World Journal of Microbiology & Biotechnology 19: 101–105, 2003.

101
 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions

of constant pH

S.N. Freer1,*, B. Dien1 and S. Matsuda2

1
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA ,
Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA
2
Kumamoto Industrial Research Institute, Higashimachi 3-11-38, Kumamoto 862-0901, Japan
*Author for correspondence: Tel.: +1-309-681-6472, Fax:+1-309-681-6427, E-mail: freersn@ncaur.usda.gov

Received 13 December 2001; accepted 18 October 2002

Keywords: Acetic acid, bioreactor, constant pH, Dekkera

Summary

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture,
from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the
ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at
28
C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l
as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the
two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose
concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.

Introduction requirement of Acetobacter, the process is energy

Acetic acid is an important industrial chemical with an
annual domestic production of about 1.83 · 106 metric
tons in 1998 (Tullo 1999). Currently, the commercial
production of glacial acetic acid is exclusively by
petrochemical routes. Expanded potential industrial
uses of acetic acid include the production of environ-
mentally friendly road de-icers and airport runway de-
icers that are non-corrosive (e.g., calcium magnesium
acetate (CMA) and potassium acetate and sodium
acetate) (Dunn & Schenk 1980). In addition, there are
reports that CMA, when used as an additive in coal-
fired combustion units, reduces sulphur dioxide emis-
sions, thus, partially mitigating the problem of acid rain
pollution (Levendis 1991; Sharma 1991).
Two microbiological approaches have been previously
proposed for the fermentative production of acetic acid/
CMA from corn (Yang et al. 1997). In one approach,
ethanol, which is produced by yeast fermentation, is
aerobically converted to acetic acid by Acetobacter. In
the other approach, glucose/cellulose is converted to
acetic acid, usually using thermophilic Clostridium spp.
High concentrations of acetic acid are produced in the
yeast–Acetobacter process, however, due to the oxygen
 
Names are necessary to report factually on available data;
however, the USDA neither guarantees nor warrants the standard of
the product, and the use of the name by USDA implies no approval of
the product to the exclusion of others that may be suitable.
intensive. The thermophilic process (55–60
C) is also
energy intensive and results in low final acetic acid
levels.
A third, as yet unexplored approach, involves using
yeasts that belong to the genera Dekkera and its
anamorph, the genus Brettanomyces (Smith et al.
1990). These yeasts are noted for spoiling cellar and
bottled wine through the production of haze, turbidity
and acetic acid (Sponholz 1993), as well as use in the
secondary fermentation of lambic beer. Recently,
60 yeast strains belonging to these, and several other
genera, were screened for the production of ethanol and/
or acetic acid from either glucose or ethanol in shaken
batch culture (Freer 2002). Several of these cultures
produced over 25 g acetic acid/l from 100 g glucose/l. In
this paper, we describe the production of acetic acid by
seven of these cultures when grown in bioreactors with
pH, temperature, and aeration control.
Materials and methods
Organisms, media and growth conditions
The yeasts tested in this study were: Brettanomyces cus-
tersianus NRRL Y-6653; Dekkera intermedia NRRL
YB-4553, YB-5164; B. intermedius NRRL Y-2395; D.
bruxellensis NRRL Y-17525, Y-17535; D. anomala
NRRL Y-17520. All cultures were obtained from the
102 S.N. Freer et al.
ARS Culture Collection, Peoria, IL. The basal medium

Time (h)
(YP) consisted of 20 g peptone/l and 10 g yeast extract/l.
The bioreactors (Biostat B, B. Braun Biotech, Inc.,

64
67

66

49

53
92

73
Allentown, PA) were fitted with 2 l reaction vessels
containing 1.2 l of YP medium. The basal medium and

C-Balc (%)
glucose solutions were sterilized (121
C, 40 min) sepa-
rately and added to the bioreactor upon cooling. The
inocula were prepared as previously described (Freer

82
92

67

53

62
61

69
2002) and the fermentations were initiated by inoculat-
ing with a 5% (v/v) inoculum. The bioreactors were

Effb
(%)

27
23

28

10

14
27

13
aerated at 0.75 v/v/m with agitation (630 rev/min) at
28
C. The pH was maintained by the addition of a

Yielda
(g/g)
solution of 30% (w/v) base (NaOH:KOH=1:1).

0.36
0.29

0.36

0.13

0.18
0.35

0.16
Growth, ethanol, carbohydrate and acetic acid analysis

ethanol (g/l)
Residual
Growth was measured by the increase in optical density

0.0
8.2

1.6

0.0

0.0
0.0

0.0
at 600 nm. Glucose, ethanol, and acetic acid were quan-
tified by high-pressure liquid chromatography (HPLC)
on a Spectra-Physics chromatograph fitted with a

Acetic acid
BioRad HP-87H column. The mobile phase was 5 mM
sulphuric acid. The compounds of interest were detected

14.9
12.3

15.0

14.6
5.4

7.4

6.8
(g/l)
with a Waters 410 differential refractometer.

Yeast dry mass is assumed to be 50% carbon. Carbon balance was based upon acetate and biomass production.
A600

50.7
47.5

31.9

63.4

38.8
36.4

35.4
Results and discussion

Production of acetic acid from either glucose or ethanol

Time (h)

Previously, 60 yeast strains were evaluated for acetic

64
67

91

67

70

67
162
acid production in shaken flask batch culture using
either 100 g glucose/l or 35 g ethanol/l as the carbon/
C-Balc

energy source. Seven of the strains that produced the

(%)

most acetic acid were further tested on these carbon

94
84

53

79

86
87

71
sources in bioreactor batch culture in which the pH was
Effb(%)

maintained at 6.5 (Table 1). D. bruxellensis NRRL Y-

Table 1. Growth, acetate production and carbon usage from glucose or ethanol.

17535 produced the most acetic acid (44.3 g/l) from

70
52

51

64
73

47
0

glucose, however, the time required to do so was

Yield is defined as the amount of product/amount of initial substrate.
Yielda

extensive (162 h). D. intermedia NRRL YB-4553 and

(g/g)

0.47
0.35

0.34

0.43
0.49

0.31
0.0

D. bruxellensis NRRL Y-17525 produced 42.8 and

39.4 g acetic acid/l, respectively, in 70 h or less. These
Efficiency is defined as amount of product/theoretical yield.
glucose (g/l)

cultures also utilized all of the 100 g glucose/l initially

Residual

present in the medium. D. intermedia NRRL YB-5164,

12.3

71.2

11.8
0.0

0.0
0.0

0.0

B. intermedius NRRL Y-2395 and D. anomala NRRL

Y-17520 all produced approximately 30 g acetic acid/l in
less than 70 h. B. custersianus NRRL Y-6653 produced
Acetic acid

no detectable acetic acid when grown on glucose and

utilized only about 30% of the initial glucose present in
42.8
31.8

31.5

39.4
44.3

29.3
0.0
(g/l)

the medium, even after 91 h of incubation.

When grown under conditions of constant pH, all of
the tested cultures produced more acetic acid than when
A600

62.0
60.3

44.0

50.5

55.8
40.9

67.5

grown in shaken flask batch cultures. For example, D.

intermedia NRRL YB-4553 and D. bruxellensis NRRL
Y-17525 produced 30.0 and 31.6 g acetic acid/l, respec-
Culture (NRRL)

tively, when grown in shaken flask batch culture (Freer

B. custersianus

D. bruxellensis
B. intermedius
D. intermedia

2002). A carbon balance analysis (Table 1) indicated

D. anomala

that these cultures converted 94 and 86% of the initial

YB-4553
YB-5164

Y-17525
Y-17535

Y-17520
Y-6653

Y-2395

carbon to acetic acid and biomass. The unaccounted

b
a

carbon was probably diverted towards respiration since

Acetate from yeasts
no other additional product was detected in the culture
broth (data not shown). Overall, the yeasts produced
50–70% of the theoretical amount of acetic acid possible
from glucose.
Although B. custersianus NRRL Y-6653 produced no
acetic acid from glucose, it produced the most acetic
acid (15.0 g/l) when ethanol was used as the carbon/
energy source. The D. intermedia strains and D. brux-
ellensis NRRL Y-17535 produced over 12 g acetic acid/
l, although the D. bruxellensis fermentation was notably
slower than the D. intermedia fermentation. However,
these cultures produced less acetic acid in the bioreactor
batch culture (14.9 and 14.6 g/l) than they did in shaken
flask batch culture (24.3 and 33.0 g/l) (Freer 2002). The
reasons for this are unknown. D. bruxellensis NRRL Y-
17525 and the B.intermedius and D. anomala strains
produced less than 8 g acetic acid/l from the 43 g
ethanol/l initially present in the medium.
Comparison of D. intermedia NRRL YB-4553
and D. bruxellensis NRRL Y-17525
The initial bioreactor batch cultures indicated that the
two most promising yeasts for acetic acid production,
when glucose is used as a carbon/energy source, are D.
intermedia NRRL YB-4553 and D. bruxellensis NRRL
Y-17525. These yeasts were simultaneously compared
for their ability to produce acetic acid in bioreactor
batch culture, in which the pH was maintained at 6.5,
from 100 g glucose/l. Results (Figure 1) are presented
for D. intermedia NRRL YB-4553. The yeasts behaved
similarly. Both utilized all of the glucose initially present
in the medium by 48 h, grew at equivalent rates and to
about the same extent, and, produced similar amounts
of acetic acid and ethanol at about the same rate.
However, since D. intermedia converted ethanol to
acetic acid more efficiently than D. bruxellensis (Ta-
ble 1), further experiments were conducted with D.
intermedia NRRL YB-4553.
Figure 1. Growth and acetate production by D. intermedia NRRL
YB-4553. The pH was maintained at 6.5: cell density (optical density at
600 nm) (s), glucose (d), acetic acid (j) and ethanol (().
103
Optimization of pH
D. intermedia was grown in YP media initially contain-
ing 100 g glucose/l. Although the initial pH was not
adjusted, the pH of the bioreactor batch cultures was
regulated at 4.5, 5.5, or 6.5 by the addition of base. The
results showed that pH 5.5 was the optimal pH for acetic
acid production under the conditions tested (Figure 2).
At pH 5.5, glucose was depleted from the medium by
48 h, and the culture produced 39.6 g acetic acid/l.
Ethanol accumulation was maximal at 36 h, and the
ethanol appeared to be subsequently converted to acetic
acid. At pH 4.5, the culture produced less than 30 g
acetic acid/l and failed to utilize all of the glucose
initially present in the medium. Also, ethanol accumu-
lated to almost 10 g/l in the medium. When the pH was
maintained at 6.5, glucose was depleted from the
medium by 48 h, and the ethanol accumulation in the
medium was transient. However, the final acetic acid
yield (36.2 g/l) was slightly lower than in the pH 5.5
fermentation (data not shown).
Effect of glucose concentration on acetic acid production
In order to determine the maximal amount of acetic
acid that D. intermedia NRRL YB-4553 is capable of
producing, bioreactor batch cultures were performed at
pH 5.5 in YP medium initially containing either 150 or
200 g glucose/l (Figure 3). In medium initially contain-
ing 150 g glucose/l, D. intermedia produced 57.5 g
acetic acid/l and utilized all of the glucose initially
present in the medium in 96 h. Ethanol accumulated in
the medium to a concentration of 17 g/l at 60 h and
then decrease to undetectable levels by 112 h. About
87% of the carbon was accounted for in a carbon
balance analysis and approximately 71% of the glucose
initially present in the medium was converted to acetic
acid (data not shown). This resulted in an acetic acid
yield of 0.38 g acetic acid/g added glucose (theoretical
Figure 2. Fermentation of glucose by D. intermedia NRRL YB-4553 at
pH 5.5: cell density (optical density at 600 nm) (s), glucose (d), acetic
acid (j) and ethanol (().
104
Figure 3. Fermentation of (A) 150 g glucose/l and (B) 200 g glucose/l by D. intermedia NRRL YB-4553 at pH 5.5: cell density (optical density at
600 nm) (j), glucose (s), acetic acid (d) and ethanol (r).
yield is 0.667 g acetic acid/g glucose) and a productivity
of 0.51 g/l/h. When grown in YP medium initially
containing 200 g glucose/l, D. intermedia did not
completely utilize all of the glucose. After 144 h of
incubation, the culture produced 65 g acetic acid/l,
however, 40 g glucose/l remained in the medium. This
suggests that acetic acid becomes inhibitory at this
concentration.
The majority of the research on the microbial pro-
duction of acetic acid (excluding the vinegar process)
revolves around the use of Clostridium thermoaceticum.
This anaerobic bacterium ferments a wide spectrum of
sugars, including glucose, xylose and/or fructose, and
has a high theoretical yield (1 g acetic acid/g glucose).
However, the reported acetic acid yields for batch
fermentations are significantly less than theoretical
(0.6–0.8 g/g) and productivities are low (0.15–0.25
g/l/h). Furthermore, C. thermoaceticum is sensitive to
acetic acid. At near neutral pH values, the acid tolerance
is 40 g/l. At pH 5.5, the acetic acid tolerance for the
bacterium decreases to 18 g/l (Han & Cheryan 1996).
Much work has been done to overcome these limita-
tions through strain development (Reed et al. 1986;
Parekh & Cheryan 1990a; Parekh & Cheryan 1991;
Parekh & Cheryan 1994) and continuous acetic acid
removal (Parekh & Cheryan 1990b; Chukwu & Cheryan
1999; Borden et al. 2000). A C. thermoaceticum mutant
selected for high acetic acid tolerance produced 45–50 g
acetic acid/l (Parekh & Cheryan 1994; Witjitra et al.
1996). However, this fermentation was run at pH 6.7,
which precludes producing the free acid and necessitates
the addition of considerable amounts of base.
In conclusion, D. intermedia NRRL YB-4553 appears
to produce slightly more acetic acid, at a higher
productivity and lower pH’s, than does C. thermoace-
ticum. However, D. intermedia has the disadvantage in
that the theoretical yield is lower. In order to evaluate
the potential that this yeast has for the large-scale
production of acetic acid, the medium must first be
optimized using industrial components.
S.N. Freer et al.
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