Tài liệu Quá trình lên men axit axetic ppt

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

for this system. The optimum condition obtained
from this batch fermentation studies was at pH 6
and temperature of 60
o
C with the maximum
product yield (Y
p/s
) of 0.88 g acetic acid /g
glucose and maximum cell yield (Y
x/s
) of 0.11 g
cell/g glucose. The maximum specific growth
rate was 0.11 hour
-1
achieved at initial glucose
concentration of 30 g/L. The acetic acid
fermentation profile from these studies showed a
mix of growth associated and non-growth
associated

Keywords: Acetic acid, Clostridium
thermoaceticum, Single step anaerobic
fermentation.


(1) INTRODUCTION

Presently, almost all acetic acid in chemical
industry products is produced synthetically.
About sixty two percent is produced by the

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

. The single step anaerobic fermentation
process has a higher product yield, could eliminate
separate fermentors for the different stages of acetic
acid production and reduces the time required. In these
experiments, the single step anaerobic fermentation
process uses Clostridium thermoaceticum which
converts glucose almost quantitatively to the theoretical
amount of acetic acid. The reaction for this process is as
follows:

C
6
H
12
O
6
3 CH
3
COOH

The acetic acid product yield from this
single step fermentation process from several
studies is around 0.80-0.90 gram acetic acid per
gram of glucose [4,5,9]. The maximum specific
growth rate (µ
max
) from previous researchers
[4,5,6,8]

was 0.1 h

-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:

Solution A: Glucose (variable), Water (150)
Solution B: Yeast extract Difco(5 g), Tryptone
difco(5 g),Sodium acetate (1.55 g) (NH
4
)
2
SO
4
(1
g), MgSO
4
. 7 H
2
O (0.25 g),Fe(NH
4
)
2
(SO
4
)
2
. 6
H
2
O (0.039 g), Co(NO
3
)
2
. 6 H
2

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

2
S.9H
2
O 5%(5 ml)
Solution C: NaHCO
3
(16.8 g), K
2
HPO
4
(7 g),
KH
2
PO
4
(5.5 g), Distilled water (300 ml).
The media was prepared by heating before
sterilization, to help driven off dissolved oxygen
Growth condition, medium and inoculums
preparation, and culture storage condition have
been previously described [3].
Analytical methods

Acetic acid production was analyzed using Gas
chromatograph. Growth of Clostridium thermoaceticum
was monitored spectrophotometrically by measuring the
optical density at 600 nm. A standard curve relating the
optical density to cell dry weight was constructed. The
glucose utilization was analyzed by DNS method
spectrophotometrically at optical density of 550 nm.

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

o
C. This is probably because of the pH
optimum conditions for the bacteria growth is near to
neutral pH while the pH of acetic acid production was
expected to be at acid pH. See Figure 1. According to
literature[8], at low pH below pH 6 undissociated acetic
acid is responsible for growth inhibition and at high pH
(above pH 6) ionized acetate is responsible for growth
inhibition
. The maximum specific growth rate (µ
max
)
obtained was 0.11 h
-1
at initial glucose concentration of
30 g/L. The specific growth rate increased with increase
in initial glucose concentration from 15 g/L to 30 g/L
before it tends to decrease up to concentration of 60
g/L. See Figure 2.This may be so as the substrate
concentration has exceeded the substrate critical
concentration (S>S
crit
)[7]. The maximum specific
growth rates were determined at the exponential
phase and through graphical data of plot ln(x/x
o
)
as a function of time (t) by following the growth
rate equation:
X

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

Fig.1 Acetic acid yield and growth yield
at different pH

0
0.02
0.04
0.06
0.08
0.1
0.12
0 20406080
Initial glucose concentration (g/L)
Specific growth rate
Fig.2 The maximum specific growth rate

The maximum kinetic parameters result at
the optimum condition are given in Table 1
Table.1 Kinetic parameters

Initial glucose
concentration Temp pH
µ
max

(h
-1
)
Y
x/s

(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

6
8
10
12
14
16
0 20406080100120140
Time (hours)
Acetic acid, Glucose (g/L)
0
2.5
5
7.5
10
Dry weight (g/L)
acetic acid
glucose
dry weight

Fig.3 Batch fermentation profile at pH 6 and 60
o
C

[4] Conclusion

The conclusions which could drawn from this
acetic acid batch fermentation process from glucose by
Clostridium thermoaceticum are:
1. Overall the optimum condition for acetic acid
production obtained from these studies was at pH 6

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.

[5] References

[1]Busche, R.M.(1991). Extractive Fermentation
of acetic acid; Economix trade off between yield
of Clostridium and concentration of Acetobacter.
Appl.Biochem.Biotech.28(29):605-621.

[2] Cheryan,M., Parekh,S., Shah,M.,
Witjitra,K.,(1997). Production of acetic acid by
Clostridium thermoaceticum.
Adv.Appl.Microbial.V.43.pp.1-31

[3] Harumi.V,
Masitah.H,Ramachandran.K.B,.(2003) “Acetic
acid production by single step anaerobic
fermentation of glucose. In; Proceeding of
Technical Postgarduate Symposium 2003
(TECHPOS’03).

[4] Ljungdahl,L.G., (1983).Formation of acetate
using homoacetate fermenting anaerobic
bacteria. In: Organic chemicals from biomass,
edited by Wise,D.L., pp.219. The
Benjamin/Cummings Publishing Company,Inc.

[5]Parekh,S.R., Cheryan,M., (1990). Acetate
Production from Glucose by Clostridium
thermoaceticum. Process Biochemistry
International.117-121.

[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