygen concentration as an easy way
to follow the course of fermentation over time.
Experiments were carried out in triplicate. Reproducibility was
good and mean values and standard deviation are shown in the
tables.
Assays
Samples of a few millilitres were periodically taken from the fer-
menters in the course of acetification, and acetic acid, acetalde-
hyde, ethanol, pH and optical density were determined. Acetic acid
was measured titrimetrically with phenolphthalein as an indicator.
It was assumed that all broth acidity was due to acetic acid. Ac-
etaldehyde was determined by colorimetry using a solution of 2%
sodium nitroprussiate and measuring the absorbance at 570 nm
[14]. The blank solution used to plot the calibration curve was a
wine/vinegar mixture containing 47 g l1 ethanol and 70 g l1 acetic
acid. Ethanol was enzymatically assayed with kit no. 176290
(Roche Molecular Biochemicals). The biomass was monitored by
measuring the absorbance within a linear range at 575 nm. A
previously prepared calibration curve was used to convert the ab-
sorbance readings into biomass dry weight.
The yield of the process was calculated by the method used in
the vinegar industry: i.e., as the quotient of the final and initial
“total concentration”, where total concentration is defined by the
sum of the alcohol (% v/v, ) and acetic acid (grams per 100 ml)
concentrations (1 alcohol provides 1.011 g per 100 ml1 acetic
acid in this type of fermentation).
Statistical analysis
Statistical analysis was performed using SPSS statistical software
(SPSS, Chicago, USA).
Results and discussion
Six-litre fermenter
To study the influence of the oxygen partial pressure in
the air supplied to the reactor during semicontinuous
acetification for wine vinegar production (from 70 to
115–120 g l1 acetic acid, as performed industrially),
fermentations were carried out with aerating gases con-
taining different oxygen concentrations, which ranged
from 21% (air) to 63%. The rate of aeration was low:
3.7 h1 (vvm).
The results from the acetification cycles using air and
oxygen-rich air up to an oxygen content of 36% are
shown in Table 1. The overall acetic acid productivity
improved with the oxygen content, from 0.59 g l1 h1 at
21% to 1.29 g l1h 1 at 36%. The maximum acetification
rate behaved in the same way, so the total process time
was reduced in proportion to the increase in productivity,
from 78 h with air to 36.5 h with an aerating gas mixture
containing 36% oxygen. The observed lag phase de-
creased from 2.3 h with air to 0.5 h with 26% oxygen,
remaining constant at higher oxygen contents. Under all
acetification conditions, the process yield was about 96%;
the final concentration of acetic acid reached was about
116–118 g l1, and the residual ethanol at the end of the
fermentation was 7.0–8.0 g l1, corresponding to 0.85–
1.0 ethanol (% v/v). The substrate yield coefficient based
on ethanol metabolised was higher with oxygen-rich air
than with air.
At oxygen concentrations in the aerating gas higher
than 40%, acetification was not satisfactory, as reported in
Fig. 1. Overall acetic acid productivity was lower at 42%
oxygen content than at 26% oxygen; moreover, there was
practically no acetic acid bacteria growth and no product
formation during the second cycle. When the aerating gas
contained 63% oxygen, the process ended during the first
acetification cycle, with a final concentration of acetic
acid of only 78–80 g l1.
The results may appear contradictory in that oxygen is
a substrate of the acetification reaction and an increase in
 395
Table 1 Semicontinuous acetifications performed in the 6-l labo- with different letters within the same row are statistically different
ratory fermenter with different oxygen-rich air compositions in the (p0.05). Initial conditions: 47.0€2.0 g l1 EtOH, 70.0€2.0 g l1
aerating gas (n3; aeration rate 3.7 h1, vvm). n is the number of acetic acid (AcH). Acetic acid productivity (AcH p); maximum
stable semicontinuous acetification cycles performed. Mean values acetification rate (AcH pmax); metabolised ethanol (EtOHmet)
Oxygen content (%)
21 26 31 36
1 1 a b c
AcH p (g l h ) 0.59€0.03 0.90€0.04 1.14€0.09 1.29€0.11c
AcH pmax (g l1 h1) 0.87€0.06a 1.20€0.06b 1.41€0.01c 1.73€0.01d
Total process time (h) 78.2€4.5a 52.0€2.8b 40.5€3.5c 36.5€3.5c
Lag phase (h) 2.3€3.2a 0.5€0.7a 0.5€0.7a 0.5€0.7a
Yield (%) 95.5€0.6a 96.8€0.4a 96.2€1.1a 96.6€0.2a
Final AcH (g/l) 116.3€0.4a 116.5€0.1a 116.5€0.1a,b 118.0€0.9b
Final EtOH (g/l) 6.9€0.9a 8.0€0.7a 7.5€1.4a 7.2€0.1a
Biomass/EtOHmet (mg g1) 1.63€0.10a 1.71€0.10a 2.14€0.33a 1.78€0.11a

Fig. 1 Acetic acid formation during the time course of semicon-

tinuous acetification cycles performed at an oxygen content higher
than 40%

Fig. 2 Acetic acid, acetaldehyde and biomass formation during the

the oxygen partial pressure in the aerating gas improves
the volumetric oxygen transfer coefficient and hence the
productivity of the process [6]. However, it has been es-
tablished that a high dissolved oxygen value can inhibit
the growth of acetic acid bacteria, and that an optimum
concentration for this type of process is 1–3 ppm [12].
Some authors have managed to increase productivity us-
ing 40%+ oxygen content in the aerating gas supplied to
the fermenter [4, 9, 10, 11, 15]; however, in most of these
cases immobilised bacteria were used and the concen-
tration of acetic acid was lower than 40 g l1, which is
very different from the conditions employed in industry
and in this study.
Under the different conditions of oxygen partial pres-
sure employed in the aerating gas supplied to the fer-
menter, an inverse relationship was observed between the
acetic acid formation rate profile in the course of aceti-
fication and the amount of dissolved acetaldehyde in the
time course of semicontinuous acetification cycles performed at
different oxygen-rich air compositions of the aerating gas
fermentation broth formed by the acetic bacteria, as de-
picted in Fig. 2.
At 21% and 36% oxygen content, where the perfor-
mance of the process was high, in both cases the con-
centration of acetaldehyde decreased to nil at the end of
any of the several cycles performed from an initial con-
centration of 100–110 mg l1. When 42% oxygen-rich air
was used, two stages in the acetaldehyde profile could be
distinguished during the first cycle. At the outset, during
the first 30–35 h the concentration of this intermediate
metabolite of acetification was almost constant and the
acetic acid production rate was lower than when the
process was performed with 26% oxygen. Later, when the
product concentration was about 85–90 g l1, the con-
centration of dissolved acetaldehyde started decreasing to
396
Table 2 Semicontinuous aceti- Oxygen content (%)
fications performed in the 100-l
reactor with different oxygen- 21 26 36
rich air compositions in the 1 1 a b
aerating gas (n3; aeration rate AcH p (g l h ) 0.72€0.02 0.95€0.02 1.35€0.03c
3.7 h1, vvm). Mean values with AcH pmax (g l1 h1) 1.07€0.03a 1.23€0.03b 1.80€0.05c
different letters within the same Total process time (h) 65.0€3.0a 48.0€2.0b 35.0€1.0c
row are statistically different Lag phase (h) 5.0€1.0a 1.0€1.0b 0,1€0.3c
(p0.05). Initial conditions: Yield (%) 98.9€0.3a 98.9€0.2a 98.7€0.2a
47.0€2.0 g l1 EtOH, Final AcH (g l1) 115.5€0.5a 116.2€0.2a 115.7€0.5a
70.0€2.0 g l1 AcH Final EtOH (g l1) 9.0€0.8a 10.4€1.0a 9.5€1.0a
Biomass/EtOHmet (mg g1) 1.62€0.02a 1.73€0.03b 1.72€0.02b

nil; the production rate rose, with an observed maximum

value of 1.25 g l1 h1, although the overall acetic acid
productivity was only 0.69 g l1 h1, which is lower than
was recorded at 26% oxygen (0.9 g l1 h1). When the
aerating gas contained 63% oxygen, the cells apparently
did not grow and the first cycle stopped at an acetic acid
concentration of only 78–80 g l1; the bacteria generated a
large amount of acetaldehyde, which increased exponen-
tially, probably owing to the inhibition of acetaldehyde
dehydrogenase, as suggested but not demonstrated ex-
perimentally by Allgeier and Hildebrandt in 1960 [16].
In order to reduce the consumption of pure oxygen,
semicontinuous fermentations were carried out with in-
termittent addition of pure oxygen. Under these experi-
mental conditions the rate of production increased (by
about 15–20% compared with the case for constant air),
especially in an acetic acid concentration range of 80-
90 g l1. Two types of procedures gave the best results,
both similar: 5 min with oxygen-rich air (26% oxygen)
and 10 min with air; or 5 min with oxygen-rich air and
15 min with air. These results suggest that acetic acid
productivity might be improved under these working con- Fig. 3 Acetic acid formation during the time course of semicon-
ditions, which reduce the cost of added pure oxygen; tinuous acetification cycles performed at different oxygen-rich air
compositions of the aerating gas in the 100-l acetator
however, more detailed experiments are needed to con-
firm the feasibility of this procedure in industrial pro-
duction. The use of oxygen-rich air could be of interest in the
industrial production of wine vinegar, since productivity
can be greatly increased with low aeration. This technique
One-hundred-litre fermenter can only be applied in industrial reactors that operate with
compressed air, and therefore not in the Frings aceta-
Acetification was also carried out with oxygen-rich air tor, which is equipped with a turbine (aerator). Under
(up to 63%) in a 100-l pilot acetator. These experiments these conditions, the yield is close to 100%, and the wine
confirmed the behaviour observed with the 6-l reactor, vinegar should possess better sensory properties, since the
and are reported in Table 2 and Fig. 3. loss of volatile compounds is minimal. In a sensory study
The overall acetic acid productivity increased from on the differences between vinegars obtained with air and
0.72 g l1 h1 with air to 1.35 g l1 h1 with a 36% oxygen-rich air, the trained tasters preferred vinegars
oxygen-rich air (85%). As also observed in the previous obtained using oxygen-rich air because their flavour was
experiments using the 6-l laboratory fermenter, semi- more pleasant and less sour [17]. This is particularly
continuous acetification cycles were not feasible with an important for wine vinegar, given that its peculiar orga-
oxygen content higher than 40%, and with 63% oxygen noleptic characteristics make it much more attractive than
the process came to a halt with little production of acetic vinegars made from other substrates such as ethanol.
acid (Fig. 3). The use of oxygen-rich air reduced the lag The use of oxygen-rich air naturally increases the price
phase and increased the substrate yield based on metab- of the final product (by about 0.02–0.03 euros per litre),
olised ethanol. No statistically significant differences because pure oxygen is more costly. Nevertheless, an
were observed in the process yield or the final concen- overall improvement in productivity will reduce process
trations of product and substrate. costs (electricity, refrigeration, etc.) by cutting down the
total process time. In short, the use of oxygen-rich air on

an industrial scale has advantages, but more extensive and
detailed research is required, among other things into the
specific process conditions at the different vinegar fac-
tories.
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