Available online at www.sciencedirect.

com

Bioresource Technology 99 (2008) 27242735

Optimization of acetic acid production from synthesis gas

by chemolithotrophic bacterium Clostridium aceticum using
statistical approach
Jia Huey Sim, Azlina Harun Kamaruddin

School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, Nibong Tebal, 14300 Penang, Malaysia
Received 9 March 2007; received in revised form 3 July 2007; accepted 3 July 2007
Available online 13 August 2007

Abstract
Eorts in optimizing reducing agents, cysteine-HCl H2O and sodium sulde in order to attain satisfactory responses during acetic
acid fermentation have been carried out in this study. Cysteine-HCl H2O each with ve concentrations (0.000.50 g/L) was optimized
one at a time and followed by sodium sulde component (0.000.50 g/L). Response surface methodology (RSM) was used to determine
the optimum concentrations of cysteine-HCl H2O and sodium sulde. The statistical analysis showed that the amount of cells produced
and eciency in CO conversion were not aected by sodium sulde concentration. However, sodium sulde is required as it does inuence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl H2O and
sodium sulde respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO
conversion.
 2007 Elsevier Ltd. All rights reserved.
Keywords: Clostridium aceticum; Acetic acid; Synthesis gas; Reducing agents; Statistical analysis

1. Introduction
Acetic acid is an important industrial feedstock that is
produced mainly from mineral oil and natural gas either
through methanol carbonylation or acetaldehyde oxidation
(Spath and Dayton, 2003). At present, high petroleum cost
due to substantially depletion of fossil fuel resources has
stimulated the development of new technologies based on
renewable resources. Consequently, fermentation and
catalysis processes that change resource entry from nonrenewable (petroleum) to renewable (biomass) feedstocks
have drawn great attention (Klasson et al., 1992). In addition, fermentation process is of great interest for researchers because it is economically feasible due to low energy
and pressure requirement and high durability of biocatalyst

Corresponding author. Tel.: +60 4 599 6417; fax: +60 4 594 1013.
E-mail address: chazlina@eng.usm.my (A.H. Kamaruddin).

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.07.004

as compared to catalytic processes (Probstein and Hicks,

1985). Thus, the focus of many researchers has changed
towards employing acetogenic bacteria as biocatalyst via
fermentation process to produce acetic acid almost stoichiometrically from renewable resources.
The direct utilization of cheap and abundantly available
biomass into fermentation process for acetic acid production includes acid or enzymatic hydrolysis of the cellulosic
biomass to fermentable sugar and followed by bacteria fermentation (Slapack et al., 1985). Acid hydrolysis is hindered due to low glucose yields and corrosion of the
equipment. Enzymatic hydrolysis employs enzymes to
break down the lignocellulose to fermentable sugars and
subsequently fermented to acetic acid. This process may
achieve higher substrate conversion yield but its production is very expensive (Parisi, 1989; Vallender and Eriksson, 1990). Direct conversion of cellulosic biomass to
acetic acid by single fermenting organism is economical
but formed a variety of by-products which include ethanol

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

and some lactic acids (Ravinder et al., 2001; Florenzano

and Poulain, 1984). These potential anaerobic bacteria
are Clostridium lentocellum SG6 (Ravinder et al., 2001)
and Clostridium thermocellum (Florenzano and Poulain,
1984) for a single step fermentation of cellulose to acetic
acid.
The employment of microorganism in fermenting COcontaining gas like synthesis gas into chemical products
like acetic acid is another alternatives and ecient route
(Klasson et al., 1992). Synthesis gas that was fermentable
by bacteria (Natarajan et al., 1998; Reed and Jantzen,
1979) could be obtained renewably through the incomplete
combustion of biomass and municipal wastes or known as
gasication technology (Najafpour et al., 2004). Eubacterium limosum KIST 612 (Chang et al., 2001) and Peptosteptococcus productus U-1 (Vega et al., 1988) are among the
acetogenic bacteria that grow and produce acetic acid
under CO gaseous substrate. These bacteria are less favorable to be used as biocatalysts due to a considerably low
CO tolerance by both bacteria (less than 2.0 atm CO partial pressure) (Chang et al., 2001). Clostridium aceticum
that has high CO tolerance (beyond 2.60 atm CO partial
pressure), achieve product yield stoichiometrically
(0.25 mol acetic acid/mol CO) (Sim et al., 2007) with optimum growth in alkaline medium (pH 8.5) lead to its selection as acetic acid producer in this work.
Obligately anaerobic bacteria like C. aceticum, are
dened as bacteria which are unable to grow under high
redox potential environment. In fermentation media, oxygen is primarily responsible for raising the oxidation
reduction potential (redox potential Eh) that caused the
growth inhibition of obligately anaerobic bacteria. The oxidationreduction (redox potential Eh) is a measure of the
tendency of a solution to be oxidized or reduced (Hungate,
1969). Most anaerobic bacteria are inhibited at Eh values
higher than 100 mV. Reducing agents thus become an
essential chemical component in the growth medium as it
is responsible to depress and poise the redox potential at
optimum levels. The reducing agents must be nontoxic
and at optimum concentration to ensure a satisfactory level
of nal redox potential for the anaerobic organism under
study. The commonly used reducing agents in the anaerobic cultures includes cysteine-HCl H2O, Dithiothreitol,
H2with Palladium Chloride, Na2S 9H2O (Costilow,
1981). As in the CO fermentation by strict anaerobic bacteria like Peptostreptococcus productus, 1.5 mL of sodium
sulde solution was added into fermentation media to
ensure a low redox potential (Vega et al., 1988). The redox
potential must be as low as 300 and 360 mV in order to
impose the growth of Clostridium thermoaceticum (Schwartz and Keller, 1982). Wieringa (1940) observed that a
low redox potential by the addition of 0.1% sodium sulde
(Na2S) in the medium was advantageous to the growth of
C. aceticum under CO2and H2 inorganic substrate. Therefore, this study involves the used of reducing agents at optimum conditions to provide a reducing environment to
ensure C. aceticum growth.

2725

Fermentation time is another key factor that inuences

cell growth in batch system due to depletion in nutrient
sources with time and thus aected acetic acid production
directly. Therefore, ultimate goal in batch process is to
ensure maximum acetic acid product to be harvested at
optimum cultivation time. Optimization of processing
parameters that included fermentation time for maximum
yield of dry cell weight and extracellular polysaccharide
content produced by the fungus Boletus spp. ACCC
50328 were investigated by Wang and Lu (2005). The main
focus of this work is to optimize three process parameters:
(1) cysteine-HCl H2O, (2) sodium sulde and (3) fermentation time in order to enhance maximum acetic acid production and high CO conversion in batch fermentation.
2. Methods
2.1. Microorganism and cultivation
C. aceticum (DSMZ 1496) in the freezed-dried pellets
form which was obtained from Braunschweig, Germanys
culture collection (DSMZ) was used throughout the experimental studies. The pellet was rehydrated and the growth
was propagated in the DSMZ recommended growth medium 1496. Strain C. aceticum was examined routinely using
microscope to check for purity. The chemical compositions
in DSMZ medium 1496 were weighed accordingly for the
preparation of cultivation medium. Dissolved oxygen must
be eliminated from anaerobic growth medium by degassing
the boiling medium under N2 gas for a few minutes. Serum
bottles with N2 headspace were equally lled with 50 mL
working volume and sealed with gas impermeable butyl
rubber septum-type stoppers and aluminium crimp seals.
The liquid media was then autoclaved at 120 C and
15 min prior to inoculation. 2.5 mL of 200 g/L fructose
solution instead of H2/CO gaseous substrate was added
as sole carbon source to initiate the dense growth of C.
aceticum at the early cultivation process prior to batch
studies. 0.25 mL of sterilized reducing agent was used for
lowering the redox potential of the media. Cultivation
medium was accomplished by adjusting the basal medium
to pH 8.5 (optimum pH for C. aceticum) by adding either
2 M HCl or 2 M NaOH.
2.2. Batch fermentation start-up
Three independent variables: Cysteine-HCl H2O concentration, sodium sulde concentration and fermentation
time with three dependent responses: cell concentration,
CO residue inside the broth and the acetic acid concentration were utilized in the experimental study. In dening an
optimized reducing agents for acetic acid fermentation, a
total of two sets of experiments were carried out with each
consisted of one reducing agents: cysteine-HCl H2O or
sodium sulde and fermentation time. The employed studied levels for three concerned parameters of cysteine-

2726

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

HCl H2O, sodium sulde and fermentation time are

clearly stated in Sections 2.2.1 and 2.2.2.
Glass serum bottle with an average volume of 163 mL
was used as a reactor for batch fermentation. Experimental medium was prepared approximately in the same
way as cultivation media as described in Section 2.1.
The experimental medium compositions were similar as
DSMZ medium 1496 except that fructose was being omitted and substituted by mixed gas (4% H2:18% Argon:78%
CO) as sole carbon source for chemolithotrophic growth
of C. aceticum. The serum bottles were ushed with
1.80 atm mixed gas or equivalent to 1.40 atm CO partial
pressure which acted as carbon source throughout the
experimental studies. The reactors were incubated at
30 C and shaken at 200 rpm for 20 min prior to inoculation. Ten percent inoculum at exponential phase (v/v)
equivalent to 5 mL inoculum discharge from 50 mL working volume were transferred antiseptically to initiate batch
fermentation with operating conditions at 30 C and
200 rpm. For every changes in the fermentation variable,
liquid sample and gas sample were withdrawn from bottles
continuously for ve days at 12 h time interval. The withdrawn liquid sample was used for cell density measurement and acetic acid detection. Two hundred microliters
of gas sample was withdrawn and analysed by gas chromatograph to determine CO gaseous substrate utilization.
All the reactions were performed in duplicates and the
results were reported as mean values. The standard deviations for all the experimental results were within 0.05%
of the mean values and are not shown due to the small
error.
2.2.1. Eect of cysteine-HCl H2O
The eect of independent variables: cysteine-HCl H2O
concentration and fermentation time over acetic acid production were studied. In this experiment, the concentrations studied for cysteine-HCl H2O were varied from
0.0 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L to 0.5 g/L. Zero concentration of cysteine-HCl H2O acted as the control run in
the experiment. The sodium sulde concentration was
maintained at 0.5 g/L (the concentration used in DSMZ
medium 1496) throughout the experiment. The eect of
cysteine-HCl H2O concentration on cell growth, total
CO consumed and acetic acid produced were investigated
for 120 h cultivation time. Desirable cysteine-HCl H2O
that was quantied from the response surface plot was used
in the experiment of sodium sulde optimization.
2.2.2. Eect of sodium sulde
Sodium sulde and the fermentation time were the two
independent variables to be optimized while cell concentration, CO uptake and amount of acetic acid produced were
the dependent responses to be maximized. Dierent concentrations of sodium sulde: 0.0 g/L, 0.2 g/L, 0.3 g/L,
0.4 g/L and 0.5 g/L were employed in the work and the
eect towards fermentation parameters was monitored
for 120 h.

2.3. Analytical methods

2.3.1. Cell concentration measurement
A calibration curve consisted of absorbance reading at
y-axis against the cell density of C. aceticum at x-axis
was constructed for the purpose of biomass measurement.
A half ml of collected liquid sample was diluted to 10-fold
dilution with distilled water. The solution was measured
for its optical density by using spectrophotometer at
400 nm wavelength. The resulting absorbance reading
was then compared with the generated calibration curve
to obtain the corresponding cell concentration (g/L).
2.3.2. Gas measurement
In the study, argon component in the mixed gas acted as
an inert component to calculate the total pressure changes
in batch system. The gas compositions were analysed using
gas chromatography (GC) equipped with a thermal conductivity detector (TCD). Packed column, Carboxene
1000 (Supelco, USA) with dimension of 15 ft 1/8 in was
used for detecting hydrogen, argon and carbon monoxide
in the experiment. The injector and detector temperature
were both at 200 C. The initial oven temperature was
40 C, with a rate of 20 C/min until it reached 180 C.
Helium (Air Products, Malaysia) was the carrier gas to
be utilized and was set at 30 mL/min owrate. The gas concentration calculation was done using TotalChrom Workstation Version 6.2 (Perkin Elmer, USA).
2.3.3. Acetic acid measurement
Syringe lter with 0.45 lm pore size (Whatman, England) was used to lter liquid sample from the cells before
acetic acid analyses by gas chromatography. The 0.25 mL
of ltrate was added with 20 lL of 1% 2-propanol (internal
standard). The solution was then acidied with 30 lL of
pure formic acid for acetic acid analysis. 0.4 lL from the
mixture was used for gas chromatography analysis which
is equipped with a ame ionization detector (FID). The
column used was Carbopack B-DA/4% Carbowax 20 M
(Supelco, USA) with dimensions of 2.0 m length and
0.2 cm I.D. The detector and injector temperature were
both at 225 C. The initial oven temperature was 100 C,
with a rate of 10 C/min until it reached 175 C. Helium
(Air Products, Malaysia) is the carrier gas with a owrate
of 30 mL/min.
2.4. Reducing agents optimization
The responses monitored in the fermentation are: cell
concentration (g/L), CO concentration (mmol) and acetic
acid concentration (g/L) resulted from varying concentrations of cysteine-HCl H2O and sodium sulde were performed graphically to ease the visual evaluation. In order
to obtain the optimum operating conditions, the response
surface methodology (RSM) was carried out using the
Design-Expert software (version 6.0.6) during the batch
studies.

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

3. Results and discussion

2727

0.7

3.1. Eect of cysteine-HCl H2O on acetic acid fermentation

Cysteine-HCl H2O complements sodium sulde as the
reducing agents for anaerobic media. Reducing agent in
appropriate concentration is fairly important to anaerobic
bacteria so as to poise and depress the redox potential in
medium to the levels that would initiate cell growth. The
experiment was conducted with the aim to obtain the optimum concentration of cysteine-HCl H2O and fermentation time through RSM.
Fig. 1 presents the cell growth in dierent concentrations
of cysteine-HCl H2O as a function of time. All cells were
experiencing the same and typical sigmoid growth curve
where lag phase occurred for the initial 12 h incubation
period. Eventually, there appeared to be small variation
within the maximum cells concentration achieved at each
levels of cysteine-HCl H2O (Table 2). The specic growth
rate as indicated by the steepness of the slope increased
accordingly with descending cysteine-HCl H2O concentration. During exponential phase of growth, the biomass produced was inversely proportional to the cysteine-HCl H2O
concentration. High cysteine-HCl H2O concentration,
0.5 g/L was less favorable to the cell growth which lead
to the lowest cell concentration, 0.49 g/L. Therefore, the
inhibition of cysteine-HCl H2O to the cell growth was
likely to occur at 0.5 g/L as indicated by apparently lowest
cell growth curve in Fig. 1. In other words, 0.00.4 g/L of
cysteine-HCl H2O concentration were sucient to reduce
Table 1
pH of medium at various cysteine-HCl H2O and sodium sulde
concentration during initial and nal fermentation time
Cysteine (g/L)

0.0

0.2

0.3

0.4

0.5

pH medium without cysteine at 0 h

pH medium at 120 h

8.98
7.08

8.84
7.13

8.96
7.13

8.64
7.18

8.75
7.23

Sodium sulde (g/L)

pH medium without sodium at 0 h
pH medium at 120 h

0.0
8.80
7.11

0.2
8.59
7.27

0.3
8.93
7.13

0.4
8.70
7.09

0.5
8.66
7.20

0.6

Cell concentration, g/ L

Section 2.1 in method mentions the steps taken during

strain propagation and medium preparation for C. aceticum cultivation while Section 2.2 is mainly on the startup process for experimental studies. The pH usually
decreases during cultivation due to acetogenic activity (acetic acid production). The initial pH of medium has been
adjusted to optimum pH 8.5, whereas the nal pH after
120 h at various cysteine and sodium sulde concentrations
were recorded and shown in Table 1. The nal pH recorded
at dierent cysteine and sodium sulde concentrations were
similar, ranging between pH 7.087.27. This indicated that
all experiments were subjected to small pH variations
throughout the experimental study, thus any variation in
responses due to pH can be neglected.

0.5
0.4
0.3
Cysteine-HCl.H2O Concentration

0.2

0.0 g/ L
0.2 g/ L
0.3 g/ L
0.4 g/ L
0.5 g/ L

0.1
0.0
-0.1
0

20

40

60

80

100

120

140

Time, h

Fig. 1. Eect of cysteine-HCl H2O on cell concentration.

the redox potential to the extend that would permit anaerobic cell growth.
The use of cysteine-HCl H2O ranging between 0.0 and
0.4 g/L resulted in minimum CO residue inside the system.
After 48 h of incubation period, two regions were clearly
dened based on the rapidity in the amount of CO consumed. Cysteine-HCl H2O of 0.5 g/L with low CO consumption rate was situated at higher region while others
with similarly high CO consumption rate gathered at lower
region. CO uptake rate at 0.5 g/L cysteine-HCl H2O
decreased sharply after 48 h as compared to other cysteine-HCl H2O concentration and the CO consumption
gradually retarded (Fig. 2). The total CO consumed at
0.5 g/L cysteine-HCl H2O was 4.97 mmol whereas the
total CO uptake at other cysteine-HCl H2O concentrations were between 5.77 and 6.29 mmol (Table 2). From
Fig. 2 and Table 3, the CO consumption rates attained
by all concentrations of cysteine-HCl H2O (ranging from
2.53 to 3.19 mmol/L h) were not signicantly dierent.
The curves for CO consumption rate as a function of time
at various cysteine-HCl H2O concentration were of similar quantitative trend that resemble a bell shape.
In general, the production of acetic acid at each concentration of cysteine-HCl H2O does not dier signicantly
from each other (Fig. 3). This means that cysteineHCl H2O with concentrations of 0.00.5 g/L did not exert
signicant eect to the acetic acid production. Within 84 h
fermentation time, the acetic acid production was the highest at 0.3 g/L cysteine-HCl H2O compared to other concentrations. Although cells in 0.5 g/L cysteine-HCl H2O
reached the highest peak of acetic acid concentration of
2.35 g/L at 108 h (Table 2) but the acetic acid production
before 108 h was mostly maintained at low concentration
compared to other cysteine-HCl H2O concentrations
(Fig. 3). Therefore, 0.3 g/L cysteine-HCl H2O was the
desirable concentration to be applied in the batch system
since it maintained a relatively high level of cell concentration throughout the experimental study and achieved
nearly 100% CO conversion.

2728

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

Table 2
Maximum cell concentration, CO uptake and acetic acid produced under dierent cysteine-HCl H2O and sodium sulde concentration
Reducing agents
(g/L)

Cell concentration

CO concentration

Acetic acid concentration

Maximum produced
(g/L)

Fermentation
time (h)

Maximum consumed
(mmol)

Fermentation
time (h)

Maximum produced
(g/L)

Fermentation
time (h)

Cysteine-HCl H2O
0.0
0.2
0.3
0.4
0.5

0.56
0.55
0.59
0.51
0.49

48
48
60
48
48

6.29
5.82
6.04
5.77
4.97

120
120
120
120
120

1.52
1.52
2.08
1.87
2.35

108
108
108
120
108

Sodium sulde
0.0
0.2
0.3
0.4
0.5

0.81
0.78
0.80
0.71
0.79

36
36
36
48
36

6.45
6.45
6.41
6.44
6.43

48
48
48
60
60

1.42
1.72
1.89
1.46
1.43

120
60
60
120
108

3.5

2.5
Cysteine-HCl.H2O Concentration

Cysteine-HCl.H2O Concentration

0.0 g/L
0.2 g/L
0.3 g/L
0.4 g/L
0.5 g/L

2.5

Acetic acid concentration, g/L

CO consumption rate, mmoles/L.h

3.0

2.0

1.5

1.0

0.0 g/L
0.2 g/L
0.3 g/L
0.4 g/L
0.5 g/L

2.0

1.5

1.0

0.5

0.5

0.0

0.0
0

12

24

36

48

60

72

84

96

108

120

132

12

24

36

48

60

72

84

96

108

120

132

Time, h

Time, h

Fig. 2. Eect of cysteine-HCl H2O on CO consumption rate.

Fig. 3. Eect of cysteine-HCl H2O on acetic acid concentration.

Table 3
Comparison of fermentation parameters under dierent cysteine-HCl H2O and sodium sulde concentration
Reducing
agents (g/L)

CO
Consumption rate
(mmol/L h)

Acetic acid
a

Acetic acid yield

a

Fermentation
time (h)

Formation rate
(g/L h)

Fermentation
time (h)

Yp/s

Cysteine-HCl H2O
0.0
3.19
0.2
2.62
0.3
2.82
0.4
2.92
0.5
2.53

24
24
36
36
36

0.041
0.033
0.070
0.051
0.112

48
48
108
36
108

0.31
0.61
0.69
0.73
1.06

Sodium sulde
0.0
0.2
0.3
0.4
0.5

24
24
24
24
24

0.053
0.100
0.056
0.058
0.048

48
48
60
24
24

0.18
0.18
0.25
0.19
0.19

5.76
5.37
5.14
4.84
5.29

Fermentationa
time (h)

Yp/x

Fermentationa
time (h)

12
12
12
12
12

2.33
2.59
3.43
3.29
5.26

108
108
108
120
108

120
96
60
120
108

2.63
1.83
2.60
2.61
2.36

120
96
120
120
108

a
Fermentation time reported was referring to time consumed for maximum CO consumption rate, maximum acetic acid formation rate and maximum
acetic acid yield, respectively.

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

3.1.1. Response surface method (RSM) analysis

The relationship between responses: cell concentration
and acetic acid concentration with factors: cysteineHCl H2O and fermentation time were well tted with
quadratic model as expressed by Eqs. (1) and (2) while
the CO residue response can be predicted by cubic

2729

model as in Eq. (3). The CO concentration and acetic

acid concentration were transformed to square root in
order to solve the abnormal response problems. Empirical
models were in good t to the experimental results (R2
ranging from 0.84 to 0.97, data not shown) for all the
responses
120

90

0.42

Fermentation Time (h)

Cell concentration (g/L)

0.59

0.36

0.25
0.07
-0.10
0.50
0.38

0.48

60

0.36
30

0.25

0.25
120

0.13
0.02

0.13

90

60

30

Fermentation Time (h)

0.00

0.00

Cysteine-HCl.H2O
(g/L)

0.13

0.25

0.38

0.50

0.38

0.50

90

120

Cysteine-HCl.H2O (g/L)

120

0.87

7.40
90

Fermentation Time (h)

CO concentration (mmoles)

5.61
3.82
2.04
0.25
0.50
0.38

1.24
1.61
1.98
2.35

60

2.74
3.37
30

0.25
0.13

120

90

60

30

Fermentation Time (h)

0.00

0.00

0.13

Cysteine-HCl.H2O
(g/L)

0.25

Cysteine-HCl.H2O (g/L)

0.50

1.57
1.18
0.38

0.41

Cysteine-HCl.H2O (g/L)

Acetic acid concentration (g/L)

0.80

0.02

0.50

0.22

0.56
0.38

0.25

0.91

0.73

1.09

0.13

0.38
0.25
0.13

Cysteine-HCl.H2O
(g/L)

0.00

60

90

30
Fermentation Time (h)

120

0.00
0

30

60

Fermentation Time (h)

Fig. 4. Response surface plot and contour plot on cysteine-HCl H2O and fermentation time for (a) cell concentration; (b) CO residue; (c) acetic acid
produced.

2730

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

Cell concentration 0:017528  0:23341  Cysteine

0:015248  Fermentation time  1:00676E  004
 Fermentation time2 ;

SqrtAcetic acid concentration 0:02 0:20233

0:022475  Fermentation time  1:18977E  004
 Fermentation time2 ;

SqrtCO concentration 2:66915 1:32369  Cysteine

 0:028229  Fermentation time  9:70078  Cysteine2
8:47015E  005  Fermentation time2 0:010704
 Cysteine  Fermentation time 14:49455  Cysteine3 :
3

The response surface for cell concentration is presented in

Fig. 4a. The eect of cysteine-HCl H2O on cell growth
was less pronounced during early fermentation time (less
than 30 h). However, after 45 h of fermentation, the cells
in higher cysteine-HCl H2O concentration took longer
time to achieve 0.48 g/L cell concentration as clearly seen
from the response surface plot in Fig. 4a. The same quantitative trend for cell concentration response surface plot
(Fig. 4a) occurred in the response surface plot of CO concentration in Fig. 4b. The eect of cysteine-HCl H2O on
the CO uptake by C. aceticum was becoming signicant
when incubated for more than 30 h. Approximately 90%
of CO conversion was located at around 90 h fermentation
time over a wide range of cysteine-HCl H2O. Fermentation time was the key parameter for acetic acid production
(Fig. 4c). Long fermentation time (for more than 50 h) was
necessary for higher acetic acid production by C. aceticum

Table 4
Analysis of variance (ANOVA) for the regression model and the respective model terms on the studies of cysteine-HCl H2O and sodium sulde
concentration
Source

Model terms

Cysteine-HCl H2O
Cell concentration
Quadratic
A
B
B2
CO concentration
Cubic
A
B
A2
B2
AB
A3
Acetic acid concentration
Quadratic
A
B
B2
Sodium sulde
Cell concentration
Cubic
A
B
B2
B3
CO concentration
Quadratic

Sum of squares

1.69
0.081
0.72
0.90
17.84
3.245E003
16.56
0.050
0.64
0.24
0.083

Mean square

0.56
0.081
0.72
0.90
2.97
3.245E003
16.56
0.050
0.64
0.24
0.083

F Value

Prob > F

Remarks

81.49
11.69
104.80
130.58

<0.0001
0.0013
<0.0001
<0.0001

Signicant
Signicant
Signicant
Signicant

275.29
0.30
1533.70
4.60
59.02
22.63
7.65

<0.0001
0.5864
<0.0001
0.0376
<0.0001
<0.0001
0.0083

Signicant
Signicant
Signicant
Signicant
Signicant
Signicant

6.05
0.02
4.84
1.26

3.02
0.02
4.84
1.26

252.79
1.89
405.01
105.16

<0.0001
0.1762
<0.0001
<0.0001

Signicant
Insignicant
Signicant
Signicant

2.67
6.02E004
0.18
1.71
0.54

0.89
6.02E004
0.18
1.71
0.54

317.97
0.21
65.88
611.95
193.93

<0.0001
0.6474
<0.0001
<0.0001
<0.0001

Signicant
Insignicant
Signicant
Signicant
Signicant

A
B
B2

180.23
0.44
150.71
29.53

90.12
0.44
150.71
29.53

308.92
1.53
516.61
101.22

<0.0001
0.2221
<0.0001
<0.0001

Signicant
Insignicant
Signicant
Signicant

Acetic acid concentration

Quadratic
A
B
B2

15.95
0.67
13.34
1.93

5.32
0.67
13.34
1.93

43.85
5.56
110.05
15.93

<0.0001
0.0222
<0.0001
0.0002

Signicant
Signicant
Signicant
Signicant

A = cysteine-HCl H2O or sodium sulde, B = fermentation time.

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

over the entire cysteine-HCl H2O concentration. As stated

earlier, cysteine-HCl H2O concentration seems to exert
minor eect on acetic acid productivity from the graphical
presentation of acetic acid production as a function of
time. However, the signicance test performed during ANOVA on cysteine-HCl H2O to acetic acid concentration
response shows that (Prob > F) far exceeds 0.05 (Table
4). This means that the eect from cysteine-HCl H2O
alone on acetic acid produced was insignicant to be
included in the empirical model for process optimization.
Therefore, cysteine-HCl H2O alone was insignicant on
the acetic acid concentration. It can be concluded that
the eect of cysteine-HCl H2O (A) to acetic acid produced
was insignicant while fermentation time (B) remain to be
the key factor for the three responses (Table 4).

3.1.1.1. System optimization within designated constraints.

The objectives of the study were to maximize cell concentration and acetic acid produced with minimum CO residue

2731

left inside the system (Table 5). Therefore, the most desirability experimental region was found at 0.30 g/L cysteine-HCl H2O and at 60 h (Fig. 5). Thus, when the
system was operating under the optimum conditions, the
CO residue was 1.71 mmol and the acetic acid produced
was 1.20 g/L as compared to the predicted values of
1.93 mmol CO concentration and 1.23 g/L acetic acid concentration. The cysteine-HCl H2O concentration was said
to be successfully reduced from initial 0.5 g/L to 0.3 g/L
while still retaining high acetic acid productions.

3.2. Eect of sodium sulde on acetic acid fermentation

Sodium sulde as mentioned earlier in Section 3.1, is
part of the reducing agent that is responsible in initiating
the growth for anaerobic bacteria. Sodium sulde is the
nal component chosen to be minimized while cysteineHCl H2O were constant at its optimum concentrations.

Table 5
The preset goal with the constraints for all the independent factors and responses in numerical optimization
Variables

Ultimate goal

Experimental region
Lower limit

Eect of cysteine-HCl H2O

Factor
Cysteine-HCl H2O, A (g/L)
Fermentation time, B (h)
Response

Eect of sodium sulde

Factor
Response

Upper limit

In range
Minimized

0.00
0

0.50
120

Cell concentration (g/L)

CO concentration (mmol)
Acetic acid concentration (g/L)

In range
Minimized
Maximized

0.00
0.16
0.024

0.69
6.46
5.51

Na2S 9H2O, A (g/L)

Fermentation time, B (h)

In range
Minimized

0.00
0

0.50
120

Cell concentration (g/L)

CO concentration (mmol)
Acetic acid concentration (g/L)

In range
Minimized
Maximized

0.00
0.00
0.71

0.81
6.45
1.89

120
0.263

0.175
0.351

0.526

Fermentation Time (h)

0.395

Desirability

0.263
0.132
0.000

0.439

90

0.506
0.506

60

0.439

30

120
0.50

90
60

Fermentation Time (h)

0.351
0.263
0.088 0.175

0.38
0.25
30

0.13
0 0.00

Cysteine-HCl.H2O
(g/L)

0
0.00

0.13

0.25

0.38

Cysteine-HCl.H2O (g/L)

Fig. 5. Response surface plot of the desirability region across cysteine-HCl H2O and fermentation time.

0.50

2732

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

The desirable level of sodium sulde was identied by

response surface method analysis (RSM).
Generally, cells in 0.0 g/L and 0.5 g/L sodium sulde
possesses the highest and second highest cell growth rate
respectively while cells replicated at about the same rate
in the mid studied range of sodium sulde (0.2 g/L, 0.3 g/
L and 0.4 g/L). In addition, cells cultured at this range of
sodium sulde (0.2 g/L, 0.3 g/L and 0.4 g/L) exhibited
growth curve with lag phase for the rst 12 h before entering exponential phase. As seen from Table 2, the cell concentration reached the peak values of 0.780.81 g/L within
36 h fermentation time throughout the whole range of
sodium sulde except for 0.4 g/L. The lowest cell concentration i.e. 0.71 g/L was obtained at 0.4 g/L sodium sulde.
Apparently, sodium sulde when employed at high concentration (0.5 g/L) did not show toxicity to the cell growth.
The sodium sulde within the studied range was sucient
to depress and poise the redox potential of the medium that
allowed the anaerobic bacteria propagation.
It was interesting to note that the CO reduction for all
the sodium sulde levels were nearly of the same rate.
Low sodium sulde concentration (0.0 g/L, 0.2 g/L and
0.3 g/L) tends to achieve 100% CO conversion at shorter
fermentation time, 48 h as compared to high sodium sulde.
In addition, the CO consumption rate increased with a
decrease in sodium sulde concentration except at 0.5 g/L
sodium sulde (Table 3). The maximum CO being consumed and maximum CO consumption rate achieved at
varying concentration of sodium sulde were almost similar
as clearly observed from Tables 2 and 3. Therefore, the
application of sodium sulde within the studied range in
batch system encourages 100% CO uptake by C. aceticum.
Although sodium sulde did not seem to exert signicant
eects on cell growth and CO uptake rate, but sodium sulde concentration aects appreciably the acetic acid production (Fig. 6). At 0 h fermentation, the concentration of
acetic acid which were close to 1 g/L in all cases resulted
from the transfer during inoculation process. Acetic acid
was initially present in relatively high concentrations due

to the preculture production from inoculum broth. Sodium

sulde at 0.3 g/L produced the highest net acetic acid concentration, 1.89 g/L in shortest fermentation time i.e. 60 h
compared to others as shown in Table 2. Table 3 summarizes the maximum substrate uptake rate, maximum production rate and maximum product yield under dierent
sodium sulde concentration to ease the investigation of
the sodium sulde eect. C. aceticum in 0.3 g/L sodium sulde fermented CO stoichiometrically to acetic acid with 4 g
of CO completely converted to 1 g of acetic acid as suggested
in the stoichiometric reaction (Eq. 1) and resulted in
0.25 gacetic acid/gCO product yield, Yp/s (Table 3). In contrast,
sodium sulde at concentrations higher or less than 0.3 g/L
were inhibiting C. aceticum to convert CO gaseous substrate
to acetic acid and caused low production yield of 7576% or
Yp/s = 0.180.19 compared to 100% product conversion
(Yp/s = 0.25) being achieved at 0.3 g/L. The eciency of cell
in producing acetic acid which was denoted by Yp/x (2.36
2.63) were less inuenced by the applied sodium sulde concentrations except at 0.2 g/L. Therefore, 0.3 g/L sodium sulde was the most suitable concentration to be applied in the
fermentation medium that resulted in maximum cell concentration, complete CO conversion (100%) and maximum production within short cultivation time (Table 2).
3.2.1. Response surface method (RSM) analysis
Eqs. (4)(6) were the empirical models used for generating response surface plot. Cell concentration was best presented with cubic model (Eq. (4)) while quadratic model
was used to correlate CO concentration and acetic acid
concentration to the key factors (Eqs. (5) and (6)). Cell
concentration was transformed to square root scale while
CO concentration was transformed to natural log scale
and resulting in relatively high R2, 0.9492 and 0.9224,
respectively (data not shown).
SqrtCell concentration 0:01 0:085147 0:038915
 Fermentation time  5:75372E  004
 Fermentation time2 2:42571E  006
 Fermentation time3 ;

Acetic acid concentration, g/L

3.0

Ln CO concentration 0:06 2:56819  0:11276

 Fermentation time 5:76131E  004

2.5

 Fermentation time2 ;

2.0

Acetic acid concentration 0:13921  0:64369

 Na2 S  9H2 O 0:030659  Fermentation time

1.5

 1:47339E  004  Fermentation time2 :

Na2S.9H2O Concentration
1.0

0.0 g/ L
0.2 g/ L
0.3 g/ L
0.4 g/ L
0.5 g/ L

0.5

0.0
0

12

24

36

48

60
72
Time, h

84

96

108

120

Fig. 6. Eect of sodium sulde on acetic acid concentration.

132

The impact of sodium sulde on cell growth and CO uptake were completely eliminated as clearly observed from
the response surface in Fig. 7a and b. In other words, cell
concentration and CO concentration at xed fermentation
time can be determined at any point along the line or fermentation time is the key factor for both responses. Therefore, acetic acid fermentation has to be cultivated within

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

optimum period in order to achieve dense cell at late exponential phase and targeted to zero CO residue. However,
the amount of sodium sulde used in the study exerted
minor inuence on the acetic acid produced as clearly
viewed from Fig. 7c. Acetic acid production was said to
be proportional to the length of the fermentation time
but inversely proportional to the applied sodium sulde

2733

during batch fermentation as clearly viewed from Fig. 7c.

In others word, acetic acid production was preferably enhanced at reduced amount of sodium sulde concentration
but longer incubation time. The maximum acetic acid production of 1.14 g/L was presumed to be occurring at moderate fermentation time over wide range of sodium sulde
but less than 0.50 g/L.
0.50

0.79
0.60
0.38

0.20

Na2S.9H2O (g/L)

Cell concentration (g/L)

0.40

-0.00

0.50

0.36
0.22

0.25

0.63

0.49

0.76

0.76

0.63

0.49

0.13

0.38

Na2S.9H2O 0.25
(g/L)
0.13
0.00

30

0.00

120

90

60

30

60
Fermentation time (h)

90

120

90

120

0.38

0.50

Fermentation time (h)

0.50

12.98

0.38

Na2S.9H2O (g/L)

CO concentration (mmoles)

9.73
6.48
3.24
-0.01

4.32
6.48

0.25

0.06
0.82 0.36

0.11

0.02

2.15

0.13
0.50

0.38

30

0.00

0.25

60
0.13

90

Fermentation time (h)

120

0.00

Na2S.9H2O (g/L)

30

60

Fermentation time (h)

120

90

0.98

Fermentation time (h)

Acetic acid concentration (g/L)

1.46

0.50
0.02
-0.46

1.14

60
0.82

0.50

30

0.50
120

0.18

0.38
-0.14

90

0.25

60

Fermentation time (h)

0.13

30
0 0.00

Na2S.9H2O (g/L)

0
0.00

0.13

0.25

Na2S.9H2O (g/L)

Fig. 7. Response surface plot and contour plot on sodium sulde and fermentation time for (a) cell concentration; (b) CO residue; (c) acetic acid produced.

2734

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

120

0.340

0.226
0.453

0.679

Fermentation time (h)

0.340

Desirability

0.566

90

0.510

0.170
0.000

0.651

60

0.566

30
0.453

120
0.50

90

Fermentation time (h)

0.340

0.38
60

0.25
30

0.13

Na2S.9H2O (g/L)

0.113 0.226

0
0.00

0.13

0.25

0.38

0.50

Na2S.9H2O (g/L)

0 0.00

Fig. 8. Response surface plot of the desirability region across sodium sulde and fermentation time.

3.2.1.1. System optimization within designated constraints.

The optimum working conditions for all responses is given
in Table 5. The most desirable operating conditions that
optimized all the responses simultaneously were located
at 0.30 g/L sodium sulde and at 60 h (Fig. 8). The amount
of CO residue was 0.0 mmol and the acetic acid being produced in the system was 1.28 g/L. The predicted results
were 0.0 mmol CO concentration and 1.17 g/L acetic acid
concentration. Therefore, the optimum reductants for C.
aceticum were at 0.3 g/L each for cysteine-HCl H2O and
sodium sulde. 0.3 g/L were proven again as optimum
reductants when drastic improvement of 89% in specic
growth rate, l was observed in optimum medium compared to medium DSMZ 1496. The specic growth rate,
l for medium DSMZ 1496 before and after process optimization of reductants was 0.0326 h1 and 0.0617 h1,
respectively.
4. Conclusion
Both reducing agents have shown to be signicantly
aecting the acetic acid fermentation by C. aceticum. Normal graphical plot and response surface plot illustrated
that inhibition of cysteine-HCl H2O over cell concentration and the CO uptake ability existed when high cysteine-HCl H2O concentration was employed (0.5 g/L or
above). In contrast to cysteine-HCl H2O, sodium sulde
on its own did exert certain eect on acetic acid produced
but has no inuenced on cell concentration and CO uptake
based on the 2-D and 3-D surface plot. Optimum operating conditions were found to be at 0.3 g/L for both cysteine-HCl H2O and sodium sulde and this study has
successfully reduced 40% of the initial reducing agents
employed, from 0.5 g/L (concentration suggested in
DSMZ growth media) to 0.3 g/L. The optimum operating
region which corresponded to 0.3 g/L cysteine-HCl H2O
and 0.3 g/L sodium sulde for 60 h fermentation time produced 1.28 g/L acetic acid and attained 100% CO
conversion.

Acknowledgements
The present research was made possible through an
IRPA grant project (01-02-05-32230EA011) sponsored by
Ministry of Science, Technology and Innovations (MOSTI), Malaysia and graduate assistant scheme allowance
awarded by Universiti Sains Malaysia (USM). Dr. Habibollah Younesi and Dr. Long Wei Sing are acknowledged
for their assistance and comments.
References
Chang, I.S., Kim, B.H., Lovitt, R.W., Bang, J.S., 2001. Eect of CO
partial pressure on cell-recycled continuous CO fermentation by
Eubacterium limosum KIST612. Process Biochemistry 37, 411
421.
Costilow, R.N., 1981. Biophysical factors in growth. In: Gerhardt, P.,
Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg,
N.R., Phillips, G.B. (Eds.), Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, USA.
Florenzano, G., Poulain, M., 1984. A study of acetate production from
cellulose using Clostridium thermocellum. Biomass 4, 295303.
Hungate, R.E., 1969. A roll tube method for cultivation of strict
anaerobes. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in
Microbiology. Academic Press, New York, USA.
Klasson, K.T., Ackerson, M.D., Clausen, E.C., Gaddy, J.L., 1992.
Bioconversion of synthesis gas into liquid or gaseous fuels. Enzyme
Microbial Technology 14, 602608.
Najafpour, G.D., Younesi, H., KuSyahidah, K.I., Mohamed, A.R.,
Kamaruddin, A.H., 2004. Performance of biological hydrogen production process from synthesis gas, mass transfer in batch and
continuous bioreactors. International Journal of Engineering 17 (2),
105120.
Natarajan, E., Nordin, A., Rao, A.N., 1998. Overview of combustion and
gasication of rice husk in uidized bed reactors. Biomass and
Bioenergy 14, 533546.
Parisi, F., 1989. Advances in lignocellulosics hydrolysis and in the
utilization of the hydrolyzates. Advances in Biochemical Engineering
and Biotechnology 38, 5387.
Probstein, R.F., Hicks, R.E., 1985. Synthetic Fuels. McGraw-Hill,
Singapore.
Ravinder, T., Swamy, M.V., Seenayya, G., Reddy, G., 2001. Clostridium
lentocellum SG6-a potential organism for fermentation of cellulose to
acetic acid. Bioresource Technology 80, 171177.

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

Reed, T.B., Jantzen, D., 1979. Biomass gasication: principles and
technology. Energy Technology Review 67, 2790.
Schwartz, R.D., Keller, F.A., 1982. Acetic acid production by Clostridium
thermoaceticum in pH-controlled batch fermentations at acidic pH.
Applied and Environmental Microbiology 43 (6), 13851392.
Sim, J.H., Kamaruddin, A.H., Long, W.S., Najafpour, G., 2007.
Clostridium aceticum A potential organism in catalyzing carbon
monoxide to acetic acid: Application of response surface methodology.
Enzyme Microb. Technol. 40, 12341243.
Slapack, G.E., Russell, I., Stewart, G.G., 1985. Thermophilic bacteria
and thermotolerant yeasts for ethanol production. Project Report
submitted to Division of Energy, NRC, Ottawa, NRCC No. 24410, 1404.
Spath, P.L., Dayton, D.C., 2003. Preliminary Screening Technical and
Economic Assessment of Synthesis Gas to Fuels and Chemicals with

2735

Emphasis on the Potential for Biomass-Derived Syngas. NREL/TP510-34929.

Vallender, L., Eriksson, K.E.L., 1990. Production of ethanol from
lignocellulosic materials: state of the art. Advances in Biochemical
Engineering and Biotechnology 2, 6995.
Vega, J.L., Clausen, E.C., Gaddy, J.L., 1988. Study of gaseous substrate
fermentations: carbon monoxide conversion to acetate. 1. Batch
culture. Biotechnology and Bioengineering 34, 774784.
Wang, Y.X., Lu, Z.X., 2005. Optimization of processing parameters
for the mycelial growth and extracellular polysaccharide production
by Boletus spp. ACCC 50328. Process Biochemistry 40 (34), 1043
1051.
Wieringa, K.T., 1940. The formation of acetic acid from carbon dioxide
and hydrogen by anaerobic spore-forming bacteria. Journal of
Microbiol Serology 6, 251262.