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Journal of Biotechnology 76 (2000) 83 – 92

www.elsevier.com/locate/jbiotec

Anaerobic thermophilic fermentation for acetic acid


production from milk permeate
Mylène Talabardon *, Jean-Paul Schwitzguébel, Paul Péringer
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, CO2 and H2. The lactate yield was affected by cell growth. During the
exponential phase, acetate, ethanol, CO2 and H2 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

1. Introduction used for concentrating milk in several large cheese


producing plants (e.g. Feta cheese) as well as in
In Switzerland, cheese industry produces large manufacturing special milk products. This cheese-
amounts of lactose in the form of milk permeate making technology produces, instead of whey, a
or whey permeate. Ultrafiltration is frequently deproteinated permeate which needs further pro-
cessing. The permeate contains about 5% lactose,
* Corresponding author. Present address: Department of 1% salts, and 0.1–0.8% lactic acid; it is practically
Chemical Engineering, Ohio State University, 140 West 19th free of N-compounds and thus not comparable
Avenue, Columbus, OH 43210, USA. Fax: + 1-614-292-3769.
with whey which contains up to 0.8% protein
E-mail address: mylene.talabardon@epfl.ch (M. Talabar-
don) (Käppeli et al., 1981). Because of its lack of

0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 8 0 - 7
84 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92

protein, it is unsuitable for animal or human a thermophilic fermentation process could be


feeding. It has a high chemical oxygen demand of more interesting, since it has generally a higher
57 to 65 g l − 1 or even higher, depending on the production rate, should be more resistant to con-
cheese manufacturing process and is a major dis- tamination and more convenient to maintain
posal problem of overloading to sewage treatment anaerobic conditions required for acetogens.
plants. This lactose source, being directly fer- In this work, several potential ways for lactose
mentable by many bacteria and presently being a fermentation to acetic acid under anaerobic ther-
negative value waste stream on account of the mophilic conditions ( 60°C) were studied. Dif-
expensive wastewater treatment before discharge, ferent heterofermentative and acetogenic bacteria
could serve as an excellent feedstock for the pro- were evaluated for their potential use to produce
duction of acetic acid. acetate, and a co-culture of two bacteria, Clostrid-
Anaerobic acetogenesis conserves all the carbon ium thermolacticum and M. thermoautotrophica,
of glucose in the product acetic acid, thus increas- was found to give high acetate yield from lactose.
ing overall yield per glucose molecule by 50% over
the aerobic vinegar process (Busche, 1991). Acetic
acid production from glucose by Moorella ther- 2. Materials and methods
moacetica under thermophilic conditions appears
to be feasible (Shah and Cheryan, 1995). With 45 2.1. Microorganisms
g l − 1 of glucose in the feed of a fed-batch bioreac-
tor and a two-stage CSTR, the productivity and The heterofermentative and acetogenic bacteria
the concentration of acetic acid are 1.12 g used in this study are listed in Tables 1 and 2,
l − 1·h − 1 and 38 g l − 1, respectively. Although respectively. The freeze-dried strains were first
most thermophilic acetogens can convert glucose hydrated in a minimal volume of fresh culture
to acetate with a product yield as high as 90% medium in an anaerobic chamber, and then trans-
(Wiegel, 1994), there is no known thermophilic ferred anaerobically in serum bottles. Bacteria in
acetogen able to produce acetate from lactose spore phase (or in the exponential growth phase
directly. Bream (1988) has isolated a mutant of for non-sporulating species) were stored at 4°C
M. thermoacetica able to grow on lactate as the and used as stock cultures. The purity of cultures
only source of carbon and energy, whereas the was routinely checked under microscope (phase
parent strain consumes lactate only in the pres- contrast).
ence of a second fermentable substrate. With the The heterofermentative bacterium C. thermo-
mutant strain, it is possible to produce acetate lacticum DSM 2910 and the acetogenic bacterium
from lactose through lactate as an intermediary M. thermoautotrophica DSM 7417, used in this
fermentation step. study, were isolated from a mesophilic digester
Anaerobic fermentations to produce acetic acid fed with Lemna mina (France) by Le Ruyet et al.
from whey lactose have been studied under (1984), and from a pectin-limited culture of
mesophilic conditions. Tang et al. (1988) have Clostridium thermosaccharolyticum by van Rissjel
reported the use of Lactobacillus lactis and et al. (1992), respectively.
Clostridium formicoaceticum on sweet whey per-
meate. The former is a homolactic bacterium, 2.2. Culture media
which converts lactose to lactate, and the latter
can produce acetate from lactate. A new fermen- Each bacterial strain was cultivated in the
tation process has recently been developed by medium as specified in the DSM or ATCC cata-
Huang and Yang (1998) using this co-culture logues. Unless otherwise noted, the medium used
immobilized in a fibrous-bed bioreactor. Under in the fermentation study was prepared as follows.
fed-batch fermentation conditions, a final acetate The basal medium (see medium 326 in the DSM
concentration of 75 g l − 1 and an overall produc- catalogue) contained (per liter in deionized water):
tivity of 1.23 g l − 1·h − 1 were obtained. However, K2HPO4, 0.348 g; KH2PO4, 0.227 g; NH4Cl, 2.5
Table 1
Summary of screening results for various thermophilic heterofermentative bacteria grown on milk permeate

Species Clostridium thermolacticum Thermoanaero- Thermoanaerobacter ethanolicus Thermoanaero- Thermoanaerobacter thermohy- Thermoanaero-
bacter brockii ssp bacter finnii drosulfuricus bacterium ther-
brockii mosaccha-
rolyticum

Strain DSM 2910T DSM 2911T DSM 1457T DSM 2246T DSM 2355T DSM 3389T DSM 2247T DSM 567T DSM 571T

From the refer- Le Ruyet et al., 1984, 1985 Zeikus et al., Wiegel and Ljungdahl, 1981; Schmid et al., Wiegel et al., 1979; Hollaus and Wiegel, 1992
ences 1979 Zeikus et al., 1980 1986 Klaushofer, 1973
Temperature 50–70 (60–65) 50–70 (65) 35–85 (65–70) 37–78 (69) 40–75 (65) 37–78 (67–69) 35–67 (55)

M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92


range (°C)
(optimal tem-
perature)
pH range for 6.0–7.8 (7.0–7.2) 6.0–7.8 (7.2–7.4) 5.5–9.5 (7.5) 4.4–9.8 (5.8–8.5) (6.5–6.8) 5.5–9.2 (6.9–7.5) 7.0–8.5
growth (opti-
mal pH for
growth)
From the present study
Lactose fer- 13.04 15.02 25.57 31.42 29.74 20.02 42.65 35.38 56.50
mented (mmol
l−1)
Temperature 60 65 65 65 65 65 65 65 60
(°C)
Initial pH 7.68 7.42 7.40 7.57 7.28 7.41 7.52 7.77 7.70
Final pH 5.90 5.84 4.76 4.71 4.80 4.76 4.70 4.90 4.82
Product yield
(mol mol−1)
Lactate 2.45 2.02 1.14 0.96 1.24 2.38 0.69 0.79 0
Acetate 0.73 1.00 0.19 0.30 0.23 0.26 0.20 0.58 0.29
Ethanol 0.73 1.00 1.76 1.63 2.06 0.98 2.87 1.26 1.38
CO2 1.46a 2.00a 3.94 4.56 4.7 2.33 3.5 4.80 8.41
H2 1.46a 2.00a 0.53 0.27 0.24 0.59 0.34 3.67 7.44
Other fermenta- – – – – – – – – +
tion products
detected by
HPLC but
not identified
Biomass (by dry 0.40 0.43 0.98 0.88 1.31 0.61 1.87 0.97 0.64
weight in g
l−1)
% carbon 97.00 97.00 93.83 94.23 98.00 97.5 97.58 90.42 97.98
recoveryb
Ratio mol lac- 3.36 2.02 0.65 0.59 0.60 2.43 0.24 0.63 0
tate/mol
ethanol

85
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.
86 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92

g; NaCl, 2.25 g; FeSO4 · 7H2O, 0.002 g; yeast pyridoxin-HCl, 10; thiamine-HCl · 2H2O, 5; ri-
extract (Difco), 2 g; resazurin, 0.001 g; trace ele- boflavin, 5; nicotinic acid, 5; D-Ca-pantothenate,
ment solution, 1 ml. The trace element solution 5; vitamin B12, 0.1; p-aminobenzoic acid, 5; lipoic
SL-10 (see medium 320 described in the DSM acid, 5. The pH of the medium was adjusted to
catalogue) contained (per liter in 0.077 mmol l − 1 the desired value with a filter-sterilized NaOH or
HCl): FeCl2 · 4H2O, 1.5 g; ZnCl2, 70 mg; HCl solution.
MnCl2 · 4H2O, 100 mg; H3BO4, 6 mg; It is noted that the spores of thermophiles are
CoCl2 · 6H2O, 190 mg; CuCl2 · 2H2O, 2 mg; heat resistant and all medium bottles used in this
NiCl2 · 6H2O, 24 mg; Na2MoO4 · 2H2O, 36 mg. study were not mixed for different strains, which
Each serum bottle (1 l) containing 250 ml of the allowed us to use the less stringent sterilisation
basal medium was flushed with 20% CO2/80% N2 conditions without the risk of cross contamina-
gas to remove oxygen, then autoclaved at 121°C tion. However, for the stock cultures, media con-
for 20 min. After autoclaving, additional nutrients taining all components were autoclaved for 45
contained in a concentrated solution were added min at 121°C to ensure complete sterilisation. Any
to the basal medium, by passing through a mi- medium components that were heat labile were
crofilter (0.45 mm pore size), to the following final sterilised with a sterile 0.2 mm filter.
concentrations (per liter of basal medium): 0.5 g
MgSO4 · 7H2O, 0.25 g CaCl2 · 2H2O, 4.5 g 2.3. Batch culture fermentations
KHCO3, 0.3 g cysteine-HCl · H2O, 0.3 g
Na2S · 9H2O, 10 ml vitamin solution (see below), All batch fermentation studies were performed
and 20 g of a carbon source selected from lactose, in 1 l screw-capped serum bottles, with 250 ml of
milk permeate, glucose, galactose, or DL-sodium medium, and fitted with gas-impermeable black
lactate. The milk permeate was prepared from a butyl rubber septa under anaerobic and non-con-
frozen, concentrated sweet milk permeate contain- trolled pH conditions, in a constant temperature
ing 200 g l − 1 lactose (Cremo, Fribourg, Switzer- incubator (58°C, agitation speed: 100 rpm). Fif-
land), which was sterilized by ultrafiltration teen milliliters of a spore or cell (in the exponen-
(UFP-10-c-ss column, MM cutoff 10 000, A/G tial growth phase) suspension were added as
Technology, USA) and stored in a 250 l vat at inoculum to each serum bottle. For the spore
10°C under CO2 atmosphere. The vitamin solu- inoculum, a heat treatment (5 min at 105°C) was
tion (see medium 141 in the DSM catalogue) used to kill vegetative cells and to activate spores.
contained (in mg l − 1): biotin, 2; folic acid, 2; 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 5463T –a –


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

a
No growth is indicated by –.
M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92 87

syringes throughout the batch fermentation for calculated by dry weight. A calibration curve, OD
optical density (OD) reading, pH measurement, versus dry weight, was done for each strain.
and HPLC analysis.

2.4. Analytical techniques 3. Results

Lactose, glucose, galactose, lactate, acetate and 3.1. Screening


ethanol were identified and quantified by high-per-
formance liquid chromatography (HPLC). The Two major groups of bacteria, including hetero-
HPLC system consisted of a pump (Varian 9012), fermentative and acetogenic bacteria, that might be
an automatic injector (Varian 9100), and a differ- involved in the acetic acid fermentation were
ential refractometer at 45°C (ERC-7515, Erma screened (Tables 1 and 2). All anaerobic acetogens,
CR-INC). Samples were deproteinated, centrifuged carrying out a homoacetogenic fermentation, can
and finally filtered through a 0.2 mm membrane utilize glucose and CO2 and H2 to produce acetate,
filter to remove bacterial cells. Then, 20 ml of filtrate but none can grow on lactose. However, lactose can
were injected onto an organic acid column (Inter- be readily hydrolyzed to glucose and galactose by
action ORH-801) at 60°C. Elution was done by many fermentative bacteria or the b-galactosidase
0.005 mmol l − 1 sulfuric acid at a flow rate of 0.8 enzyme. Thus, 12 known thermophilic homoaceto-
ml min − 1. Calibration curves for standards of each gens were screened for their abilities to ferment
compound were done. The accuracy of this analysis galactose. Table 2 shows that only M. thermoau-
was higher than 95% with daily control of the totrophica DSM 1974 was able to use galactose
calibration. when this substrate was present as the only source
H2 and CO2 were determined using a type F20H of carbon and energy, producing  2.5 mol acetic
Perkin-Elmer gas chromatograph with thermal acid per mol of galactose consumed. However, this
conductivity detector and 2-m glass column con- bacterium fermented only glucose when both glu-
taining 5A molecular sieve (E. Merck, AG, Switzer- cose and galactose were present in the medium.
land). To analyze the gases solubilised in the culture Consequently, this bacterium was not suitable to
fluid, 1 ml sample was transferred to a 4.5 ml produce acetic acid from hydrolyzed milk perme-
stoppered serum bottle containing 1 ml concen- ate.
trated sulfuric acid to liberate CO2. After the bottle Among the 12 acetogens screened, two strains
had been shaken to equilibrate the gas phase with produce acetate from lactate. M thermoau-
the acidified sample, a 200 ml sample of the gas totrophica DSM 7417 and M. thermoacetica DSM
phase was analyzed as described above. The total 2955 produced  1.4 mol acetic acid per mol lactic
pressure inside the serum bottle was measured with acid consumed (0.93 g g − 1). The growth and
a digital pressure meter (Galaxy). The amount of degradation rates were very similar for both strains:
gas (H2 or CO2) produced per unit volume of the the pH range for cell growth was between 5.0 and
liquid medium (mol per liter) was then calculated 7.8, with an optimal pH at  6.5. The optimal
from the gas composition (%), total pressure (Pa) temperature was reported to be at 58°C, although
and gas volume (m3) inside the bottle, and temper- they can grow at a temperature as high as 68°C
ature (K) as follows: (Wiegel, 1992).

total pressure [Pa] · volumegas[m3] 1


PercentageH2 or CO2 · ·
8.31[J mol-1 K-1]·temperature [K] volumemedium[L]
Cell density was monitored by measuring the Meanwhile there are many mesophilic and
optical density at 650 nm (OD650) in a spectropho- thermo-tolerant homolactic bacteria that can con-
tometer (Hitachi, U-2000). Samples were diluted vert lactose to lactate, such as Lactobacillus bulgari-
when OD was greater than 0.5. The biomass was cus, Bifidobacterium thermophilum, L. lactis and L.
88 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92

ethanol, CO2 and H2. 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, H2 and
CO2 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, CO2 and H2. In such batch cultures, cell
growth stopped when only 18 mmol l − 1 of
Fig. 1. Batch culture of Clostridium thermolacticum DSM 2910 lactose had been consumed, probably because of
grown on lactose, at 58°C, initial pH 7.32, and 100 rpm an effect of pH on cell growth. Actually, pH was
agitation speed (with 6% inoculation in exponential phase). not controlled during fermentation, and the
medium pH dropped from an initial value of 7.32
hel6eticus; there is only one known thermophilic to  5.9 when cell growth stopped. During the
homolactic bacterium, Streptococcus (Lactobacil- exponential phase of growth, acetate, ethanol,
lus) thermophilus. However, the production of CO2 and H2 were produced, while lactate forma-
lactic acid from sugars by this bacterium at ther- tion was relatively small and was delayed. How-
mophilic temperatures (\ 50°C) is poor (Wiegel ever, neither ethanol nor acetate was produced
and Ljungdahl, 1986) because of its fastidious once cells reached the stationary phase, indicating
growth requirements. Thus, S. thermophilus is that their production was growth-associated. On
usually considered as unsuitable for thermophilic the other hand, more lactate was produced in the
production of lactic acid and has only been used stationary phase. The drop of pH generally coin-
at mesophilic temperatures (up to 45°C) in co-cul- cided with acids production. It was also obvious
ture with the mesophilic L. hel6eticus (Boyaval et that lactose was hydrolyzed to glucose and galac-
al., 1988). Therefore, various anaerobic ther- tose, which accumulated in the broth, when cell
mophilic strains, belonging to the saccharolytic or growth was low. Hydrolysis of lactose, continued
cellulolytic group of bacteria, were screened for even after the fermentation had stopped, possibly
their abilities to produce acetic acid from lactose catalyzed by the b-galactosidase enzyme released
present in milk permeate. Results are summarized during the sporulation. Based on the carbon bal-
in Table 1: all were obtained from batch fermen- ance calculation, about 97% of the lactose fer-
tation experiments without pH control or any mented was converted into the various
other attempt to optimize the fermentation condi- metabolites and only 3% was incorporated into
tions. Among these strains, there was no ther- cell biomass. The final product molar ratio in this
mophilic homolactic bacterium, and the main fermentation was approximately: lactate (1), ac-
fermentation products were lactate, acetate, etate (1), ethanol (1), CO2 (5), H2 (5).
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 CO2 and H2 (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 (CO2 and
H2) 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), CO2 (2), H2 (2). Because
of its content in phosphate ( 1.5 g l − 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).

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. 2. Batch culture of Clostridium thermolacticum DSM 2910 Fig. 3 shows a typical time course of batch
grown on milk permeate, at 58°C, initial pH 7.68, and 100 rpm fermentation of lactose by the co-culture of C.
agitation speed (with 6% inoculation in spore phase). thermolacticum DSM 2910 and M. thermoau-
90 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83–92

totrophica DSM 7417. As can be seen in this culture experiments without pH control, an ac-
figure, acetic acid was the major product, with a etate yield of 4.83 mol per mol lactose fermented
yield of 4.83 mol mol − 1 (0.81 g g − 1) lactose was obtained. The overall acetate yield from lac-
fermented, and ethanol was only produced at the tose can be further improved by optimizing the
beginning of the fermentation, with a final yield fermentation conditions (e.g. pH and medium
of 0.77 mol mol − 1. There was no lactic acid composition) that may affect cell growth and the
accumulation in the fermentation broth, suggest- fermentation pathway used in the heterofermen-
ing all lactate produced by the heterofermenta- tative bacterium.
tive bacterium was completely converted to As shown on Figs. 1 and 2, acetate and
acetate by the acetogen. Because the medium pH ethanol were produced from lactose by C. ther-
was not controlled and dropped below 6.0 in the molacticum only during the exponential phase of
medium, C. thermolacticum stopped its lactose growth, whereas the production of lactate oc-
consumption, as evidenced by the accumulation curred mainly in the stationary phase. Appar-
of glucose and galactose in the broth. However ently, there was a metabolic shift from
using pH-controlled fermentation should solve heterofermentative to homolactic pathway de-
this problem. It was noted that even after lactose pendent on the growth phase. The production of
and lactate had been completely utilized, there both acetate and ethanol was growth associated,
was continued production of acetate, which was with the same yield of 1–2 mol per mol lactose
attributed to the acetogenic growth of M. ther- fermented in the exponential phase. For each
moautotrophica on CO2 and H2. mol of acetate produced, there would be 2–5
mol of CO2 and H2 released. The production of
CO2 and H2 also seemed to stop soon after cells
4. Discussion entered the stationary phase, and only lactate
was thus produced from lactose, with a product
4.1. Ways for acetic acid fermentation from yield close to the theoretical maximum of 4 mol
lactose per mol lactose. It is thus concluded that the
heterofermentative bacterium, C. thermolacticum,
Numerous anaerobic bacteria produce acetate could perform homolactic acid fermentation
as one of the fermentation products. However, when its growth was limited and cells were in
there is no known strain able to produce acetate the stationary phase. Work is underway to opti-
as the only fermentation product directly from mize the conditions to favor lactate production
lactose. Thus, it is necessary to convert lactose from lactose by this bacterium.
to lactate and then to acetate using a mixed
culture consisting of two different groups of 4.2. Benefits of fermentation with co-culture
thermophilic anaerobic bacteria. All the screened
thermophilic bacteria also produced acetate, In the fermentation with a co-culture, interac-
ethanol, CO2 and H2 from lactose, although lac- tions between both bacterial species might have
tate was the major fermentation product in sev- also helped to shift the heterofermentative path-
eral strains. However, these by-products, way to favor the transient production of lactate
including CO2 and H2, from the heterofermenta- and the accumulation of acetate, instead of
tion can be readily converted to acetate by most ethanol, CO2 and H2. The yield of acetic acid
acetogens. In this work, the possibility to pro- observed in the co-culture, 0.81 g g − 1, was
duce acetic acid from milk permeate in anaero- higher than the yield obtained (0.73 g g − 1) when
bic thermophilic fermentation was demonstrated lactose was sequentially converted to lactic acid,
with a mixed culture of C. thermolacticum and then to acetic acid in two successive batch fer-
M. thermoautotrophica; the former for lactic acid mentations. As already seen in Fig. 3, lactate
production from milk permeate, the latter for served as a good intermediary product: all lac-
acetic acid production from lactic acid. In batch 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 Acknowledgements


should be noted that high concentrations of lac-
tate could inhibit both C. thermolacticum and The authors are grateful to Professor ST
M. thermoautotrophica. This double (product Yang (Department of Chemical Engineering,
and substrate) inhibition problem was avoided if Ohio State University, USA) for his suggestions
both bacteria were cultivated in the same vessel. and the revision of the paper. We thank Julia
Thus, it should be advantageous to use a one- Reichwald for her skillful assistance. This work
stage co-culture for acetate production from was supported by the Swiss Federal Office for
milk permeate. Education and Science, in the framework of
It is clear that more than 95% of lactose COST Action 818.
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- References
tion. To also convert ethanol to acetic acid
Beaty, P.S., Ljungdahl, L.G., 1991. Growth of Clostridium
would require a tri-culture to complete the fer- thermoaceticum on methanol, ethanol, or dimethylsulfox-
mentation. It is known that Moorella ther- ide. Annu. Meet. Am. Soc. Microbiol. Abstr. K-131, p.
moacetica ATCC 39073, although unable to 236.
couple the oxidation of ethanol to acetogenesis, Boyaval, P., Terre, S., Corre, C., 1988. Production d’acide
is competent in ethanol-dependent growth when lactique à partir de perméat de lactosérum par fermenta-
tion continue en réacteur à membrane. Le Lait 68, 65 – 84.
ethanol oxidation is coupled to the reduction of Bream, P., 1988. Fermentation of single and mixed substances
dimethylsulfoxide or thiosulfate (Beaty and by the parent and an acid-tolerant, mutant strain of
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