Appl Microbiol Biotechnol (2003) 61:157–162
DOI 10.1007/s00253-002-1197-z
ORIGINAL PAPER
M. G. Aguilar Uscanga · M.-L. Dlia · P. Strehaiano
Brettanomyces bruxellensis: effect of oxygen on growth
and acetic acid production
Received: 26 July 2002 / Revised: 4 November 2002 / Accepted: 8 November 2002 / Published online: 14 January 2003
Springer-Verlag 2003
Abstract The influence of the oxygen supply on the
growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0–300 l h–1
(0–0.5 vvm). This study shows that growth of this yeast is
stimulated by moderate aeration. The optimal oxygen
supply for cellular synthesis was an oxygen transfer rate
(OTR) of 43 mg O2 l–1h–1. In this case, there was an air
flow rate of 60 l h–1 (0.1 vvm). Above this value, the
maximum biomass concentration decreased. Ethanol and
acetic acid production was also dependent on the level of
aeration: the higher the oxygen supply, the greater the
acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong
inhibition of the ethanol yield. Over 180 l h–1 (0.3 vvm,
OTR =105 mg O2 l–1 h–1), glucose consumption was
inhibited and a high concentration of acetic acid (6.0 g l–1)
was produced. The ratio of “ethanol + acetic acid”
produced per mole of consumed glucose using carbon
balance calculations was analyzed. It was shown that this
ratio remained constant in all cases. This makes it
possible to establish a stoichiometric equation between
oxygen supply and metabolite production.
Introduction
Oxygen plays a key role in the alcoholic fermentation of
sugars by yeasts. Glucose fermentation by Saccharomyces
cerevisiae is generally inhibited by oxygen, but small
amounts of dissolved oxygen enhance ethanol production
M.-L. Dlia ()) · P. Strehaiano
ENSIACET, Laboratoire de Gnie Chimique,
UMR-CNRS 5503,
118 route de Narbonne, 31077 Toulouse cedex 4, France
e-mail: [email protected]
Tel.: +33-5-62885874, +33-5-34615251
M. G. Aguilar Uscanga
Depto. de Ing. Qumica y Bioqumica / UNIDA,
Instituto Tecnolgico de Veracruz,
Av. Miguel A. de Quevedo 2779. cp 91860, Veracruz, Mexico
compared to anaerobic conditions (Strehaiano 1984). The
genus Brettanomyces is well known as sensitive to the
availability of oxygen. In 1940, Custer found that resting
cells of Brettanomyces claussenii fermented glucose at a
higher rate under aerobic conditions than in the absence
of oxygen (cited in Scheffers and Wiken 1969). Scheffers
and Wiken (1969) introduced the term “Custer effect”
(negative Pasteur effect). This effect is attributed to the
inhibition of alcoholic fermentation under anaerobic
conditions, and to the strong tendency of Brettanomyces
to produce acetic acid from glucose with the parallel
reduction of NAD+ (Carrascosa et al. 1981). In particular,
yeasts of the genera Brettanomyces and Dekkera show a
strong Custer effect (Scheffers 1979). Gaunt et al. (1988)
provided evidence that organic hydrogen acceptors (acetaldehyde, acetone and 3-hydroxy-butan-2-one) alleviate
the Custer effect by restoring the redox balance.
Brettanomyces yeasts are associated with spoilage of
both industrial alcoholic fermentation and alcoholic
beverages. De Miniac (1989) and Dlia-Dupuy et al.
(1995) described contamination of alcohol fermentation
plants by Brettanomyces yeasts and explained that the
observed inhibitory effect on Saccharomyces was due to
the synthesis of acetic acid. The effect of the acetic acid
upon growth of both S. cerevisiae and Brettanomyces
bruxellensis was evaluated (Phowchinda et al. 1997) and
it was found that the metabolism of these yeast strains is
inhibited by high concentrations of acetic acid (4.5–6 g l–1).
Ciani and Ferraro (1997) showed that oxygen concentration exerted a strong influence on growth and acetic acid
production by Brettanomyces yeasts in winemaking.
Therefore, the aim of this work was to evaluate the
influence of oxygen supply (available in air) on the
metabolism of B. bruxellensis. It should be possible to
determine the dissolved oxygen transfer rate (OTR)
required for maximum growth and end-product synthesis,
and to propose a general stoichiometric equation that
quantifies the metabolic balance of Brettanomyces under
the conditions tested.
158
Materials and methods
Microorganism
The yeast strain used was isolated from an alcoholic fermentation
during the contamination of an industrial plant (distillery). It was
identified as B. bruxellensis by the Institute of Hygiene and
Epidemology Mycology of Brussels (IHEM), and registered under
the number 6037.
Culture media
B. bruxellensis was preserved at 4C on a medium composed of
agar-agar, glucose and yeast extract (2, 2, 1%). A minimal culture
medium was used, which consisted of 5% glucose, 0.2%
(NH4)2SO4, 0.5% KH2PO4, 0.04% MgSO4·7H2O and 0.1% yeast
extract. The pH was adjusted to 4.0 using 10% (v/v) orthophosphoric acid.
Culture conditions
The experiments were carried out in a 15 l bioreactor (Applikon,
3100 AC Schiedam, The Netherlands) connected to a bio-controller
(ADI 1030, Applikon) to record pH and dissolved oxygen (Ingold
O2 probe). For each batch culture, the temperature was regulated at
30C, the pH was not controlled and the stirrer speed was set at
250 rpm. The culture medium (10 l) was sterilized for 60 min at
120C. Inoculum was added to attain 3106 viable cells/ml.
The gas-liquid transfer coefficients (kLa) were determined at
different air flow rates [30 (0.05 vvm), 60 (0.1 vvm), 90 (0.15 vvm),
120 (0.20 vvm), 180 (0.3vvm) and 300 l h–1 (0.5 vvm)] by the static
gassing out method (dissolved oxygen probe and measurement in a
non respiring system) (Moo Young and Blanch 1987). The resulting
kLa values are 3.5, 6.4, 9.8, 13.5, 15.6 and 21.7 h–1, respectively.
Dissolved OTR was calculated using the equation OTR=kLa (C*–
C).
Fig. 1 Batch growth of Brettanomyces bruxellensis under anaerobiosis (0 l h–1) (h glucose, biomass) and aerobiosis (300 l h–1;
0.5 vvm). n Glucose, l biomass, u acetic acid
Elementary formula of B. bruxellensis
The elementary formula of the biomass was determined at two
different growth stages (t=48 h and t=87 h). Three measurements
were performed at each stage. The concentration (%) of elements
(C, H, N, and O) obtained was unchanged; the average of the data
gave the elementary formula of B. bruxellensis: C H1.54 O0.83 N0.107.
The Inter-University Service of Microanalyses of ENSIACET
carried out composition analyses.
Results
Influence of oxygen on growth and end-products
Analysis of exit gases
Oxygen uptake and CO2 production were measured on-line by
means of a paramagnetic gaseous oxygen analyzer (570/711 A
model Servomex, Crowborough, UK) and an IR CO2 analyzer
(series 1410 model Servomex), respectively.
The general expression of the oxygen uptake rate (OUR, mol
l–1 h–1) and CO2 production rate (CPR, mol l–1 h–1) proposed by
Cooney et al. (1977) were simplified according to the conditions
employed. For each exit gas, oxygen uptake OU(t) and CO2
production CP(t) were found by integrating [OUR(t) and CPR(t)],
respectively.
Analytical techniques
Growth was followed by measuring the optical density of yeast cell
suspensions at 620 nm. This measurement has previously been
correlated to cell dry weight.
Glucose, ethanol, glycerol and acetic acid concentrations were
determined by HPLC with a TPS Spectra System apparatus using a
Bio-Rad Aminex HPX-87H column heated to 40C and a refraction
index detector (TPS RefractoMonitor V). The mobile phase was
sulfuric acid (0.005 M) flowing at 0.4 ml min–1. Specialized
software (BORWIN V 1.2) allowed the surface area of detected
peaks to be calculated. A calibration curve was prepared in the
concentration range 0–20 g l–1 for each component. Experimental
error was estimated at less than 3%.
To investigate the influence of oxygen concentration on
the growth and activity of B. bruxellensis, batch cultures
were run on glucose under both aerobic (300 l h–1;
OTR=145.6 mg O2 l–1 h–1) and (not strict) anaerobic
conditions (dissolved O2 present at the beginning of
fermentation). Figure 1 shows biomass concentration,
glucose consumption and acetic acid production curves
obtained under both conditions. The final biomass
concentration increased from 3.76 g l–1 (anaerobic
conditions) to 5.16 g l–1 (air supply 300 l h–1). With
oxygen provided to the medium, growth was faster and
better compared to anaerobic conditions. Under both
conditions, this strain presented a lag phase of approximately 12 h. It is worth noting that under anaerobic
conditions all the glucose was consumed, while with
300 l h–1 aeration glucose consumption was partial
(residual glucose 24 g l–1). A high concentration of acetic
acid was reached (9.4 g l–1) only under aerobic conditions; the minimum amount of acetic acid was synthesized under anaerobic conditions (0.025 g l–1, not shown
in Fig. 1).
Table 1 shows the kinetic parameters observed under
both conditions. It is clear that the presence of oxygen in
the medium brought about a strong increase in specific
growth rate and acetic acid production compared to
159
Fig. 2 Carbon balance at different times for a culture of B.
bruxellensis aerated at 300 l h–1 (0.5 vvm)
anaerobiosis. In contrast, the ethanol yield dropped
from 0.48 to 0.06 g g–1.
In yeast metabolism, glucose is completely converted to end-products and biomass. Indeed, in order to
check the consistency of the experimental values and to
verify if a secondary product of the fermentation was
produced but not taken into account, we performed a
molar carbon balance on the substrate (glucose) and on
the end-products (biomass, ethanol, acetic acid and
CO2) obtained during the fermentation. The analysis of
metabolite products during these experiments showed
that very little glycerol was produced (0.36 g l–1 under
anaerobic, and approximately 0.03 g l–1 under aerobic
conditions).
Figure 2 shows the evolution of the carbon balance
between glucose and the different measured endproducts for a culture performed at an aeration rate
of 300 l h–1.
The calculation of the instantaneous carbon balance
at an aeration rate of 300 l h–1 confirmed that it is well
balanced throughout the fermentation. The maximum
error remained less than 1.4%. The same calculation was
made for anaerobic conditions (performed in the bioreactor).The carbon balance was equally good for all
products present in the two experiments.
As large differences were observed between nonaerated and aerated cultures–and in order to better
understand the role of oxygen in the metabolism of this
yeast–different air flow rates were tested (in the range
30–300 l h–1; 0.05–0.5 vvm; OTR=23.5–145.6 mg O2
l–1 h–1). Figure 3 illustrates the effect of different aeration
conditions on the growth of B. bruxellensis.
Fig. 3 Effect of air flow rates (l h–1) on B. bruxellensis growth
Fig. 4 Effect of aeration conditions on acetic acid and ethanol
production, and glucose consumption by B. bruxellensis at the end
of the fermentations (200, 76, 64 and 90 h, respectively)
Biomass concentration curves revealed the optimum
aeration rate for B. bruxellensis growth within the range
tested. To obtain maximum concentration of biomass
(8.5 g l–1) the most favorable aeration condition was 60 l
h–1 (OTR=43 mg O2 l–1 h–1). Beyond this level of
aeration, the final biomass produced begins to decrease,
reaching a value of approximately 5 g l–1 at 300 l h–1.
Figure 4 presents results concerning ethanol and acetic
acid production and glucose consumption obtained for all
the aeration levels. The substrate was completely consumed in a range of aeration between 0 and 120 l h–1, (in
this case, the maximum concentration of acetic acid was
4.5 g l–1 in the final phase). However, beyond these
Table 1 Values of the kinetic parameters obtained from different aeration conditions
Aeration condition
(l h–1)
Cell growth maximum
(g l–1)
Ethanol maximum
(g l–1)
Acetic acid maximum
(g l–1)
Ethanol yield
(g g–1)
mmax (h–1)
0
300
3.76
5.16
24.0
3.0
0.15
10.0
0.48
0.06
0.24
0.62
160
Fig. 5 pH variation along the culture under different air flow rates
(l h–1)
Fig. 7 Linear correlations between the oxygen (b), ethanol (d) and
acetic acid (e) coefficients
Table 2 Value of the oxygen (b), ethanol (d) and acetic acid (e)
coefficients for each batch culture
Fig. 6 Dissolved oxygen concentration under different air flow
rates (l h–1)
aeration rates, glucose was not totally consumed (e.g., 7%
of the sugar is not used at an aeration rate of 180 l h–1).
Moreover, the lowest pH value (2.31) was observed under
these conditions (Fig. 5).
When the medium was sparged with air flows in the
range 30–120 l h–1, the OTR became limiting for growth.
The dissolved oxygen concentration fell to zero as soon as
the lag phase ended and remained at zero during the
growth and stationary phases (Figs. 6, 3). An increase in
air flow rate (180 l h–1) slightly reduced the limitation but
did not cancel it out. Only a large increase in the partial
pressure of oxygen in the inlet gas made it possible to
provide the medium with sufficient dissolved oxygen; the
concentration did not fall below 16% with an air-flow rate
of 300 l h–1 (0.5 vvm).
Determination of ethanol and acetic acid ratio coefficients
As a decrease in ethanol production and an increase in
acetic acid production were observed when aeration
conditions varied, the stoichiometric coefficients were
calculated for each condition as a function of the oxygen
uptake. The stoichiometric general equation is:
glucoseþb O2 ! a biomassþc CO2 þd ethanol
þe acetic acid:::
Aeration (l h–1)
(b)
Oxygen
(d)
Ethanol
(e)
Acetic acid
0
30
60
90
120
180
300
0
0.04
0.08
0.09
0.12
0.25
0.70
0.60
0.57
0.56
0.47
0.45
0.40
0.16
0
0.02
0.03
0.11
0.14
0.20
0.43
Table 2 and Fig. 7 show the variations obtained in
seven batch cultures with different air flow values. Acetic
acid and ethanol production varied proportionally with
oxygen uptake. Acetic acid increased with oxygen uptake
while ethanol production decreased.
Linear correlations were established between the
coefficients of both oxygen (b) and ethanol (d) with that
of acetic acid (e) (Fig. 7); the first being positive and the
second negative (r2=0.94 in both cases). This equation
can be written in the following way: ethanol coefficient
d=–0.6 oxygen +0.5692, and acetic acid coefficient e=0.6
oxygen +0.0224.
Discussion
The positive effects of oxygen on growth and acetic acid
production were clear. However, an inhibitory effect on
substrate consumption was observed when oxygen concentration increased (180 l h–1 and 300 l h–1); in both
cases, acetic acid concentration was at its highest level (7
and 10 g l–1, respectively). This behavior suggests that the
observed inhibitory effect on glucose metabolism by this
yeast is due to the presence of the acetic acid produced
161
during fermentation. Different investigations carried out
on the inhibitory effect of acetic acid (Pampulha and
Loureiro-Dias 1989; Rasmussen et al. 1995; Taherzadeh
et al. 1997) show that its toxicity acts at the cytoplasmic
level where the enzymes involved in glycolysis are
present. They also mention that the toxicity of this acid is
dependent on the external pH, because the active form is
the undissociated form, which penetrates the cellular
membrane more easily and, once inside, can dissociate,
causing modification of the internal pH of the cell.
Ciani and Ferrara (1997) tested the effect of oxygen by
keeping the dissolved oxygen concentration between 40
and 80% of saturation. Under these conditions, they found
that Brettanomyces sp. produced 12 g l–1 acetic acid at a
final pH of 1.91, and residual glucose was 40%. Blondin
et al. (1982) reported a similar fermentation with Dekkera
and Brettanomyces intermedius. They observed that for a
high aeration rate and 120 g l–1 initial glucose, the sugar
was never totally consumed (quantity used: 55 g l–1) and
the acetic acid production was 30 g l–1. These results are
also consistent with previous reports by Scheffers and
Wiken (1969), and Peynaud and Domercq (1956). All
these data indicate that, under aerobic conditions, Brettanomyces yeast is able to produce acetic acid depending
on the air flow. The molar carbon balance confirmed that
this yeast produces low amounts of glycerol under
anaerobic conditions and only traces under aerobic
conditions.
These observations are consistent with previous work
by Wijsman et al. (1984a) and Van Dijken and Scheffers
(1984). These authors did not find glycerol production
during their studies (under aerobic conditions) on B.
intermedius. They proposed that the Custer effect in this
yeast is due to the tendency of the organism to produce
acetic acid in combination with its inability to restore the
redox balance via production of reduced metabolites such
as glycerol.
Moderate aeration did however have an effect on
ethanol production; above 30 l h–1 the quantities of
ethanol produced decreased when the air flow rate
increased. For 90 l h–1 the final ethanol concentration
and yields were strongly affected (under anaerobic
conditions the theoretical yield was 96%, but only 72%
was observed, 24.4 g l–1 and 18 g l–1 ethanol, respectively). In this case, it was possible that the decrease in
ethanol production observed was linked to the increase of
acetic acid production. As already stated, the switch from
ethanol to acetic acid was a progressive phenomenon,
with oxygen uptake and the decrease in ethanol production compensated by the synthesis of acetic acid.
The oxygen effect was clearly demonstrated on each
end-product concentration, and the general equation for
glucose breakdown by the B. bruxellensis strain was
deduced from experimental data as follows:
glucoseþb O2 ! a biomassþc CO2 þð0:6b
þ0:569Þ ethanol þ ð0:6bþ0:022Þ acetic acid
Some previous studies on glucose metabolism kinetics
(Blondin et al. 1982; Wijsman et al. 1984b) showed that
the sugar fermentation rate in this yeast increases under
aerobic conditions. However, our data proves that the
dissolved OTR is a very important factor for growth and
fermentative activity of B. bruxellensis. Optimum biomass production is achieved at an OTR of 43 mg O2 l–1
h–1. Under highly aerated conditions (OTR‡104.8 mg
O2 l–1 h–1) it appears that the sugar is never totally
consumed and large quantities of acetic acid are synthesized before the inhibition effect appears. In this case the
behavior observed in this work (concerning acetic acid
and glycerol formation) is similar to that described by
Wijsman et al. (1984a).
Moreover, the accumulation of acetate in the culture
medium explains the inhibition of glucose metabolism
and decreased ethanol. The strain appears to be able to
form large quantities of acetic acid when the dissolved
OTR increases. The abilities of B. bruxellensis to react
with oxygen seem to be linked to acetic acid production.
Furthermore, Eq. 2 provides some helpful information on
glucose metabolism by B. bruxellensis.
Acknowledgement The authors thanks Patricia M. Hayward
Jones, M.Sc. for the critical reading of the manuscript for English
language.
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