Acetic Acid Batch Fermentation Process by
Clostridium Thermoaceticum Harumi Veny, Masitah Hasan
,
Department of Chemical Engineering Faculty of Engineering
University of Malaya ABSTRACT

Acetic acid was produced through
anaerobic batch fermentation process by using
Clostridium thermoaceticum
as microorganism.
The kinetic parameters (i.e.
µ
max
, Y
p/s,
Y
x/s
)

and

the relationship between growth and acetic acid
product formation were determined for all
environmental effects studied in batch
fermentation to establish the optimum condition

carbonylation of methanol and the rest by the
liquid phase oxidation on n-butane and direct
oxidation of acetaldehyde (Wacker process) [1]
The major disadvantages of the synthetic process
are the need of high temperature and pressure, a good
agitation, the threat of explosion, the high cost of
catalyst, and the dependence on non-renewable sources
of raw material (crude oil). Hence, acetic acid
productions to be carried out by biological
(fermentation) mean become more attractive. The
production of acetic acid via fermentation by using
renewable biomass feedstock has also been studied as
alternatives to acetic acid production from petroleum or
natural gas feedstock [2]. The fermentation process
could reduce the high cost since it uses renewable
resource as raw material and low energy utilization. The
problem with the conventional fermentation process is
that the two step aerobic fermentation could only
produce low acetic acid product yield (around 0.67 g
acetic acid/g glucose). This process also involved two
stages for converting glucose into ethanol followed by
oxidation of the ethanol to acetic acid. Hence, it needs
two separate fermentors. A possible solution to this
problem is a single step anaerobic fermentation which
is described in this paper.
The single step anaerobic process requires the
absence of oxygen; hence the condition during all
fermentations should be strictly anaerobic by gassing
with CO
2

-1
to 0.14 h
-1
in batch
fermentation system.

(2) MATERIAL AND METHODS

Microorganism

Clostridium thermoaceticum bacteria from
DSMZ, Germany was used in this experiment.

Media and growth condition

RCM and a modification of DSMZ
recommended medium.
RCM medium is used for maintenance and stock
cultures in solid and semi solid form, which then
is sub cultured for every 2 weeks. The RCM
composition is: Yeast extract (3 g/l), Lab–Lemco
Powder (10 g/l), Peptone (10 g/l), Glucose ( 5
g/l), Soluble starch (1 g/l), Sodium Chloride
(5g/l), Sodium acetate (3 g/l), Cysteine
hydrochloride (0.5 g/l), Agar (0.5 g/l) or (15 g/l
for solid medium).
Medium for cultivation of Clostridium
thermoaceticum is a modification of some
medium compositions from earlier researches [4,
5,10]. The compositions are:

O (0.03 g),
Na
2
WO
4
. 2H
2
O (3.30 mg), Na
2
MoO
4
. 2H
2
O
(2.4 mg), ZnCl
2
(1.4 mg),Na
2
SeO
3
. 5H
2
O(0.2
mg), NiCl
2
. 6H
2
O (0.2 mg), Resazurin 0.1% (1
ml), Distilled water (550 ml), Cysteine
hydrochloride (0.25 g), Na

Detail analytical methods have been previously
described[3].

Batch fermentation studies

The batch fermentation studies were carried out in
2.5 L KO biotech fermentor with a working volume of
1.5 L. The inoculums for all batch fermentation runs
were incubated for 48 hours at 60
o
C. The anaerobic
condition for fermentation process was maintained by
continuous supply of sterile CO
2
gas. The pH was
maintained by automatic addition of 2N KOH and 2N
HCl. The foam formed during the process was reduced
by automatic addition of antifoam agent and with the
help of foam breaker in the stirrer. The stirrer rate for
all batch fermentation was maintained at a low rate of
50 rpm.

(3) RESULTS AND DISCUSSIONS

The environmental effects on batch fermentations
were studied by varying the pH (i.e. pH 5 to 7) and
initial glucose concentration (15 to 60 g/L). The
environmental effect studies achieved the maximum
acetic acid yield of 0.88 g/g and growth yield of 0.1 g/g
at pH 6 and 60

d
t
dx
.
µ
=

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
45678
pH
0
0.02
0.04
0.06
0.08
0.1
0.12
Product Yield
Growth yield


(g.g
-1
)
Y
p/s

(g.g
-1
)
15 g/L 60
o
C 6 0.082 0.108 0.88
30 g/L 60
o
C 6 0.11 0.11 0.815

The batch fermentation profile at optimum
condition of pH 6 and 60
o
C from this study is given in
Figure 3. This fermentation profile showed that the
acetic acid concentration increased with the increase in
biomass/dry cell. However, the acetic acid still
increased even when the dry cell had already ceased to
grow. This phenomenon illustrates a mixed growths
associated which also have been reported previously in
some literature [6, 8].
0
2
4

and temperature of 60
o
C.
2. The maximum production yield (Y
p/s
) achieved was
0.88 g of acetic acid/g glucose which is within the
range of earlier reported researches on this process.
Y
p/s

Y
x/s

3. The maximum biomass yield (Y
x/s
) of 0.11 g
biomass/g glucose and maximum specific
growth rate (µ
max
) of 0.11 hour
-1
were obtained
at initial glucose concentration of 30 g/L.
4. The relation between the growth and acetic
acid production from these studies showed a
mix of non growth and growth associated
where the acetic acid was still produced even
when growth has ceased.


[6] Parekh,S.R., Cheryan.M., (1991). Production
of acetate by mutant strain of Clostridium
thermoaceticum.Appl.Microbiol.Biotechnol.36:3
84-387

[7] Sugaya,K., Tuse,D., Jones,J.L., 1986.
Production of acetic acid by Clostridium
thermoaceticum in batch and continous
fermentations. Biotech.Bioeng. v.xxviii.pp.678-683.

[8] Pirt, S.John, (1975). Principles of microbe and cell
cultivation, Halsteed Press Book.UK.

[9] Wang,I.C.D., Fleishchaker.J., Wang,Y.G., (1978) A
novel route to the production of acetic acid by
fermentation. AIChE Symposium series, No.181, vol
74.

[10] Witjitra,K., Shah,M.M., Cheryan,M., (1996).
Effect of nutrient sources on growth and acetate
production by Clostridium thermoaceticum. Enzyme &
Microbiol.Technol.19:322-327