Biotechnology Letters 21: 349–353, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

349

Production of Oenococcus oeni biomass to induce malolactic fermentation

in wine by control of pH and substrate addition

Sergi Maicas1,2 , Pilar González-Cabo1,3 , Sergi Ferrer1 & Isabel Pardo1,∗

1 Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, C/ Dr Moliner, 50,
E46100 Burjassot-València, Spain
Present addresses: 2 Departament de Biotecnologia, Institut d’Agroquı́mica i Tecnologia d’Aliments (CSIC), Pa-
terna, Spain
3 Unitat de Genètica. Hospital Universitari ‘La Fe’, València, Spain
∗ Author for correspondence (Fax: +34-963864372; E-mail: Isabel.Pardo@uv.es)

Received 18 January 1999; Accepted 26 February 1999

Key words: biomass production, growth control, lactic acid bacteria, Oenococcus oeni, pH control

Abstract
To increase the commercial production of Oenococcus oeni strains to be used for biological deacidification of
wines, substrates addition and pH control have been optimized. The highest biomass yield of Oenococcus oeni
(Y = 6.9 mg mmol−1 sugar) was obtained when 55 mmol glucose l−1 and 30 mmol fructose l−1 were added both
to the culture medium, and the pH was controlled at 4.8. Fructose was used as carbon and energy source, but also
as electron acceptor improving the ability to reoxidize NAD(P)H.

Introduction efficiently metabolized sugar. The use of fructose in

Oenococcus oeni is the major bacterial species found
in wines during the malolactic fermentation (MLF),
and is well adapted to the low pH and high ethanol
concentration of wine (Wibowo et al. 1985, Ramos
et al. 1995). The natural conditions of the malolac-
tic conversion are unfavourable, and in recent years,
winemakers have used selected strains of this mi-
croorganism to ensure a better control of the process.
Inoculating the wine with a high concentration of
O. oeni enhances the probability of obtaining rapid
and complete MLF. The inoculation of wines or musts
with optimal numbers of cells that perform the MLF
requires the use of media and cultural conditions that
optimize growth rate, biomass yield, and the ability to
conduct the MLF in wine. Studies on the utilization
of the major hexoses in wine (glucose and fructose)
have been previously reported in non-pH controlled
experiments (Tracey & van Rooyen 1988, Salou et al.
1994, Serpa et al. 1994). Glucose is used as the carbon
and energy source by all strains of O. oeni, but it has
also been shown that fructose is the most rapidly and
the growth medium as an electron acceptor has usually
been seen as beneficial for most strains (Salou et al.
1994). However, some exceptions have been reported
(Tracey & van Rooyen 1988).
A primary effect of sugar fermentation is the low-
ering of pH of the medium. Internal pH decreases as
well and the effects inside the cell are (1) a higher
consumption of energy (ATP) to pump out protons,
and (2) a decrease in the cytoplasmic enzymatic ac-
tivities as the internal pH decreases. When the excess
of protons in the medium is neutralized by addition of
the alkali, the pH remains constant outside and inside
the cell and more energy can be used to increase bio-
mass. Several authors have demonstrated the benefits
of the pH control in other lactic acid bacteria cultures
(Beal et al. 1989, Champagne et al. 1989), but no
references have been found about its utilization to ef-
fectively produce O. oeni biomass able to develop the
MLF in wine.
In this paper, we describe the growth of O. oeni
M42 in a basal medium supplemented with various
carbohydrates, and the beneficial effect of the pH con-
350
trol to improve its commercial production. The use
of this technological approach enables a high produc-
tion of well-adapted biomass that can be used as an
effective starter to induce the MLF in wines.
Materials and methods
Cultural conditions. Oenococcus oeni M42 (Pardo
& Zúñiga 1992, Pardo et al. 1988), was grown
on Medium for Leuconostoc oenos (MLO medium)
(Caspritz & Radler 1983) without tomato juice or
citrate and supplemented with glucose (55 mmol glu-
cose l−1 ), or fructose (55 mmol fructose l−1 ), or
glucose (55 mmol glucose l−1 ) plus fructose (30 mmol
fructose l−1 ). pH was adjusted to 4.8 with 1 M KOH.
After autoclaving, the medium was supplemented with
pantothenic acid (0.01 g l−1 ). Fermenters were inoc-
ulated with 2% (v/v) washed cells harvested at the
late phase of growth from a MLO preculture. Cul-
tures were grown at 28 ◦ C stirring at 250 rev/min for
3 to 10 days. pH was monitored and, when specified,
maintained at pH 4.8. Buffer assays were performed
in 40 mM phosphate/20 mM tartrate buffer (Henick-
Kling et al. 1986). For vinification assays, cultures
were grown in basal MLO with glucose (55 mmol glu-
cose l−1 ) and fructose (30 mmol fructose l−1 ) with pH
control (pH 4.8). Cells were then collected and inoc-
ulated in wines at combinations of two different cell
densities (about 107 and 108 cfu ml−1 ), two pH values
(3.1 and 3.5), and two malic acid concentrations (3.5
and 7.0 g l−1 ).
Analytical methods. Samples were collected asepti-
cally through the fermenter sampling port, and bac-
terial growth was estimated by dry weight of cells
and following O.D. at 600 nm against non-inoculated
medium. For chemical analysis, supernatants from
centrifuged samples were filtered through a C-18 car-
tridge and then through a 0.22 µm membrane filter.
Sugars, organic acids and ethanol were quantified by
HPLC as described by Frayne (1986). External stan-
dards were used to quantify the required compounds.
ATP concentrations were determined as described by
Firme et al. (1994).
Results and discussion
Effect of medium composition (sugars) on biomass
yield. The fermentation of hexoses by heterofer-
mentative lactic acid bacteria theoretically produces
equimolecular amounts of lactate, carbon dioxide, and
acetate or ethanol (Kandler 1983). However, modifi-
cations in culture conditions or substrate composition
may result in variable stoichiometric data (Ragout
et al. 1994). When glucose was used as sole sub-
strate, growth was poor. Under these conditions, sugar
degradation was not detected and the pH was dropped
by less than 0.1 units. Fructose was completely me-
tabolized after ten days when it was the only carbon
source in the medium (Figure 1a). The degradation
was accomplished through two alternative pathways:
fifty seven percent of the fructose was reduced to
mannitol to reoxidize NAD(P)H, while the reminder
was degraded by the heterolactic pathway, essentially
yielding lactate and acetate, i.e. 1.4 mmol ATP/mmol
of sugar (Table 1). Additional redox power, sup-
plied by fructose reduction, allowed the production
of six times more biomass, molar growth yield Y =
5.61 mg mmol−1 of sugar, than did a glucose based
medium. Small amounts of ethanol and glycerol were
also detected, showing the non effective contribution
of their production pathways to NAD(P)H regener-
ation (Tables 1 and 2). Maximum biomass levels,
Y = 1.8 mg mmol−1 sugar, were achieved dur-
ing the co-metabolism of glucose and fructose. In a
batch culture without pH control, eight times more
biomass (180 µg mmol−1 sugar) occurred than with
glucose alone (data not shown). Fructose was almost
completely converted to mannitol while only 37% of
glucose was metabolized (Table 2). The simultaneous
consumption of both substrates led to increased rela-
tive yields of lactate, acetate and ethanol, compared to
yields in the reference culture (i.e. fructose alone) (Ta-
ble 1). After complete degradation of fructose, O. oeni
was not able to metabolize the remaining glucose,
which was due to a lack of oxidized cofactors. During
a co-fermentation of glucose and fructose, O. oeni may
use glucose by the heterolactic pathway, generating
energy (1.84 mmol ATP mmol−1 of sugar) and bio-
mass (Y = 6.89 mg/mmol of sugar), while fructose
can be reduced to mannitol by mannitol dehydroge-
nase. This provides cells of additional oxidized redox
power to that obtained from glucose culture alone (Sa-
lou et al. 1994) and explains the improved results
when both sugars were present. pH changes in the me-
dia were coupled with growth of O. oeni M42. When
pH was not controlled it decreased to 4.1 (fructose
alone) and 4.0 (cofermentation glucose and fructose)
and halted hexoses metabolism. These results indicate
the benefits to operate a fermenter under pH control to
improve the production of O. oeni starter biomass.

Fig. 1. Growth of Oenococcus oeni and carbohydrate fermentation kinetics in a culture with fructose alone or glucose plus fructose. (a) no pH

control; (b) under pH control. Dotted lines, dry weight; , fructose alone; , glucose-fructose mixture. Lines, substrate concentrations; , 
M #
fructose alone; , fructose (in a glucose-fructose mixture); , glucose (in a glucose-fructose mixture).
Effect of pH control on biomass yield. As described
earlier, growth was poor in glucose-based media, and
the pH decreased less than 0.1 units from the initial
value (4.8). Therefore, we did not perform a pH-
controlled batch with glucose alone, given that the
pH control activation threshold was over this value.
In a fructose based system, the results achieved when
the pH was not controlled (decrease of 0.7 units) and
in the pH controlled assay were similar (Figures 1a,
b). All the fructose in the medium was metabolized
in ten days with or without pH control. Under pH
control, increased lactate and acetate yield was de-
tected when compared to those in a non pH controlled
assay (Table 2). Our results suggest that in O. oeni
M42 the enzymes involved in lactic acid production
may be partially inhibited by a low pH. This has been
previously described by Ramos et al. (1995), who
showed that O. oeni lactate dehydrogenase activity de-
creased at a reduced pH. Acetate production was also
affected by pH but to a lesser extent. To elucidate the
effect of external pH in the lactate/acetate ratio we as-
sayed the degradation of fructose in tartrate phosphate
buffer at different initial values (ranging from 3.0 to
5.0). As the initial pH decreased, the lactate/acetate
ratio dropped from 0.81 to 0.68. These values, ob-
tained in a buffered system, were similar to those in
a synthetic medium, supporting the decrease of lactate
351
dehydrogenase activity as pH decreased. Acetate pro-
duction was also affected, but at a lesser extent. The
increase in production of lactate (20%) in a growth
medium when pH was controlled yielded 1.2-folds
ATP and consequently a 10% increase of biomass, up
to 160 µg/mmol of sugar. However, generation times
were both about 8.7 h−1 . These results are significa-
tively better (2–3 folds) than those previously reported
(Beelman et al. 1980, Naouri et al. 1989).
Co-fermentation of glucose and fructose revealed
greater pH dependence. In the incubation time, 30%
more glucose was degraded under pH control, yield-
ing higher quantities of final products. This led to an
increase in acetate production (ATP linked), producing
38% more biomass (220 µg mmol−1 of sugar).
These differences were possible because of the in-
creased metabolic activity of the cells when pH was
controlled (Ragout et al. 1994). While fructose was
completely metabolized in less than 30 h under pH
control, it took more than 50 h in its absence (Fig-
ures 1a, b). The kinetics of mannitol production was
coupled to fructose consumption under both condi-
tions. Glucose degradation, which is fructose depen-
dent, was also influenced by pH control (Figures 1a,
b). In any case, remaining quantities of glucose were
detected after stopping the experimentation due to the
restriction in oxidized cofactors (Table 2).
352

Table 1. Influence of sugar substrate and pH control on stoichiometrya of final products in growth of Oenococcus oeni M42. T = 28 ◦ C;
pH0 = 4.8; stirring = 250 rev/min.

Substrate (mmol l−1 ) Products (mmol l−1 )

Hexoseb Lactate Acetate Ethanol Glycerol Fructose converted
to mannitol (%)

Fructose. Without pH control 1 0.65 0.74 0.04 0.06 57

Fructose. With pH controlc 1 0.72 0.80 0.04 0.06 56
Glucose + Fructose. Without pH control 1 0.81 1.07 0.31 0.02 91
Glucose + Fructose. With pH controlc 1 0.95 1.13 0.56 0.07 99
a Results are means of determinations of duplicate cultures.
b Hexoses represents total degraded glucose plus degraded fructose not converted to mannitol.
c pH was controlled by addition of 5 M KOH and 5 M HCl.

Table 2. Effect of pH control and medium composition on substrate utilization and fermenta-
tion of Oenococcus oeni M42 in batch culture. T = 28 ◦ C; pH0 = 4.8; stirring = 250 rev/min.
Medium A: basal MLO with 55 mmol fructose l−1 ; medium B: basal MLO with glucose
(55 mmol glucose ml−1 ) plus fructose (30 mmol fructose ml−1 ).

Final concentration (mmol l−1 )a

Medium A (after 10 days) Medium B (after 3 days)
No pH control pH controlb No pH control pH controlb

Glucose (residual) 0.0 0.0 34.4 28.1

Fructose (residual) 1.4 0.4 0.5 0.2
Lactate 15.1 17.1 18.7 25.6
Acetate 17.5 18.5 24.9 30.6
Ethanol 0.9 0.9 7.3 15.1
Glycerol 1.4 1.5 0.5 1.9
Mannitol 30.5 30.8 26.8 29.6
a Results are means of determinations of duplicate cultures.
b pH was controlled by addition of 5 M KOH and 5 M HCl.

Table 3. Malolactic fermentation in red wine following inoculation with Oenococcus oeni M42
after 3 weeks of incubation

Viable bacteria in Malic acid concentration (g l−1 ) Malic acid pH

wine (cfu m−1 ) Initial Final degradation (%) Initial Final
after 3 weeks

1.2 × 107 3.5 2.3 34 3.10 3.18

1.1 × 107 7.0 5.9 16 3.10 3.12
1.2 × 107 3.5 1.0 71 3.50 3.57
1.3 × 107 7.0 4.2 40 3.50 3.57
8.2 × 107 3.5 0.8 77 3.10 3.20
9.4 × 107 7.0 3.9 44 3.10 3.17
1.1 × 108 3.5 0.1 97 3.50 3.64
9.6 × 107 7.0 1.0 86 3.50 3.74

Vinification assays. To determine the usefulness of
the starter cultures produced under optimal conditions,
several vinifications have been conducted, and two
malic acid concentrations were tested (3.5 and 7.0
g l−1 ) (Table 3). The rate of malolactic activity was
directly related to the number of bacteria added, and
when 1 × 108 cfu ml−1 were inoculated, the MLF
was generally completed in 1–3 days. MLF was devel-
oped in red wines within 2–3 weeks when inoculating
about 107 cfu ml−1 . The vinifications were assayed
at various pH values to assess the produced biomass
to degrade the malic acid present in wine. Best re-
sults were observed, as expected, at pH 3.5. However,
good degradation taxes were obtained at pH 3.1 by
using 1 × 108 cfu ml−1 . Thus, the inhibitory effect
of low pH (Bousbouras & Kunkee 1971, Davis et al.
1986) can be overcome by using high numbers of cells.
Reductions of about 60–90% of the initial concen-
trations of malic acid are here described (Table 3).
These results offer the possibility for developing the
malic acid degradation under unfavourable conditions
(low pH and high quantities of malic acid). Very few
approaches have been successfully reported to solve
these adverse situations for an effective MLF in wine
(Davis et al. 1986).
The data obtained in this study have shown that
differences in the sugar composition of the culture
medium influences growth kinetics and biomass yields
of O. oeni M42. We suggest the utilization of two
combined sugar substrates (glucose and fructose) in
culture media for O. oeni. This co-fermentation per-
mits production of up to eight times more biomass than
with the fermentation of one sugar alone (glucose).
The pH control, which avoids the acidification of the
culture medium, also gave a 38% increase in biomass
production. The utilization of this biomass to per-
form the MLF under several experimental conditions
produced improved results.
353
Acknowledgements
This work has been partially supported by grants
from the Comisión Interministerial de Ciencia y Tec-
nología (ALI93-0246) and by a grant from the M.E.C.
(Spanish Government) to S.M.
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