Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 21062110

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Journal of the Taiwan Institute of Chemical Engineers

journal homepage: www.elsevier.com/locate/jtice

An integrated in situ extraction-gas stripping process

for AcetoneButanolEthanol (ABE) fermentation
Kuan-Ming Lu, Si-Yu Li *
Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 17 March 2014
Received in revised form 24 June 2014
Accepted 29 June 2014
Available online 31 July 2014

A novel integrated in situ extraction-gas stripping process (Process 3 in this study) is proposed for
running batch AcetoneButanolEthanol (ABE) fermentation. The process involves two butanol
separation processes in which butanol is rst extracted by oleyl alcohol during ABE fermentation and gas
stripping is carried out on butanol in the oleyl alcohol phase at the same time. The butanol productivity
and yield of Process 3 is 0.28  0.01 g/L/h and 0.226  0.001 g-butanol/g-glucose. The ABE productivity and
yield are 0.46  0.01 g/L/h and 0.374  0.002 g-ABE/g-glucose. Glucose utilization was 97% and initial
glucose consumption was 121  2 g/L. Butanol and ABE concentrations of 93113 and 166204 g/L in the
condensate can be achieved. This study demonstrates that Process 3 as described here enhances ABE
fermentation and also results in the production of high purity solvents.
2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords:
Clostridium acetobutylicum
AcetoneButanolEthanol (ABE)
fermentation
Extraction
Gas stripping
Oleyl alcohol

1. Introduction
1-Butanol has been proposed as an alternative fuel that is both
renewable and sustainable and can be produced by traditional
AcetoneButanolEthanol fermentation using Clostridium acetobutylicum or Clostridium beijerinckii [1]. One obstacle for ABE
fermentation is the toxicity of 1-butanol which results in low
productivity and high recovery costs for conventional distillation
[24]. One promising way to tackle butanol toxicity is by the use of
in situ product recovery [5]. Many bio-compatible separation
processes, such as gas stripping [6,7], extraction [810], and
pervaporation [11,12] have been proposed and investigated for in
situ butanol recovery during ABE fermentation. Gas stripping and
extraction are two well known methods that are easy to maintain
and scale-up. However, both gas stripping and extraction have low
selectivity for butanol during the ABE fermentation process. Also,
the low selectivity of the extraction further leads to the need for
large volumes of solvent in the batch reactor to keep the butanol
concentration in the aqueous phase below the threshold of around
10 g/L. In practice, the best ABE fermentation performance was
achieved with a solvent to fermentation broth volume ratio of 1.75
[13]. However, this dramatically decreases the volume available
for ABE fermentation in the batch reactor.

In this study, a combination of extraction and gas stripping as

an integrated process is proposed for in situ butanol recovery in
ABE fermentation as shown in Fig. 1. The non-volatile solvent oleyl
alcohol acts as the extraction solvent and nitrogen is used for gas
stripping. Oleyl alcohol is biocompatible and a feasible extraction
agent for in situ butanol recovery under these conditions [13,14].
The performance of integrated in situ extraction-gas stripping
during ABE fermentation is investigated and discussed.
2. Materials and methods
2.1. Strains, culture media, and growth conditions
The bacterial strain used in this study was C. acetobutylicum
ATCC 824. The culture was in spore form and stored at room
temperature. Each liter of the fermentation medium used in this
study contained: 38 g Reinforced Clostridial Medium (RCM), 0.11 g
FeSO47H2O, 0.6 g MgSO47H2O, 0.008 g CaCl2, and 125 g glucose.
For batch ABE fermentation, 32 mL of C. acetobutylicum spore stock
solution was added aseptically to 300 mL of fresh medium without
using a heat-induced germination process. Batch ABE fermentation
was carried out at 37 8C with stirring at 150 rpm.
2.2. Integrated in situ extraction-gas stripping ABE fermentation

* Corresponding author. Tel.: +886 4 2284 0510x509.

E-mail address: syli@dragon.nchu.edu.tw (S.-Y. Li).

As shown in Fig. 1, the equipment used in the integrated in situ

extraction-gas stripping process includes a 500-mL serum bottle, two

http://dx.doi.org/10.1016/j.jtice.2014.06.023
1876-1070/ 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 21062110

2107

Fig. 1. Schematic of integrated in situ extraction-gas stripping process for ABE fermentation.

cold traps (1 and 2) in parallel for the condensation of stripped

solvents, and a cylinder of nitrogen gas. A 500-ml serum bottle was
used as the reactor throughout the study. The serum bottle contained
300 mL of fresh medium and 100 mL of oleyl alcohol (8085%, Alfa
Aesar1). Cold trap 3 was used for security to ensure condensation of
all stripped solvents. All cold traps were immersed in liquid nitrogen
at 196 8C. In all the experiments the nitrogen gas for gas stripping
was sterilized using a 0.2 mm Millipore membrane lter and
delivered through a single 18-G syringe needle at a ow rate of
0.5 L/min. 0.2 mm lters were used to lter-sterilize the nitrogen gas
stream throughout the experiment. Gas stripping was performed on
the oleyl alcohol phase and was initiated after a fermentation time of
48 h. Condensates from the cold trap were analyzed by gas
chromatography (GC) at 12 hourly intervals as described below.
2.3. Analytical methods
The cell density was measured at 600 nm using a UVvis
spectrophotometer (GENESYS 10S, Thermo Scientic, USA). The
concentration of residual sugars was determined by the DNS
method [15]. The concentrations of acetone, butanol, ethanol,
acetate, and butyrate in the fermentation culture solution were
determined by GC (Hewlett Packard HP 5890 Series II) using a
ame ionization detector and a capillary column DP-FFAP
(30 m  0.32 mm  0.25 mm) as described earlier [16].
3. Results and discussion
3.1. Performance of ABE fermentation using an in situ extraction-gas
stripping separation process
ABE fermentation was carried out using the in situ extractiongas stripping separation process described here, while batch ABE

fermentation as well as batch ABE fermentation with in situ

extraction were used as controls. It can be seen in Fig. 2a that
without the heat-induced germination treatment, a 36-h lag phase
was observed for batch ABE fermentation. Despite this, the
clostridial culture still reached a peak OD600 of 9.6  1.5 after
72 h. The glucose concentration in Process 1 remained steady for the
rst 36 h and then decreased strongly up to 72 h, see process 1 in
Fig. 2b. The total glucose consumption in process 1 was 57  8 g/L in
72 h. The concentration of butanol in Process 1 (see Fig. 2c) started
accumulating after 48 h when bacterial growth entered the
exponential phase and the nal butanol titer reached a concentration
of 13.0  0.7 g/L. Note that the nal acetone and ethanol titers were
9.0  0.4 and 2.1  0.3 g/L, respectively (data not shown). This data
suggests that the direct use of spores is feasible and can be used for
initiating ABE fermentation.
Both bacterial growth and glucose consumption can be
enhanced by incorporating in situ butanol extraction with oleyl
alcohol. It can be seen in Fig. 2a that the peak OD600 in Process 2
reached 12.2  0.7 by 72 h and total glucose consumption reached
96  9 g/L in 96 h. Process 2 showed signicant improvements in ABE
fermentation over Process 1 and this can be attributed to the efcient
in situ extraction of toxic butanol from the fermentation broth into the
oleyl alcohol phase [8,13,14]. At 96 h the butanol concentrations in
the aqueous and organic phases were 9  1 and 40  5 g/L,
respectively. On the other hand, while butanol was selectively
extracted into the oleyl alcohol, this was not the case with acetone
and ethanol. It was found that the concentration of acetone and
ethanol in the organic phase was lower than in the aqueous phase
where their partition ratios were 0.4 and 0.2, respectively (data not
shown). These are consistent with previous data [13,14]. For example,
Ishizaki et al. reported that the partition ratios of acetone and ethanol
were 0.3 and 0.2, respectively [14]. Although 60% of the butanol
produced was extracted from the aqueous phase to the organic phase,

Table 1
Characteristics of ABE fermentation for Process 1, 2, and 3.a
Characteristics

Process 1

Initial glucose (g/L)

Final glucose (g/L)
Glucose consumption (g/L)
Glucose utilization (%)
Glucose consumption rate (g/L/h)b
Max. optical density (time)
Butanol production (g/L)
Total ABE production (g/L)
Butanol productivity (g/L/h)b
Total ABE productivity (g/L/h)b
Butanol yield (g/g)
Total ABE yield (g/g)

125  1
65  6
57  8
48  5
0.79  0.11
9.61 (72 h)
12.5  0.3
23.1  0.7
0.18  0.01
0.33  0.02
0.21  0.02
0.39  0.03

Process 2
115  2
19  10
96  9
83  9
1.00  0.09
12.17 (72 h)
22  3
34  4
0.23  0.03
0.36  0.04
0.23  0.02
0.36  0.03

Process 3
121  2
3.6  0.2
117  2
97.0  0.2
1.22  0.02
14.57 (96 h)
26.5  0.5
43.9  0.6
0.28  0.01
0.46  0.01
0.226  0.001
0.374  0.002

a
Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ
extraction-gas stripping ABE fermentation. Error bars represent standard deviation.
b
Fermentation time frames for calculating the glucose consumption rate and solvent productivities are 72, 96, and 96 h for Process 1, 2, and 3,
respectively.

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K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 21062110

Fig. 2. (a) Bacterial growth, (b) glucose consumption, (c) butanol production of three fermentation processes, and (d) partition ratios of butanol in the organic phase to butanol
in the aqueous phase. Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ
extraction-gas stripping ABE fermentation. Error bars represent standard deviations.

it was found that 9  1 g/L of butanol remained and had a negative

effect on clostridial growth. The cell density in Process 2 decreased
and there was a 19  10 g/L of glucose residue. Although a lower
volume ratio of fermentation broth to oleyl alcohol could be achieved,
the ABE fermentation capacity of a single fermentor would be less.
The dilemma encountered in Process 2 can be resolved by using
integrated in situ extraction-gas stripping, Process 3 in this study.
While it can be seen from Fig. 2a that all three processes had
similar specic growth rates during the early exponential phase,
bacterial growth in Process 3 had an extended growth prole and
reached an OD600 value of 14.6  0.7 at 96 h. An examination of
Fig. 2b shows that bacterial culture consumed 97% of the available
glucose in 96 h with a total glucose consumption of 117  2 g/L. This
suggests that there was no obvious inhibition of bacterial growth and
glucose consumption over the total fermentation time of 96 h. This
nding is supported by the data in Fig. 2c which shows that the
highest butanol concentration in the aqueous solution, during Process
3 fermentation, was only 6.4  0.6 g/L at 96 h. Also, the butanol
concentrations in the organic phase reached 16.6  2.3 and
20.9  1.3 g/L at 72 and 96 h, respectively. These high butanol
concentrations served as a strong driving force for the mass transfer of
butanol from liquid to the gas bubbles during gas stripping. Note that
the partition of butanol between the organic and aqueous phases
dropped from 4.64.9 (Process 2) to 3.23.5 (Process 3) after the
initiation of gas stripping at 48 h, see Fig. 2d. This suggested that the
organic phase in Process 3 was not saturated with butanol during
fermentation. Therefore, a mass transfer resistance for butanol
extraction in Process 3 was noticeable [17]. Nevertheless, ABE
fermentation using Process 3 is still signicantly better than in
Process 1 and 2. Table 1 shows that the glucose utilization in Process 3
is 102% and 17% higher than that in Process 1 and Process 2,
respectively. It can be seen in Fig. 3 that nal titers of butanol,

acetone, and ethanol in Process 3 are higher than that in Processes 1

and 2, where the nal titers of butanol and ABE from Process 3 at 96 h
are 26.5  0.5 and 43.9  0.6 g/L, respectively. Note the calculation of
solvent titers for Process 3 is based on the total amount of solvents in
the fermentation broth, oleyl alcohol, and condensates with respect to
the volume of fermentation broth. While the three processes have
similar butanol and ABE yields, the butanol and ABE productivity of
Process 3 is signicantly better. The butanol productivity in process 3

Fig. 3. Comparisons of ABE fermentation performance using processes 1, 2, and 3.

Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ
butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas
stripping ABE fermentation. Note: the total solvent concentration in Process 3 is the
sum of that in the aqueous, organic, and condensate phases with respect to the
volume of fermentation broth. Error bars represent standard deviation.

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2109

is 0.276  0.005 g/L/h, indicating a 22% and 56% higher than that of
Process 2 and 1, respectively. Also, the ABE productivity in Process
3 is 0.458  0.007 g/L/h, which is 28% and 39% higher than that of
Process 2 and 1, respectively. The enhanced productivity achieved in
Process 3 results from the fact that more glucose is consumed in a
single batch because toxic butanol is continuously removed.
Fermentation Process 3 as presented in this study is very competitive
compared to others [6,7,1820]. Table 1 summarizes the performance of Process 1, 2, and 3.
3.2. Separation efciency of the integrated extraction-gas stripping
process
Performance of in situ gas stripping of butanol from oleyl
alcohol during ABE fermentation is discussed. It can be seen in
Fig. 4a that concentrations of acetone, ethanol, and butanol in the
rst 12 h condensate were 45  17, 12  1, and 82  14 g/L,
respectively. During the stripping process the concentration of
acetone, ethanol, and butanol reached peaks of 71  5, 19  1, and
113  5 g/L in the condensate of the third batch. This increase in
solvent concentration simply reects an increase in solvent
concentrations in the oleyl alcohol layer during fermentation. A
total ABE solvent concentration of 160200 g/L in the condensate can
be achieved with Process 3. It has been shown in previous studies that
common ABE concentrations in the condensate spanned a range of
between 20 and 80 g/L [2124]. The ndings of this study represent a
great improvement in terms of the purity of the recovered solvents
while the butanol ux was maintained. It is also notable that no oleyl
alcohol, butyric acid, nor acetic acid is detected in the condensate, and
this is an indication that short chain fatty acids are difcult to strip
from either the aqueous phase [7,18,19,22] or the oleyl alcohol during
ABE fermentation.
It was interesting to see that there were 800850 g/L of water in
the condensate even though gas stripping of the solvents was
performed directly in the organic phase layer. This can be
attributed to out-gassing from bacterial growth where the released
gas carries water from the fermentation broth to the condensate.
Considering the nal composition of the condensate, the average
water removal rate was roughly four times higher than the average
removal rate of ABE, see Fig. 4b. This suggests a way to further
increase the purity of solvents using this process would be to
increase the ux of solvents, where it has been shown previously
that the solute removal rate (g-solute/h) is directly proportional to
the stripping gas ow rate [2527]. As shown in Fig. 4b, ABE
removal rates of 0.140.17 g/h were achieved over 1248 h with a
stripping gas ow rate of 0.5 L/min. Clearly, the solvent removal
rate could be improved by increasing the ow rate of the stripping
gas. Furthermore, the solvent purity in the condensate can be
improved by an increase in the amount of solvent in the
condensate while the amount of water it contains remains steady.
Two other techniques can be employed with Process 3 to further
improve the degree of solvent purity in the condensate. It has been
shown that a high ABE concentration of 195.9 g/L can be achieved
by using the fractional condensation during recycled in situ gas
stripping [6]. By using cooling water at 2 8C, the butanol, which has
a relatively low vapor pressure, was condensed while water
remained in the vapor [6]. Another promising technique that could
be used with Process 3 is two-stage in situ gas stripping [7]. Xue
et al. demonstrated that a twofold increase in ABE concentration in
the condensate could be achieved [7].
The separation efciency of each step of Process 3 was
evaluated by using the separation factor. The separation factor
for butanol extraction is dened as follows:

ab;ao

yb;ao =1  yb;ao
xb;ao =1  xb;ao

(1)

Fig. 4. Performance of butanol gas stripping from oleyl alcohol during ABE
fermentation. (a) solvent concentrations in the condensate, (b) solvent removal
rates, and (c) average butanol selectivity of extraction, gas stripping, and the
combination of extraction and gas stripping. Gas stripping was initiated after 48 h of
ABE fermentation. Error bars represent standard deviation.

where yb,ao is the mass fraction of butanol in the organic phase

while xb,ao is the mass fraction of butanol in the aqueous phase.
The separation factor for gas stripping of butanol is dened as
follows:

ab;oc

yb;oc =1  yb;oc
xb;oc =1  xb;oc

(2)

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K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 21062110

where yb,oc is the mass fraction of butanol in the condensate while

xb,oc is the mass fraction of butanol in the organic phase. It can be
seen in Fig. 4c that in the rst 24 h stripping period, the butanol
selectivity for extraction and gas stripping were 5.48 and 6.45,
respectively. The overall selectivity of Process 3 was 35.31. In the
second 24 h-stripping period, the butanol selectivity for extraction
and gas stripping dropped to 3.96 and 5.06, respectively. The high
separation factor of 35.31 suggests that the integrated in situ
extraction-gas stripping process has a good separation factor that
can be compared to pervaporative butanol separation where the
separation factor typically lies in a range between 30 and 120 [12].

4. Conclusions
The butanol productivity and yield of 0.28  0.01 g/L/h and
0.226  0.001 g-butanol/g-glucose were achieved with a novel
integrated in situ extraction-gas stripping process. The ABE productivity and yield were 0.46  0.01 g/L/h and 0.374  0.002 g-ABE/gglucose. The glucose utilization was 97% where the initial glucose
consumption was 121  2 g/L. Butanol and ABE concentrations of 93
113 and 166204 g/L in the condensate can be achieved. This study
demonstrated that an enhanced ABE fermentation can be achieved
with Process 3 while a high purity of solvents is simultaneously
obtained.
Acknowledgement
This work was funded by National Science Council Taiwan,
NSC102-2221-E-005-064.
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