Bioresource Technology 100 (2009) 34033409

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Butyric acid fermentation in a brous bed bioreactor with immobilized Clostridium

tyrobutyricum from cane molasses
Ling Jiang a, Jufang Wang a,*, Shizhong Liang a, Xiaoning Wang a, Peilin Cen b, Zhinan Xu b,*
a
b

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China
Institute of Bioengineering, Dept. of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR China

a r t i c l e

i n f o

Article history:
Received 16 December 2008
Received in revised form 17 February 2009
Accepted 17 February 2009
Available online 17 March 2009
Keyword:
Butyric acid
Clostridium tyrobutyricum
Fibrous-bed bioreactor
Cane molasses
Immobilization

a b s t r a c t
Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a brous
bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the inuence of
pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate
production of 26.2 g l 1 with yield of 0.47 g g 1 and reactor productivity up to 4.13 g l 1 h 1 at pH 6.0.
When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and
it was found that sulfuric acid treatment gave better results regarding butyrate concentration
(34.6 0.8 g l 1), yield (0.58 0.01 g g 1), and sugar utilization (90.8 0.9%). Also, fed-batch fermentation
from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of
butyrate up to 55.2 g l 1.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Butyric acid is a specialty chemical with many applications in
chemical, foodstuff, and pharmaceutical industries. It can be used
as the pure acid to enhance butter-like notes in food avors or in
the form of esters as additives for increasing fruit fragrance, and
as aromatic compounds for the production of perfumes (Playne,
1985). The role of this acid in the treatment of hemoglobinopathies, cancer, and gastrointestinal diseases is also well known
(Pouillart, 1998). Currently, butyric acid is synthesized commercially via petrochemical routes. However, the demand for butyric
acid from microbial fermentation is high due to a strong preference
by consumers and manufacturers for using bio-based natural
ingredients in foods, cosmetics, and pharmaceuticals. For economical production of butyrate from biomass, it is critical to get high
butyrate yield, concentration and reactor productivity in the
fermentation.
Several fermentation processes have been studied for bio-production of butyric acid from glucose (Wu and Yang, 2003;
Michel-Savin et al., 1990a; Liu et al., 2006; Crabbendam et al.,
1985; Van Andel et al., 1985), xylose (Liu and Yang, 2006), sucrose
(Vandk et al., 1995), cheese whey (Alam et al., 1988), hydrolysate
of corn bre (Zhu et al., 2002), corn meal (Huang et al., 2002) and
wheat our (Fayolle et al., 1990). In general, carbon source is usually used in relatively higher concentration compared to other
* Corresponding authors. Tel./fax: +86 571 87951220 (Z. Xu); tel./fax: +86 20
39380626 (J. Wang).
E-mail addresses: jufwang@scut.edu.cn (J. Wang), znxu@zju.edu.cn (Z. Xu).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.02.032

medium components and thus has high share in the raw material
cost. Therefore, the exploitation of cheap, renewable carbon
sources has been strongly stimulated. Cane molasses is a byproduct of the sugar industry, which consists of water, approximately
4550% (w/w) total sugars (sucrose, glucose and fructose), suspended colloids, heavy metals, vitamins and nitrogenous compounds, etc. (Najafpour and Shan, 2003). It is a relatively cheap
raw material, readily available, and has already been used for the
production of different primary metabolites, such as succinic acid
(Liu et al., 2008), lactic acid (Kotzamanidis et al., 2002; Shukla
et al., 2004), citric acid (Haq et al., 2004), gluconic acid (Sharma
et al., 2008), propionic acid (Chanto et al., 1994), ethanol (Liu
et al., 2009), sorbitol (Cazetta et al., 2005), vitamin B12 (Chanto
et al., 1994), uricase (Lotfy Walid, 2008), as well as secondary
metabolites, such as erythromycin (El-Enshasy et al., 2008). To
our knowledge, cane molasses has only been used in one recent
study to produce butyric acid by Clostridium butyricum (Vandk
et al., 1995). However, the yield and productivity of butyric acid
were not satisfying for the industrial application.
Compared with the free-cell fermentation, immobilization of
microbial cells has some attractive characteristics, such as increasing the biomass in the reactor and the productivity of fermentation, preventing the loss of cells, facilitating continuous
fermentation, etc. (Najafpour, 2006). However, conventional
immobilized-cell fermentation usually suffer from productivity
lose over an extended operation period due to accumulation of
dead cells and poor mass transfer under high cell density
conditions. Recently, a brous bed bioreactor (FBB) with cells

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L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

immobilized in the brous matrix packed in the reactor has been

successfully used to produce several organic acids (lactic acid, acetic acid, propionic acid, and butyric acid) with signicantly improved reactor productivity, nal product yield and concentration
(Huang et al., 2002; Silva and Yang, 1995; Yang et al., 1994).
In this work, batch, repeated-batch, and fed-batch fermentations were studied to evaluate the feasibility and robustness of
the FBB system for producing butyric acid from cane molasses with
immobilized Clostridium tyrobutyricum, and its process optimization was also carried out.

pH meter
H2 , CO2

Out

fibrous
bed
bioreactor
NaOH

2. Methods
2.1. Cultures and medium

circulation pump
N

batch fermentor

C. tyrobutyricum (ATCC 25,755) was originally supplied by professor Shang-Tian Yang, from The Ohio State University, via further
adaption and selection from FBB. Details about the procedures to
obtain the mutant from FBB have been given elsewhere (Wu and
Yang, 2003). The stock culture was kept in serum bottles in a synthetic medium previously described (Huang and Yang, 1998) under
anaerobic conditions at 4 C. Unless otherwise noted, the cultivation medium contained (per liter of distilled water): 30 g glucose,
5 g yeast extract, 5 g peptone, 3 g (NH4)2SO4, 1.5 g K2HPO4, 0.6 g
MgSO4
7H2O, 0.03 g FeSO4
7H2O. All the media were sterilized
by autoclaving at 121 C, 15 psig, for 20 min.
2.2. Pretreatment of molasses
Cane molasses was obtained from Jiang-men sugar-renery
(Guangdong, PRC), and it contained 35% (w/w) sucrose, 10% (w/
w) converted sugars (glucose and fructose), 2.5% (w/w) other carbohydrates, 4.3% (w/w) crude protein, 0.06% (w/w) crude fat,
9.6% (w/w) ash, 4.6% (w/w) salt, 8.9% (w/w) metal ions such as calcium, potassium, sodium, iron, magnesium, copper, etc., and
25% (w/w) water. The crude molasses was diluted with distilled
water to obtain 4060 g l 1 total sugar concentration. For sulfuric
acid treatment, the molasses solution was adjusted to pH 3.5 with
5 M H2SO4, and heated at 60 C for 2 h. After centrifugation at
8000g for 15 min, the supernatant was adjusted to pH 6.0 with
10 M NaOH (Liu et al., 2008). Other pretreatment methods (resin,
potassium ferrocyanide, and activated carbon) described by Roukas (1998) and Kotzamanidis et al. (2002) were also studied. The
industrial grade resins used for the pretreatment of molasses were
obtained from Huazhen Tech. Co. (Shanghai, PRC).
2.3. Fermentation studies with brous-bed bioreactor
The fermentation system consisted of a 5-l stirred-tank fermentor (B. Braun, B. Braun Biotech International, Germany) connected
to a 0.5l brous-bed bioreactor through a recirculation loop and
operated at 37 C, agitated at 150 rpm, and pH controlled at 6.0
by adding NH4
OH facilitated by an on-line sensing and dosing
system (Fig. 1). The FBB was made of a glass column packed with
spiral wound cotton towel (organic; 185 mm  300 mm; 5 mm
in thickness; with >95% porosity). A detailed description of the
reactor construction has been given elsewhere (Silva and Yang,
1995). Anaerobic culture was maintained by initially sparging the
2 l medium with N2. To start the fermentation, 100 ml of cell suspension in serum bottle was inoculated to the fermentor and allowed to grow until the cell density reached 6.0. Cell
immobilization was then carried out by circulating the fermentation broth through the brous bed at a pumping rate of
25 ml min 1 to allow cells to attach and be immobilized onto
the brous matrix. After about 2 days of continuous circulation,
most of the cells were immobilized and no change in cell density

Fig. 1. Scheme of the immobilized FBB system used for the butyric acid
fermentation.

in the medium could be identied. The medium circulation rate

was then increased to 100 ml min 1 for new batch fermentation.
In the repeated batch mode, the fermentation broth was replaced
with sterile cane molasses as the carbon source to start a new
batch but the immobilized cells in the bioreactor were allowed
for continued growth batch after batch in order to investigate the
performance of butyric acid fermentation using cane molasses.
The fed-batch mode was started with 2 l of fresh medium, followed
with pulse feeding concentrated carbon source whenever its concentration in the fermentation broth was close to zero. To evaluate
the maximum butyric acid concentration achievable in the fermentation, the feeding was continued until the concentration of butyric
acid was kept stable because of product inhibition. Samples were
taken at regular intervals for the analysis of biomass, substrate,
and product concentrations.
2.4. Analytical methods
Free-cell density was analyzed by measuring the optical density
of the cell suspension at a wavelength of 600 nm (OD600) with a
spectrophotometer (Ultrospec 3300 pro, Amersham Bioscience).
Dry weight of immobilized-cell biomass was determined by centrifugation of the fermentation broth at 10,000g for 10 min, washing
the sediment with distilled water, and drying at 105 C overnight.
A high performance liquid chromatography (HPLC) system was
used to analyze the carbonhydrate compounds, including glucose,
fructose, and sucrose in the fermentation broth. The HPLC system
consisted of an automatic injector (Agilent 1100, G1313A), a pump
(Agilent 1100, G1311A), a Zorbax carbonhydrate analysis column
(250 mm  4.6 mm, 5 lm; Agilent, USA), a column oven at 30 C
(Agilent 1100, G1316A), and a refractive index detector (Agilent
1100, G1362A). The mobile phase was ethyl nitrile (ethyl nitrile/
water = 75:25) at a ow rate of 1.4 ml min 1. Quantitative analysis
of butyric acid and acetic acid was performed by gas chromatography (Agilent 6820 GE, Agilent Technologies) under the following
conditions:
glass
column
packed
with
HP-innovax
(30 m  0.320 mm), N2 as the carrier gas, temperatures of the
ame ionization detector and injection port set at 250 C, and column temperature at 250 C. The concentrations of butyric and acetic acid were determined according to a standard calibration curve.
3. Results and discussion
3.1. Utilization of different carbon sources by C. tyrobutyricum
To study the feasibility of using molasses as a low cost substrate
in butyric acid fermentation, fed-batch fermentation of glucose,

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L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

Table 1
Comparison of various carbon sources for butyric acid fermentation at pH 6.0.
Sugars

Butyric acid (g l

Glucose
Fructose
Sucrose
Mixturea

52.5 0.5
48.8 0.6
45.4 0.4
40.1 0.8

Acetic acid (g l
4.7 0.4
1.9 0.2
3.1 0.2
2.3 0.3

Butyric acid yield (g g

0.47 0.02
0.44 0.04
0.41 0.02
0.40 0.05

Reactor productivity
(g l 1 h 1)b

Butyrate/Acetate
ratio

Immobilized-cell biomass
(g l 1)

1.45 0.05
2.56 0.04
2.43 0.02
2.34 0.06

11.2 1.3
25.7 3.3
13.5 2.3
17.4 2.2

53.4 2.7
40.8 2.0
48.1 2.4
42.2 2.1

Sugar mixture: 10 g l 1 glucose, 15 g l 1 fructose, 35 g l 1 sucrose.

Reactor productivity for immobilized-cell fermentation was based on medium volume of 380 ml in the brous bed bioreactor, instead of the total liquid medium volume
of 2 l in the reactor system.
b

fructose, sucrose and their mixture as the major carbon source by

C. tyrobutyricum were studied at pH 6.0, and the results are shown
in Table 1. As can be seen in Table 1, C. tyrobutyricum could utilize
glucose, fructose, and sucrose to produce butyric acid. When the
cells were grown in the glucose-containing medium, butyric acid
production (52.5 0.5 g l 1) and yield (0.47 0.02 g g 1) were the
highest among the tested. Fructose and sucrose also yielded a considerable amount of butyric acid production (48.8 0.6 g l 1 vs.
45.4 0.4 g l 1). Since the inexpensive cane molasses consists primarily of sucrose, glucose and fructose, a mixture of these three
sugars which was approximating the ratio in molasses was used
as carbon source in the fermentation. It showed that the sugar mixture was fermented efciently by C. tyrobutyricum to produce
40.1 0.8 g l 1 butyric acid (yield 0.40 0.05 g g 1), which process
a great potential for producing butyric acid from cane molasses.
Interesting, good selectivity for butyric acid production was already achieved in these conditions with a maximum value of 96.3%
(Butyrate/Acetate ratio 25.7) when using fructose as the carbon
source. What is more, complete selectivity for butyrate production
could be achieved in glucose-limited, fed-batch fermentation with
immobilized-cell in FBB (data not shown). Also, such FBB system
with high cell densities (Table 1) would have a positive effect on
butyric acid yield and productivity, which will be discussed later
in this paper.
3.2. Effects of pH on butyric acid production in batch fermentation
It is reported that Clostridium bacteria are able to grow in a pH
range of 4.57.0 (Zigov et al., 1999). The butyric acid fermentation
was found to be best operated at pH 6.0, but a lower pH is desirable for producing free acids which are easier to be separated from
the fermentation broth (Wu and Yang, 2003). Therefore, the fermentation kinetics at various pH values (5.0, 5.5, 6.0 and 6.5) were
studied under batch fermentations utilizing cane molasses, and the
results are shown in Fig. 2. As shown in Fig. 2, the medium pH not
only affected cell growth and fermentation rate, but also changed
nal product yield and purity. The overall metabolic activity was
reduced at low pH as seen by low carbohydrate consumption
and impairment of cells. The optimal pH for cell growth on cane
molasses was 6.0 where the cell density in the medium reached
a maximum OD600 of 6.9. It is noted that there was a high density
of cells immobilized in the brous matrix and only a small portion
of the total cell population was present as suspended cells during
the fermentation. However, changes in the suspended cell concentration were the results of new cell growth activities in the reactor
system, and thus, can be used as an indicator of cell growth. Based
on the OD for the suspended cells in the medium, the maximum
specic growth rate, l, for cells grown on cane molasses was found
to be 0.073 h 1 at pH 6.0.
A maximum butyric acid concentration of 26.2 g l 1 was also
produced at pH 6.0, with only 3.9 g l 1 of acetic acid (Fig. 2). It appeared that at pH 6.0 or a little higher, acetate production was
inhibited by butyric acid when the latter reached up to 10 g l 1.

In contrast, at pH 5.5 and lower, acetate production continued with

butyrate production. When pH dropped to 5.0, acetate became the
major product and only a small amount of butyrate was produced
in the fermentation, it was obvious that there was a clear metabolic shift from butyrate-producing pathway to acetate-producing
pathway. The present results clearly indicated the optimal pH on
microorganism growth and butyric acid production was 6.0.
3.3. Repeated-batch fermentation in FBB
To evaluate the long-term performance of FBB system with cane
molasses as the substrate, repeated-batch fermentation was studied at pH 6.0. As shown in Fig. 3, there was no lag phase in all 10
batches, which represents an adaptation period necessary for cells
to live in a new environment. In contrast, the cells in the fermentation broth grew rapidly during the following 12 h after replacing
with fresh broth, indicating most cells immobilized in the brous
bed have a high vitality. This is consistent with that most reactions
occurred in the brous bed bioreactor, instead of in the fermentor
(Huang and Yang, 1998). The butyric acid was produced dramatically until reaching a maximum level of 26.2 g l 1, where the acetic acid concentration increased to a highest value of 5 g l 1 but
then dropped slightly and stayed at a relatively stable level till
the end of batch fermentations. The butyric acid yield from cane
molasses in all batch fermentations studied varied from 0.40 to
0.50 g g 1, with an average of 0.44 g g 1. The volumetric productivity ranged from 4.13 to 5.27 g l 1 h 1, with an average of
4.59 g l 1 h 1. The variations in yield and productivity were related
to the initial total sugar concentrations at each batch. The high results reached in this study are comparable or exceed to those either
from corn bre hydrolysate supplemented with corn steep liquor
by C. tyrobutyricum ATCC 25755 (2.91 g l 1 h 1 of productivity,
0.474 g g 1 of butyric acid yield) (Zhu et al., 2002) or from wheat
our hydrolysate by C. tyrobutyricum CIP I-776 (1.25 g l 1 h 1 of
productivity, 0.45 g g 1 of butyric acid yield) (Fayolle et al.,
1990). The highest reactor productivity ever published was
9.5 g l 1 h 1 with a butyrate concentration of 29.7 g l 1 in a continuous fermentation system with cell recycle by microltration,
which allowed operation with a high cell concentration maintained at a limited growth rate (Michel-Savin et al., 1990b). However, using a membrane lter for cell recycle to achieve high cell
density and reactor productivity could be a problem for long-term
operation and process scale-up because dead cells would accumulate and foul the membrane, reducing system performance with
time. In summary, the advantages of using immobilized cells in
the brous bed bioreactor for butyric acid production include high
reactor productivity and product yield, elimination of the lag
phase, and efcient continuous operation without repeated inoculation. However, there appeared to be culture degeneration in the
last batch fermentation, where the OD had a sharp decline, butyric
acid concentration decreased one half accompany with an increase
of acetic acid concentration. One reason to explain this phenomenon may be the high content of insoluble material in the cane

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L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

25

pH5.0

20

OD

-1

Concentration(g l )

30

15
10

2
5
0

10

glucose

fructose

15

20

25

Time(h)
sucrose
acetic acid

0
35

30

butyric acid

OD
8

30

pH5.5

25
20

OD

-1
Concentration(g l )

35

15
10

5
0

0
0

10

glucose

fructose

15

sucrose

20

25

Time(h)
acetic acid

30

35

butyric acid

OD
8

35

pH6.0
6

25
20

OD

-1

Concentration(g l )

30

15
10

5
0
35

0
0

10

15

20

25

30

Time(h)
glucose

fructose

sucrose

acetic acid

butyric acid

35
30

pH6.5
25

20

OD

Concentration(g l -1 )

OD

15
10

5
0

10

15

20

25

30

0
35

Time(h)
glucose

fructose

sucrose

acetic acid

butyric acid

OD

Fig. 2. Kinetics of batch fermentation of cane molasses by C. tyrobutyricum immobilized in FBB at pH 5.0, 5.5, 6.0, and 6.5.

molasses which cause clogging and increase pressure drop. However, we observed no signicant accumulation of insoluble materials. Most of these insoluble residues remained in the circulating

ow stream and were harvested through the fermentation medium. Meanwhile, the FBB could be easily recovered from the contamination by changing the contaminated medium to a new,

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L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

35
30
25

20

OD

-1

Concentration(g l )

40

15
10
5

86

glucose

158

fructose

235

Time(h)
sucrose
acetic acid

300

butyric acid

OD

Fig. 3. Kinetics of repeated-batch fermentation of cane molasses by C. tyrobutyricum immobilized in FBB at pH 6.0.

properly sterilized medium in the fermentor. The fermentation

rate and butyric acid yield returned to normal levels after replacing
the medium (data not shown). The other possibility is the accumulation of heavy metals which could be entrapped and encrusted
within the brous matrix in FBB through the circulating of cane
molasses medium one batch after another. It is in conrmation
of results by Lee et al. (1999) who reported that the glucose transport and cell growth were inhibited when the concentration of sodium ion was greater than 4 g l 1.
3.4. Effects of cane molasses pretreatment on butyric acid production
The production of butyric acid from pretreated cane molasses
using different chemical methods was carried out. As shown in Table 2, sulfuric acid treated molasses gave a marked increase in butyric acid concentration (34.6 0.8 g l 1), yield (0.58 0.01 g g 1),
and sugar utilization (90.8 0.9%), while the above values were
lower by 32.6%, 31.8%, and 12.3% with untreated molasses, respectively. The values of butyric acid yield obtained in this study
(0.58 g g 1 with Sulfuric acid treatment, 0.49 g g 1 with cation exchange resin treatment, 0.60 g g 1 with activated carbon treatment) even exceed the theoretical maximum value of 0.489 g g 1
calculated from the stoichiometric equations, which compels one
to the assumption that butyric acid is produced not only from
the sugar, but also from the substances derived from the breaking
down of albumen (amino acids), as in the analogical case of fuel
alcohol fermentation described by Thomas and Ingledew (1990),
where free amino nitrogen derived in situ through the hydrolysis
of wheat proteins by a protease could substitute for the exogenous
nitrogen source.
Although molasses normally contains most of the essential
nutrients for microorganisms, it also has a lot of metal ions (calcium, zinc, sodium, iron, magnesium, manganese, copper, etc.)
and suspended colloids, which could cause a critical problem during fermentation as they inhibit the growth of microorganisms,
inuence substrate pH, and are involved in the inactivation of enzymes associated with product biosynthesis. The superiority of sulfuric acid treatment over other methods of molasses treatment in
butyric acid fermentation may be due to the signicant removal
of heavy metals from molasses, which has been used to increase

the fermentative production of succinic acid (Liu et al., 2008). For

the pretreatment with cation exchange resin, the succinic acid concentration was comparable with that of sulfuric acid pretreatment,
yet the latter was an attractive alternative considering its low cost
as well as its signicant improvement in butyric acid production.
3.5. Fed-batch fermentation of pretreated cane molasses
Due to the inhibition of butyric acid, low product concentrations were attained in many cases. Fed-batch fermentation was
performed to adapt the bacterial culture to higher butyric acid concentrations and evaluated the maximum butyric acid concentration that can be produced from pretreated cane molasses. Fig. 4
shows typical fed-batch fermentation kinetics with immobilized
cells in the brous-bed bioreactor. As can be seen in Fig. 4, glucose,
fructose, and sucrose were readily fermented to produce butyric
and acetic acids. The bacterium metabolized glucose and fructose
simultaneously, indicating no preference for either one of the
two monosaccharides as carbon source. The bacterium grew exponentially in the rst fed-batch and then entered the stationary
phase. It continued to produce butyric acid until the butyric acid
concentration reached 55.2 g l 1, which was much higher than that
obtained in the batch fermentation (34.1 g l 1). However, the productivity and yield decreased dramatically at the butyrate concentration higher than 40 g l 1, and at the end of fermentation (90 h),
yield and productivity reached 0.46 g g 1, 3.22 g l 1 h 1, respectively, which decreased by 16.4%, 46.9% compared with the batch
fermentation, indicating a strong product inhibition at this level.
However, a remarkable difference between the batch and fedbatch fermentations was the production of acetic acid which
reached 5.3 g l 1, stopped when cell growth was inhibited by a
high concentration of butyric acid, and decreased to 2.6 g l 1 at
the end of the fed-batch fermentation. The lower nal concentration of acetic acid implied acetate reutilization and did not only result from a simple dilution effect due to the pulse feeding of the
fermentation broth, which was in conrmation of results previously obtained by Michel-Savin et al. (1990a) and Fayolle et al.
(1990), respectively. Another further positive effect of fed-batch
supply was the higher selectivity of butyric acid production discussed before, which resulted from particular of acetate reutiliza-

Table 2
Butyric acid production from pretreated molasses by C. tyrobutyricuma.
Pretreatment of molasses
Untreated molasses
Sulfuric acid treatment
732, Cation exchange resin
Potassium ferrocyanide treatment
Activated carbon treatment
a
b

Residual total sugar (g l

11.5 1.2
5.5 0.5
6.5 0.8
7.3 0.4
8.1 0.6

Butyric acid (g l
26.1 0.9
34.6 0.8
29.1 1.0
28.2 1.2
28.6 0.8

Acetic acid (g l
4.0 0.3
3.9 0.1
3.6 0.3
3.1 0.1
3.8 0.2

Cells were cultured in FBB for 30 h with an initial total sugar concentration of 60 g l 1.
Each value is an average of three parallel replicates and is represented as mean standard deviation.

Butyric acid yield (g g

0.44 0.02
0.58 0.01
0.49 0.02
0.47 0.02
0.60 0.01

Sugar utilization (%)

80.8 1.4
90.8 0.9
89.2 1.2
87.8 0.8
86.5 1.1

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L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

50
6

-1

40
4

30

OD

Concentration (g l )

60

20
2

10
0

10

glucose

20

fructose

30

40

50

60

Time (h)
sucrose
acetic acid

70

80

butyric acid

0
90

OD

Fig. 4. Kinetics of fed-batch fermentation of pretreatment cane molasses by C. tyrobutyricum immobilized in FBB at pH 6.0.

tion. The butyrate/acetate ratio (g g 1) increased from in batch culture to in fed-batch culture, a clear indication that the metabolic
pathway in the bacterial had been shifted. The production of acetate at a high growth rate in batch cultures can be readily interpreted as acetate production yields more ATP than butyrate
production and is better suited to meet the energy demands of rapid growth. However, energetic considerations do not lead to easy
interpretation for the reutilization of acetate and its conversion to
butyrate during growth in a phase of lower rate, although the reutilization of both acetate and butyrate to produce solvents is well
known in butanol/acetone fermentation (Qureshi et al., 2001). One
possibility could be that maintaining the external concentration of
acetate is also energy consuming thus leading to its re-utilization.
Noting that acetate conversion to butyrate involves the utilization
of protons, and that pH has been found to affect the selectivity of
butyrate production, may hint at a relationship with the proton
balance of metabolism.
4. Conclusions
In this study, the brous bed bioreactor system with immobilized C. tyrobutyricum for butyric acid production from cane molasses was developed for the rst time, and was proved to be
competent considering its almost perfect performance. The pH
had profound effects on butyrate fermentation, changing from a
predominant butyrate production at pH 6 to predominant acetate
production at pH 5, and allowed for the highest level of butyrate
at pH 6.0. Although all the pretreatment methods would cause
68% drop in the total sugar of cane molasses, whereas the dramatically improvement of the butyrate concentration, yield and reactor productivity made it attractive and competitive. During the
fed-batch fermentation in FBB, the maximum concentration of
butyrate could be achieved using sulfuric acid treated molasses
(55.2 g l 1), which is the best pretreatment technique for molasses.
In conclusion, cane molasses could be used as an economical
and feasible carbon source for the butyric acid production by C.
tyrobutyricum. The present study should be potentially useful for
the economical butyric acid production on industrial scale.
Acknowledgement
This work was supported by a grant from the Ministry of Science and Technology of China (National Basic Research Program
of China, 2007CB707805).
References
Alam, Shahriar, Stevens, David, Bajpai, Rakesh, 1988. Production of butyric acid by
batch fermentation of cheese whey with Clostridium beijerinckii. J. Ind.
Microbiol. 2, 359364.

Cazetta, M.L., Celligoi, M.A.P.C., Silva, R.S.F., et al., 2005. Optimization study for
sorbitol production by Zymomonas mobilis in sugar cane molasses. Process
Biochem. 40, 747751.
Chanto, A.Q., Afschar, A.S., Wagner, F., 1994. Microbial production of propionic acid
and vitamin B12 using molasses or sugar. Appl. Microbiol. Biotechnol. 41, 378
383.
Crabbendam, P.M., Neijssel, O.M., Tempest, D.W., 1985. Metabolic and energetic
aspects of the growth of Clostridium butyricum on glucose in chemostat culture.
Arch. Microbiol. 142 (4), 375382.
El-Enshasy, H.A., Mohamed, N.A., El-Diwany, A.I., et al., 2008. Improvement of
erythromycin production by Saccharopolyspora erythraea in molasses based
medium through cultivation medium optimization. Bioresour. Technol. 99,
42634268.
Fayolle, F., Marchal, R., Ballerini, D., 1990. Effect of controlled substrate feeding on
butyric acid production by Clostridium tyrobutyricum. J. Ind. Microbiol. 6, 179
183.
Haq, Ikram-ul, Ali, Sikander, Qadeer, M.A., et al., 2004. Citric acid production by
selected mutants of Aspergillus niger from cane molasses. Bioresour. Technol. 93,
125130.
Huang, Yan, Yang, Shang-Tian, 1998. Acetate production from whey lactose using
co-immobilized cells of homolactic and homoacetic bacteria in a brous-bed
bioreactor. Biotechnol. Bioeng. 60, 498507.
Huang, Y.L., Wu, Z., Yang, S.T., et al., 2002. Production of carboxylic acids from
hydrolyzed corn meal by immobilized cell fermentation in a brous-bed
bioreactor. Bioresour. Technol. 83, 5159.
Kotzamanidis, C., Roukas, T., Skaracis, G., 2002. Optimization of lactic acid
production from beet molasses by Lactobacillus delbrueckii NCIMB8130. World
J. Microbiol. Biotechnol. 18, 441448.
Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N., 1999. Effects of medium components on
the growth of Anaerobiospirillum succiniciproducens and succinic acid
production. Process Biochem. 35, 4955.
Liu, X.G., Yang, S.T., 2006. Butyric acid and hydrogen production by Clostridium
tyrobutyricum ATCC 25755 and mutants. Enzyme Microb. Technol. 38, 521528.
Liu, X.G., Zhu, Y., Yang, S.T., 2006. Kinetics of butyric acid fermentation of glucose
and xylose by Clostridium tyrobutyricum wild type and mutant. Process
Biochem. 41, 801808.
Liu, Yu-Peng, Zheng, Pu, Zhu, Lei-Lei, et al., 2008. Economical succinic acid
production from cane molasses by Actinobacillus succinogenes. Bioresour.
Technol. 99, 17361742.
Liu, Chun-Zhao, Wang, Feng, Fan, Ou-Yang, 2009. Ethanol fermentation in a
magnetically uidized bed reactor with immobilized Saccharomyces cerevisiae
in magnetic particles. Bioresour. Technol. 100, 878882.
Lotfy Walid, A., 2008. Production of a thermostable uricase by a novel Bacillus
thermocatenulatus strain. Bioresour. Technol. 99, 699702.
Michel-Savin, D., Marchal, R., Vandecasteele, J.P., 1990a. Control of the selectivity of
butyric acid production and improvement of fermentation performance with
Clostridium tyrobutyricum. Appl. Microbiol. Biotechnol. 32, 387392.
Michel-Savin, D., Marchal, R., Vandecasteele, J.P., 1990b. Butyric fermentation:
metabolic behavior and production performance of Clostridium tyrobutyricum in
a continuous culture with cell recycle. Appl. Microbiol. Biotechnol. 34, 172177.
Najafpour, G.D., 2006. Immobilization of microbial cells for the production of
organic acid and ethanol. Biochem. Eng. Biotechnol., 199227.
Najafpour, G.D., Shan, C.P., 2003. Enzymatic hydrolysis of molasses. Bioresour.
Technol. 86, 9194.
Playne, M.J., 1985. Propionic and butyric acids. In: Moo-Young, M. (Ed.),
Comprehensive Biotechnology. Pergamon Press, New York, pp. 731759.
Pouillart, P.R., 1998. Role of butyric acid and its derivatives in the treatment of
colorectal cancer and hemoglobinopathies. Life Sci. 63, 17391760.
Qureshi, N., Meagher, M.M., Huang, Jicai, Hutkins, R.W., 2001. Acetone butanol
ethanol recovery by pervaporation using silicalitesilicone composite
membrane from fed-batch reactor of Clostridium acetobutylicum. J. Membrane
Sci. 187, 93102.
Roukas, T., 1998. Pretreatment of beet molasses to increase pullulan production.
Process Biochem. 33, 805810.

L. Jiang et al. / Bioresource Technology 100 (2009) 34033409

Sharma, Amit, Vivekanand, V., Singh Rajesh, P., 2008. Solid-state fermentation for
gluconic acid production from sugarcane molasses by Aspergillus niger ARNU-4
employing tea waste as the novel solid support. Bioresour. Technol. 99, 3444
3450.
Shukla, V.B., Zhou, S., Ingram, L.O., et al., 2004. Production of D ( )-lactate from
sucrose and molasses. Biotechnol. Lett. 26, 689693.
Silva, E.M., Yang, S.T., 1995. Kinetics and stability of a brous-bed bioreactor for
continuous production of lactic from unsupplemented acid whey. J. Biotechnol.
41 (1), 5970.
Thomas, K.C., Ingledew, W.M., 1990. Fuel alcohol production: effects of free amino
nitrogen on fermentation of very-high-gravity wheat mashes. Appl. Environ.
Microbiol. 56 (7), 20462050.
Van Andel, J.G., Zouttberg, G.R., Crabbendam, P.M., Breure, A.M., 1985. Glucose
fermentation by Clostridium butyricum grown under a self generated gas
atmosphere in chemostat culture. Appl. Microbiol. Biotechnol. 23, 2126.

3409

Vandk, D., Telgarsky, M., turdk, E., 1995. Inuence of growth factor supplements
on butyric acid production from sucrose by Clostridium butyricum. Folia
Microbiol. 40, 669672.
Wu, Z., Yang, S.T., 2003. Extractive fermentation for butyric acid production from
glucose by Clostridium tyrobutyricum. Biotechnol. Bioeng. 82, 93102.
Yang, S.T., Zhu, H., Hong, G., et al., 1994. Continuous propionate production from
whey permeate using a novel brous bed bioreactor. Biotechnol. Bioeng. 43
(11), 11241130.
Zhu, Y., Wu, Z., Yang, S.T., 2002. Butyric acid production from acid hydrolysate of
corn ber by Clostridium tyrobutyricum in a brous bed bioreactor. Process
Biochem. 38, 657666.
Zigov, J., Sturdik, E., Vandak, D., Schlosser, S., 1999. Butyric acid production by
Clostridium butyricum with integrated extraction and pertraction. Process
Biochem. 34, 835843.