Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Journal of Biotechnology 76 (2000) 83–92
Anaerobic thermophilic fermentation for acetic acid
production from milk permeate
Myle`ne Talabardon *, Jean-Paul Schwitzgue´bel, Paul Pe´ringer
Laboratory for En6ironmental Biotechnology, Swiss Federal Institute of Technology of Lausanne
(
EPFL
)
, Ecublens,
CH-
1015
Lausanne, Switzerland
Received 27 January 1999; received in revised form 26 July 1999; accepted 30 July 1999
Abstract
Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions ( 60°C) was
studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one
strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. ther-
moacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of  0.93 g
g
−1
. Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium
thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also
produced significant amounts of ethanol, CO
2
and H
2
. The lactate yield was affected by cell growth. During the
exponential phase, acetate, ethanol, CO
2
and H
2

were the main products of fermentation with an equimolar
acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per
mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoau-
totrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid
concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose
was converted to ethanol by C. thermolacticum. © 2000 Elsevier Science B.V. All rights reserved.
Keywords
:
Screening; Clostridium; Moorella; Lactose; Heterofermentation; Acetogens
www.elsevier.com/locate/jbiotec
1. Introduction
In Switzerland, cheese industry produces large
amounts of lactose in the form of milk permeate
or whey permeate. Ultrafiltration is frequently
used for concentrating milk in several large cheese
producing plants (e.g. Feta cheese) as well as in
manufacturing special milk products. This cheese-
making technology produces, instead of whey, a
deproteinated permeate which needs further pro-
cessing. The permeate contains about 5% lactose,
1% salts, and 0.1–0.8% lactic acid; it is practically
free of N-compounds and thus not comparable
with whey which contains up to 0.8% protein
(Ka¨ppeli et al., 1981). Because of its lack of
* Corresponding author. Present address: Department of
Chemical Engineering, Ohio State University, 140 West 19th
Avenue, Columbus, OH 43210, USA. Fax: + 1-614-292-3769.
E-mail address
:
mylene.talabardon@epfl.ch (M. Talabar-

don)
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0168-1656(99)00180-7
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
84
protein, it is unsuitable for animal or human
feeding. It has a high chemical oxygen demand of
57 to 65 g l
−1
or even higher, depending on the
cheese manufacturing process and is a major dis-
posal problem of overloading to sewage treatment
plants. This lactose source, being directly fer-
mentable by many bacteria and presently being a
negative value waste stream on account of the
expensive wastewater treatment before discharge,
could serve as an excellent feedstock for the pro-
duction of acetic acid.
Anaerobic acetogenesis conserves all the carbon
of glucose in the product acetic acid, thus increas-
ing overall yield per glucose molecule by 50% over
the aerobic vinegar process (Busche, 1991). Acetic
acid production from glucose by Moorella ther-
moacetica under thermophilic conditions appears
to be feasible (Shah and Cheryan, 1995). With 45
gl
−1

of glucose in the feed of a fed-batch bioreac-
tor and a two-stage CSTR, the productivity and
the concentration of acetic acid are 1.12 g
l
−1
·h
−1
and 38 g l
−1
, respectively. Although
most thermophilic acetogens can convert glucose
to acetate with a product yield as high as  90%
(Wiegel, 1994), there is no known thermophilic
acetogen able to produce acetate from lactose
directly. Bream (1988) has isolated a mutant of
M. thermoacetica able to grow on lactate as the
only source of carbon and energy, whereas the
parent strain consumes lactate only in the pres-
ence of a second fermentable substrate. With the
mutant strain, it is possible to produce acetate
from lactose through lactate as an intermediary
fermentation step.
Anaerobic fermentations to produce acetic acid
from whey lactose have been studied under
mesophilic conditions. Tang et al. (1988) have
reported the use of Lactobacillus lactis and
Clostridium formicoaceticum on sweet whey per-
meate. The former is a homolactic bacterium,
which converts lactose to lactate, and the latter
can produce acetate from lactate. A new fermen-

tation process has recently been developed by
Huang and Yang (1998) using this co-culture
immobilized in a fibrous-bed bioreactor. Under
fed-batch fermentation conditions, a final acetate
concentration of 75 g l
−1
and an overall produc-
tivity of 1.23 g l
−1
·h
−1
were obtained. However,
a thermophilic fermentation process could be
more interesting, since it has generally a higher
production rate, should be more resistant to con-
tamination and more convenient to maintain
anaerobic conditions required for acetogens.
In this work, several potential ways for lactose
fermentation to acetic acid under anaerobic ther-
mophilic conditions ( 60°C) were studied. Dif-
ferent heterofermentative and acetogenic bacteria
were evaluated for their potential use to produce
acetate, and a co-culture of two bacteria, Clostrid-
ium thermolacticum and M. thermoautotrophica,
was found to give high acetate yield from lactose.
2. Materials and methods
2
.
1
. Microorganisms

The heterofermentative and acetogenic bacteria
used in this study are listed in Tables 1 and 2,
respectively. The freeze-dried strains were first
hydrated in a minimal volume of fresh culture
medium in an anaerobic chamber, and then trans-
ferred anaerobically in serum bottles. Bacteria in
spore phase (or in the exponential growth phase
for non-sporulating species) were stored at 4°C
and used as stock cultures. The purity of cultures
was routinely checked under microscope (phase
contrast).
The heterofermentative bacterium C. thermo-
lacticum DSM 2910 and the acetogenic bacterium
M. thermoautotrophica DSM 7417, used in this
study, were isolated from a mesophilic digester
fed with Lemna mina (France) by Le Ruyet et al.
(1984), and from a pectin-limited culture of
Clostridium thermosaccharolyticum by van Rissjel
et al. (1992), respectively.
2
.
2
. Culture media
Each bacterial strain was cultivated in the
medium as specified in the DSM or ATCC cata-
logues. Unless otherwise noted, the medium used
in the fermentation study was prepared as follows.
The basal medium (see medium 326 in the DSM
catalogue) contained (per liter in deionized water):
K

2
HPO
4
, 0.348 g; KH
2
PO
4
, 0.227 g; NH
4
Cl, 2.5
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
85
Table 1
Summary of screening results for various thermophilic heterofermentative bacteria grown on milk permeate
Thermoanaero- Thermoanaerobacter ethanolicus Thermoanaero-Clostridium thermolacticum Thermoanaerobacter thermohy- Thermoanaero-Species
drosulfuricus bacterium ther-bacter finniibacter brockii ssp
mosaccha-brockii
rolyticum
DSM 3389
T
Strain DSM 2247
T
DSM 2910
T
DSM 567
T
DSM 571

T
DSM 2911
T
DSM 1457
T
DSM 2246
T
DSM 2355
T
Schmid et al.,Le Ruyet et al., 1984, 1985 Wiegel et al., 1979; Hollaus andFrom the refer- Wiegel and Ljungdahl, 1981; Wiegel, 1992Zeikus et al.,
1986Zeikus et al., 1980 Klaushofer, 19731979ences
Temperature 50–70 (65) 35–85 (65–70) 37–78 (69) 40–75 (65) 37–78 (67–69) 35–67 (55)50–70 (60–65)
range (°C)
(optimal tem-
perature)
pH range for 5.5–9.5 (7.5)6.0–7.8 (7.0–7.2) 4.4–9.8 (5.8–8.5) (6.5–6.8) 5.5–9.2 (6.9–7.5) 7.0–8.56.0–7.8 (7.2–7.4)
growth (opti-
mal pH for
growth)
From the present study
25.57 31.42 29.74 20.02 42.6515.02 35.38Lactose fer- 56.5013.04
mented (mmol
l
−1
)
Temperature 65 65 65 65 65 606560 65
(°C)
Initial pH 7.417.68 7.52 7.77 7.707.42 7.40 7.57 7.28
4.76 4.70 4.90 4.824.805.90 4.71Final pH 4.765.84
Product yield

(mol mol
−1
)
2.38 0.69 0.79 01.242.45Lactate 0.961.142.02
0.230.73 0.26 0.20 0.58 0.291.00 0.19 0.30Acetate
0.98 2.87 1.26 1.38Ethanol 0.73 1.00 1.76 1.63 2.06
2.33 3.5 4.80 8.414.71.46
a
CO
2
4.563.942.00
a
0.241.46
a
0.59 0.34 3.67 7.442.00
a
0.53 0.27H
2
Other fermenta- ––––––––+
tion products
detected by
HPLC but
not identified
0.88 1.31 0.610.40 1.87Biomass (by dry 0.97 0.640.43 0.98
weight in g
l
−1
)
98.00 97.5 97.58 90.42 97.98% carbon 97.00 97.00 93.83 94.23
recovery

b
0.59 0.60 2.43 0.24 0.632.02 03.36 0.65Ratio mol lac-
tate/mol
ethanol
a
Calculated by carbon balance.
b
The percentage of carbon recovery was calculated as the ratio: total carbon present in all fermentation products/total carbon in fermented carbon sources.
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
86
g; NaCl, 2.25 g; FeSO
4
·7H
2
O, 0.002 g; yeast
extract (Difco), 2 g; resazurin, 0.001 g; trace ele-
ment solution, 1 ml. The trace element solution
SL-10 (see medium 320 described in the DSM
catalogue) contained (per liter in 0.077 mmol l
−1
HCl): FeCl
2
·4H
2
O, 1.5 g; ZnCl
2
,70mg;

MnCl
2
·4H
2
O, 100 mg; H
3
BO
4
, 6 mg;
CoCl
2
·6H
2
O, 190 mg; CuCl
2
·2H
2
O, 2 mg;
NiCl
2
·6H
2
O, 24 mg; Na
2
MoO
4
·2H
2
O, 36 mg.
Each serum bottle (1 l) containing 250 ml of the

basal medium was flushed with 20% CO
2
/80% N
2
gas to remove oxygen, then autoclaved at 121°C
for 20 min. After autoclaving, additional nutrients
contained in a concentrated solution were added
to the basal medium, by passing through a mi-
crofilter (0.45 mm pore size), to the following final
concentrations (per liter of basal medium): 0.5 g
MgSO
4
·7H
2
O, 0.25 g CaCl
2
·2H
2
O, 4.5 g
KHCO
3
, 0.3 g cysteine-HCl · H
2
O, 0.3 g
Na
2
S·9H
2
O, 10 ml vitamin solution (see below),
and 20 g of a carbon source selected from lactose,

milk permeate, glucose, galactose, or DL-sodium
lactate. The milk permeate was prepared from a
frozen, concentrated sweet milk permeate contain-
ing 200 g l
−1
lactose (Cremo, Fribourg, Switzer-
land), which was sterilized by ultrafiltration
(UFP-10-c-ss column, MM cutoff 10 000, A/G
Technology, USA) and stored in a 250 l vat at
10°C under CO
2
atmosphere. The vitamin solu-
tion (see medium 141 in the DSM catalogue)
contained (in mg l
−1
): biotin, 2; folic acid, 2;
pyridoxin-HCl, 10; thiamine-HCl · 2H
2
O, 5; ri-
boflavin, 5; nicotinic acid, 5; D-Ca-pantothenate,
5; vitamin B
12
, 0.1; p-aminobenzoic acid, 5; lipoic
acid, 5. The pH of the medium was adjusted to
the desired value with a filter-sterilized NaOH or
HCl solution.
It is noted that the spores of thermophiles are
heat resistant and all medium bottles used in this
study were not mixed for different strains, which
allowed us to use the less stringent sterilisation

conditions without the risk of cross contamina-
tion. However, for the stock cultures, media con-
taining all components were autoclaved for 45
min at 121°C to ensure complete sterilisation. Any
medium components that were heat labile were
sterilised with a sterile 0.2 mm filter.
2
.
3
. Batch culture fermentations
All batch fermentation studies were performed
in 1 l screw-capped serum bottles, with 250 ml of
medium, and fitted with gas-impermeable black
butyl rubber septa under anaerobic and non-con-
trolled pH conditions, in a constant temperature
incubator (58°C, agitation speed: 100 rpm). Fif-
teen milliliters of a spore or cell (in the exponen-
tial growth phase) suspension were added as
inoculum to each serum bottle. For the spore
inoculum, a heat treatment (5 min at 105°C) was
used to kill vegetative cells and to activate spores.
Liquid samples (5 ml each) were taken with sterile
Table 2
Screening results of various thermophilic acetogens cultivated in media containing galactose or lactate as sole carbon source
Species Strain Acetate yield from galactose (mol/mol) Acetate yield from lactate (mol/mol)
Calorimator fer6idus DSM 5463
T
–
a
–

DSM 2030
T
Acetogenium ki6ui ––
Acetomicrobium fla6idum DSM 20664
T
––
1.40–1.46Moorella thermoacetica –DSM 2955
T
DSM 521
T
––
DSM 6867
T
––
DSM 39073
T
––
ATCC 34490
T
––
ATCC 39289
T
––
Moorella thermoau-–DSM 7417
T
1.38–1.46
totrophica
2.0–2.5DSM 1974
T
–

a
No growth is indicated by –.
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
87
syringes throughout the batch fermentation for
optical density (OD) reading, pH measurement,
and HPLC analysis.
2
.
4
. Analytical techniques
Lactose, glucose, galactose, lactate, acetate and
ethanol were identified and quantified by high-per-
formance liquid chromatography (HPLC). The
HPLC system consisted of a pump (Varian 9012),
an automatic injector (Varian 9100), and a differ-
ential refractometer at 45°C (ERC-7515, Erma
CR-INC). Samples were deproteinated, centrifuged
and finally filtered through a 0.2 mm membrane
filter to remove bacterial cells. Then, 20 ml of filtrate
were injected onto an organic acid column (Inter-
action ORH-801) at 60°C. Elution was done by
0.005 mmol l
−1
sulfuric acid at a flow rate of 0.8
ml min
−1

. Calibration curves for standards of each
compound were done. The accuracy of this analysis
was higher than 95% with daily control of the
calibration.
H
2
and CO
2
were determined using a type F20H
Perkin-Elmer gas chromatograph with thermal
conductivity detector and 2-m glass column con-
taining 5A molecular sieve (E. Merck, AG, Switzer-
land). To analyze the gases solubilised in the culture
fluid, 1 ml sample was transferred to a 4.5 ml
stoppered serum bottle containing 1 ml concen-
trated sulfuric acid to liberate CO
2
. After the bottle
had been shaken to equilibrate the gas phase with
the acidified sample, a 200 ml sample of the gas
phase was analyzed as described above. The total
pressure inside the serum bottle was measured with
a digital pressure meter (Galaxy). The amount of
gas (H
2
or CO
2
) produced per unit volume of the
liquid medium (mol per liter) was then calculated
from the gas composition (%), total pressure (Pa)

and gas volume (m
3
) inside the bottle, and temper-
ature (K) as follows:
Cell density was monitored by measuring the
optical density at 650 nm (OD
650
) in a spectropho-
tometer (Hitachi, U-2000). Samples were diluted
when OD was greater than 0.5. The biomass was
calculated by dry weight. A calibration curve, OD
versus dry weight, was done for each strain.
3. Results
3
.
1
. Screening
Two major groups of bacteria, including hetero-
fermentative and acetogenic bacteria, that might be
involved in the acetic acid fermentation were
screened (Tables 1 and 2). All anaerobic acetogens,
carrying out a homoacetogenic fermentation, can
utilize glucose and CO
2
and H
2
to produce acetate,
but none can grow on lactose. However, lactose can
be readily hydrolyzed to glucose and galactose by
many fermentative bacteria or the b-galactosidase

enzyme. Thus, 12 known thermophilic homoaceto-
gens were screened for their abilities to ferment
galactose. Table 2 shows that only M. thermoau-
totrophica DSM 1974 was able to use galactose
when this substrate was present as the only source
of carbon and energy, producing 2.5 mol acetic
acid per mol of galactose consumed. However, this
bacterium fermented only glucose when both glu-
cose and galactose were present in the medium.
Consequently, this bacterium was not suitable to
produce acetic acid from hydrolyzed milk perme-
ate.
Among the 12 acetogens screened, two strains
produce acetate from lactate. M thermoau-
totrophica DSM 7417 and M. thermoacetica DSM
2955 produced  1.4 mol acetic acid per mol lactic
acid consumed (0.93 g g
−1
). The growth and
degradation rates were very similar for both strains:
the pH range for cell growth was between 5.0 and
7.8, with an optimal pH at  6.5. The optimal
temperature was reported to be at 58°C, although
they can grow at a temperature as high as 68°C
(Wiegel, 1992).
Meanwhile there are many mesophilic and
thermo-tolerant homolactic bacteria that can con-
vert lactose to lactate, such as Lactobacillus bulgari-
cus, Bifidobacterium thermophilum, L. lactis and L.
Percentage

H
2
or CO
2
·
total pressure [Pa] · volume
gas
[m
3
]
8.31[J mol
-1
K
-1
]·temperature [K]
·
1
volume
medium
[L]
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
88
Fig. 1. Batch culture of Clostridium thermolacticum DSM 2910
grown on lactose, at 58°C, initial pH 7.32, and 100 rpm
agitation speed (with 6% inoculation in exponential phase).
ethanol, CO
2

and H
2
. As can be seen in Table 1,
Clostridium thermolacticum DSM 2910 appears to
be an appropriate strain for lactate production on
account of its high lactate yield (2.45 mol per mol
lactose fermented) and high lactate/ethanol ratio
(3.36 mol mol
−1
). This bacterium had an optimal
growth temperature at 60°C and growth pH
range between 6.0 and 7.8.
3
.
2
. Fermentation of C. thermolacticum on lactose
and milk permeate
To further evaluate the fermentation way to
produce acetate from lactose via lactate, H
2
and
CO
2
using heterofermentative and acetogenic bac-
teria, detailed fermentation kinetics were studied
and the results are reported here.
Fig. 1 shows typical kinetics of fermentation of
C. thermolacticum grown on lactose. Products
from this fermentation included lactate, acetate,
ethanol, CO

2
and H
2
. In such batch cultures, cell
growth stopped when only  18 mmol l
−1
of
lactose had been consumed, probably because of
an effect of pH on cell growth. Actually, pH was
not controlled during fermentation, and the
medium pH dropped from an initial value of 7.32
to  5.9 when cell growth stopped. During the
exponential phase of growth, acetate, ethanol,
CO
2
and H
2
were produced, while lactate forma-
tion was relatively small and was delayed. How-
ever, neither ethanol nor acetate was produced
once cells reached the stationary phase, indicating
that their production was growth-associated. On
the other hand, more lactate was produced in the
stationary phase. The drop of pH generally coin-
cided with acids production. It was also obvious
that lactose was hydrolyzed to glucose and galac-
tose, which accumulated in the broth, when cell
growth was low. Hydrolysis of lactose, continued
even after the fermentation had stopped, possibly
catalyzed by the b-galactosidase enzyme released

during the sporulation. Based on the carbon bal-
ance calculation, about 97% of the lactose fer-
mented was converted into the various
metabolites and only  3% was incorporated into
cell biomass. The final product molar ratio in this
fermentation was approximately: lactate (1), ac-
etate (1), ethanol (1), CO
2
(5), H
2
(5).
hel6eticus; there is only one known thermophilic
homolactic bacterium, Streptococcus (Lactobacil-
lus) thermophilus. However, the production of
lactic acid from sugars by this bacterium at ther-
mophilic temperatures (\ 50°C) is poor (Wiegel
and Ljungdahl, 1986) because of its fastidious
growth requirements. Thus, S. thermophilus is
usually considered as unsuitable for thermophilic
production of lactic acid and has only been used
at mesophilic temperatures (up to 45°C) in co-cul-
ture with the mesophilic L. hel6eticus (Boyaval et
al., 1988). Therefore, various anaerobic ther-
mophilic strains, belonging to the saccharolytic or
cellulolytic group of bacteria, were screened for
their abilities to produce acetic acid from lactose
present in milk permeate. Results are summarized
in Table 1: all were obtained from batch fermen-
tation experiments without pH control or any
other attempt to optimize the fermentation condi-

tions. Among these strains, there was no ther-
mophilic homolactic bacterium, and the main
fermentation products were lactate, acetate,
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
89
The pattern of growth and fermentation in a
medium containing 25 mmol l
−1
lactose from
milk permeate is shown in Fig. 2. Products from
this fermentation included lactate, acetate,
ethanol, and CO
2
and H
2
(not shown). In this
batch culture, the fermentation almost stopped
when 13 mmol l
−1
of lactose had been consumed.
Similar to the previous fermentation with lactose
as the substrate, acetate and ethanol were only
produced during the exponential growth phase,
and lactate formation was delayed in the growth
phase but continued to the stationary phase.
However, more lactate and less gases (CO
2

and
H
2
) were produced in this batch as compared to
the previous one (Fig. 1). The final product molar
ratio in this batch was approximately: lactate (3),
acetate (1), ethanol (1), CO
2
(2), H
2
(2). Because
of its content in phosphate ( 1.5gl
−1
), milk
permeate has a higher buffer capacity than the
basic medium used in the previous experiment.
Therefore, the decrease of pH was lower and
slower under such growth conditions.
Fig. 3. Batch co-culture of Clostridium thermolacticum DSM
2910 and Moorella thermoautotrophica DSM 7417 grown on
milk permeate at 58°C, initial pH 7.2 with 40 mM MOPS
(buffer), and 100 rpm agitation speed (with 6% inoculation in
spore phase for each species).
Fig. 2. Batch culture of Clostridium thermolacticum DSM 2910
grown on milk permeate, at 58°C, initial pH 7.68, and 100 rpm
agitation speed (with 6% inoculation in spore phase).
3
.
3
. Acetogenic fermentation of M.

thermoautotrophica on lactate
M. thermoautotrophica DSM 7417 homofer-
mentatively converted lactate to acetate at ther-
mophilic temperature (50–65°C) and at pH
between 5.8 and 7.7 (not shown). Approximately
0.93 g of acetic acid was formed from each gram
of lactic acid. The bacterium grew at an optimal
pH of 6.35–6.85 and an optimal temperature of
58°C. This bacterium was thus chosen for use in a
fermentation with a mixed culture to produce
acetic acid from milk permeate.
3
.
4
. Fermentation of the co-culture of C.
thermolacticum and M. thermoautotrophica on
milk permeate
Fig. 3 shows a typical time course of batch
fermentation of lactose by the co-culture of C.
thermolacticum DSM 2910 and M. thermoau-
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
90
totrophica DSM 7417. As can be seen in this
figure, acetic acid was the major product, with a
yield of 4.83 mol mol
−1
(0.81 g g

−1
) lactose
fermented, and ethanol was only produced at the
beginning of the fermentation, with a final yield
of 0.77 mol mol
−1
. There was no lactic acid
accumulation in the fermentation broth, suggest-
ing all lactate produced by the heterofermenta-
tive bacterium was completely converted to
acetate by the acetogen. Because the medium pH
was not controlled and dropped below 6.0 in the
medium, C. thermolacticum stopped its lactose
consumption, as evidenced by the accumulation
of glucose and galactose in the broth. However
using pH-controlled fermentation should solve
this problem. It was noted that even after lactose
and lactate had been completely utilized, there
was continued production of acetate, which was
attributed to the acetogenic growth of M. ther-
moautotrophica on CO
2
and H
2
.
4. Discussion
4
.
1
. Ways for acetic acid fermentation from

lactose
Numerous anaerobic bacteria produce acetate
as one of the fermentation products. However,
there is no known strain able to produce acetate
as the only fermentation product directly from
lactose. Thus, it is necessary to convert lactose
to lactate and then to acetate using a mixed
culture consisting of two different groups of
thermophilic anaerobic bacteria. All the screened
thermophilic bacteria also produced acetate,
ethanol, CO
2
and H
2
from lactose, although lac-
tate was the major fermentation product in sev-
eral strains. However, these by-products,
including CO
2
and H
2
, from the heterofermenta-
tion can be readily converted to acetate by most
acetogens. In this work, the possibility to pro-
duce acetic acid from milk permeate in anaero-
bic thermophilic fermentation was demonstrated
with a mixed culture of C. thermolacticum and
M. thermoautotrophica
;
the former for lactic acid

production from milk permeate, the latter for
acetic acid production from lactic acid. In batch
culture experiments without pH control, an ac-
etate yield of 4.83 mol per mol lactose fermented
was obtained. The overall acetate yield from lac-
tose can be further improved by optimizing the
fermentation conditions (e.g. pH and medium
composition) that may affect cell growth and the
fermentation pathway used in the heterofermen-
tative bacterium.
As shown on Figs. 1 and 2, acetate and
ethanol were produced from lactose by C. ther-
molacticum only during the exponential phase of
growth, whereas the production of lactate oc-
curred mainly in the stationary phase. Appar-
ently, there was a metabolic shift from
heterofermentative to homolactic pathway de-
pendent on the growth phase. The production of
both acetate and ethanol was growth associated,
with the same yield of 1– 2 mol per mol lactose
fermented in the exponential phase. For each
mol of acetate produced, there would be 2 –5
mol of CO
2
and H
2
released. The production of
CO
2
and H

2
also seemed to stop soon after cells
entered the stationary phase, and only lactate
was thus produced from lactose, with a product
yield close to the theoretical maximum of 4 mol
per mol lactose. It is thus concluded that the
heterofermentative bacterium, C. thermolacticum,
could perform homolactic acid fermentation
when its growth was limited and cells were in
the stationary phase. Work is underway to opti-
mize the conditions to favor lactate production
from lactose by this bacterium.
4
.
2
. Benefits of fermentation with co-culture
In the fermentation with a co-culture, interac-
tions between both bacterial species might have
also helped to shift the heterofermentative path-
way to favor the transient production of lactate
and the accumulation of acetate, instead of
ethanol, CO
2
and H
2
. The yield of acetic acid
observed in the co-culture, 0.81 g g
−1
, was
higher than the yield obtained (0.73 g g

−1
) when
lactose was sequentially converted to lactic acid,
then to acetic acid in two successive batch fer-
mentations. As already seen in Fig. 3, lactate
served as a good intermediary product: all lac-
tate produced from the first bacterium was
M. Talabardon et al.
/
Journal of Biotechnology
76 (2000) 83 – 92
91
timely converted to acetate by the acetogen. It
should be noted that high concentrations of lac-
tate could inhibit both C. thermolacticum and
M. thermoautotrophica. This double (product
and substrate) inhibition problem was avoided if
both bacteria were cultivated in the same vessel.
Thus, it should be advantageous to use a one-
stage co-culture for acetate production from
milk permeate.
It is clear that more than 95% of lactose
could be converted to acetic acid in this co-cul-
ture if the ethanol produced could also be con-
verted to acetic acid or if the heterofermentation
could be shifted to homolactic acid fermenta-
tion. To also convert ethanol to acetic acid
would require a tri-culture to complete the fer-
mentation. It is known that Moorella ther-
moacetica ATCC 39073, although unable to

couple the oxidation of ethanol to acetogenesis,
is competent in ethanol-dependent growth when
ethanol oxidation is coupled to the reduction of
dimethylsulfoxide or thiosulfate (Beaty and
Ljungdahl, 1991). This possibility, however, re-
mains to be tested. It is thus better and simpler
to shift the heterofermentation to homolactic
fermentation by controlling the fermentation
conditions and growth phases, as demonstrated
in this study. Furthermore, it is possible to use
immobilized cell fermentation to reduce cell
growth and increase product yields (Huang and
Yang, 1998). Better acetic acid production from
lactose can be obtained if the co-culture of C.
thermolacticum and M. thermoautotrophica are
maintained in the stationary phase by immobi-
lizing the cells in a fibrous-bed bioreactor (Tal-
abardon, 1999). As already discussed before,
when the heterofermentative bacteria were in the
non-growing state or stationary phase, the het-
erofermentative pathway shifted to the homolac-
tic acid pathway and only lactate was produced
from lactose with a nearly 100% yield. It is thus
possible to produce acetate from lactose with a
yield higher than 95% by using this thermophilic
co-culture. Immobilized cell fermentations also
give higher productivity and higher final product
concentration (Huang and Yang, 1998), and
thus should be the choice for thermophilic pro-
duction of acetate from milk permeate.

Acknowledgements
The authors are grateful to Professor ST
Yang (Department of Chemical Engineering,
Ohio State University, USA) for his suggestions
and the revision of the paper. We thank Julia
Reichwald for her skillful assistance. This work
was supported by the Swiss Federal Office for
Education and Science, in the framework of
COST Action 818.
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