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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.
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
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)
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)
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.
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
Schmid, U., Giesel, H., Schoberth, S.M., Sahm, H., 1986. Acetogenesis. Chapman and Hall, New York, pp. 484 –
Thermoanaerobacter finnii spec. nov., a new ethanologenic 504.
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85. from soil and properties of the extreme thermophilic
Shah, M.M., Cheryan, M., 1995. Improvement of productivity Clostridium thermohydrosulfuricum. J. Bacteriol. 139, 800 –
in acetic acid fermentation with Clostridium ther- 810.
moaceticum. Appl. Biochem. Biotechnol. 51/52, 413–422. Wiegel, J., Ljungdahl, L.G., 1981. Thermoanaerobacter ethano-
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permeate in anaerobic thermophilic fermentation. Ph.D.
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Wiegel, J., Ljungdahl, L.G., 1986. The importance of ther-
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Tang, I.C., Yang, S.T., Okos, M.R., 1988. Acetic acid produc-
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