Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Anaerobic thermophilic fermentation for acetic acidproduction from milk permeate Myle`ne Talabardon *, Jean-Paul Schwitzgue´bel, Paul Pe´ringer Laboratory for En 6ironmental Biotechnolog

Anaerobic thermophilic fermentation for acetic acid

production from milk permeate Myle`ne Talabardon *, Jean-Paul Schwitzgue´bel, Paul Pe´ringer

Laboratory for En 6ironmental Biotechnology, Swiss Federal Institute of Technology of Lausanne(EPFL), Ecublens,

CH-1015Lausanne, 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

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: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 8 0 - 7

M Talabardon et al./Journal of Biotechnology76 (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

g l− 1of 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− 1were 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):

K2HPO4, 0.348 g; KH2PO4, 0.227 g; NH4Cl, 2.5

Summary of screening results for various thermophilic heterofermentative bacteria grown on milk permeate

Thermoanaero- Thermoanaerobacter ethanolicus

Thermoanaero-Species

drosulfuricus bacterium ther-bacter finnii

bacter brockii ssp

mosaccha-brockii

rolyticum

DSM 3389 T

Schmid et al.,

1986

1979 ences

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

mented (mmol

l −1 )

(°C)

4.80

Product yield

(mol mol −1 )

1.24 2.45

0.23

Acetate

4.7 1.46 a

0.24

H 2

tion products

detected by

HPLC but

not identified

weight in g

l −1 )

recovery b

Ratio mol

lac-tate/mol

ethanol

a

Calculated by carbon balance.

b

M Talabardon et al./Journal of Biotechnology76 (2000) 83 – 92

86

g; NaCl, 2.25 g; FeSO4· 7H2O, 0.002 g; yeast

extract (Difco), 2 g; resazurin, 0.001 g; trace

ele-ment solution, 1 ml The trace eleele-ment solution

SL-10 (see medium 320 described in the DSM

catalogue) contained (per liter in 0.077 mmol l− 1

HCl): FeCl2· 4H2O, 1.5 g; ZnCl2, 70 mg;

MnCl2· 4H2O, 100 mg; H3BO4, 6 mg;

CoCl2· 6H2O, 190 mg; CuCl2· 2H2O, 2 mg;

NiCl2· 6H2O, 24 mg; Na2MoO4· 2H2O, 36 mg

Each serum bottle (1 l) containing 250 ml of the

basal medium was flushed with 20% CO2/80% N2

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 (0.45mm pore size), to the following final

concentrations (per liter of basal medium): 0.5 g

MgSO4· 7H2O, 0.25 g CaCl2· 2H2O, 4.5 g

KHCO3, 0.3 g cysteine-HCl · H2O, 0.3 g

Na2S · 9H2O, 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 CO2 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 · 2H2O, 5; ri-boflavin, 5; nicotinic acid, 5; D-Ca-pantothenate, 5; vitamin B12, 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)

DSM 2030 T

1.40–1.46

Moorella thermoacetica DSM 2955 T –

totrophica

2.0–2.5

a No growth is indicated by –.

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, 20ml of filtrate

were injected onto an organic acid column

(Inter-action ORH-801) at 60°C Elution was done by

0.005 mmol l− 1sulfuric 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

H2and CO2were 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 CO2 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 (H2 or CO2) produced per unit volume of the

liquid medium (mol per liter) was then calculated

from the gas composition (%), total pressure (Pa)

and gas volume (m3) inside the bottle, and

temper-ature (K) as follows:

Cell density was monitored by measuring the

optical density at 650 nm (OD650) 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 CO2and H2to 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 produced1.4 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.

PercentageH2 or CO

2·total pressure [Pa] · volumegas[m

3] 8.31[J mol-1 K-1]·temperature [K]·

1 volumemedium[L]

M Talabardon et al./Journal of Biotechnology76 (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, 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

CO2using 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 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,

CO2 and H2 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), CO2 (5), H2 (5)

hel 6eticus; 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,

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− 1of 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).

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 Biotechnology76 (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 CO2 and H2

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, CO2 and H2 from lactose, although

lac-tate was the major fermentation product in

sev-eral strains However, these by-products,

including CO2 and H2, 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 CO2 and H2 released The production of

CO2 and H2 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, CO2 and H2 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

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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