Appl Microbiol Biotechnol
DOI 10.1007/s00253-017-8520-1
BIOENERGY AND BIOFUELS
High-efficiency acetone-butanol-ethanol production and recovery
in non-strict anaerobic gas-stripping fed-batch fermentation
Zhangnan Lin 1 & Hongjuan Liu 1 & Xiang Yan 1 & Yujie Zhou 1 & Keke Cheng 2 &
Jian’an Zhang 1
Received: 5 July 2017 / Revised: 22 August 2017 / Accepted: 7 September 2017
# Springer-Verlag GmbH Germany 2017
Abstract Conventional acetone-butanol-ethanol (ABE) fer-
mentation coupled with gas stripping is conducted under strict
anaerobic conditions. In this work, a fed-batch ABE fermen-
tation integrated with gas stripping (FAFIGS) system using a
non-strict anaerobic butanol-producing symbiotic system,
TSH06, was investigated for the efficient production of buta-
nol. To save energy and keep a high gas-stripping efficiency,
the integrated fermentation was conducted by adjusting the
butanol recovery rate. The gas-stripping efficiency increased
when the butanol concentration increased from 6 to 12 g/L.
However, in consideration of the butanol toxicity to TSH06,
8 g/L butanol was the optimal concentration for this FAFIGS
process. A model for describing the relationship between the
butanol recovery rate and the gas flow rate was developed,
and the model was subsequently applied to adjust the butanol
recovery rate during the FAFIGS process. In the integrated
* Keke Cheng
chengkeke@dgut.edu.cn
* Jian’an Zhang
zhangja@tsinghua.edu.cn
Zhangnan Lin
linzhangnan1@163.com
Hongjuan Liu
liuhongjuan@tsinghua.edu.cn
Xiang Yan
yanxiang007@mail.tsinghua.edu.cn
Yujie Zhou
zhouyj@mail.tsinghua.edu.cn
1
Institute of Nuclear and New Energy Technology, Tsinghua
University, Beijing 100084, People’s Republic of China
2
School of Chemical Engineering and Energy Technology, Dongguan
University of Technology, Dongguan 523808, China
system under non-strict anaerobic condition, relatively stable
butanol concentrations of 7 to 9 g/L were achieved by con-
trolling the gas flow rate which varied between 1.6 and
3.5 vvm based on the changing butanol productivity.
185.65 g/L of butanol (267.15 g/L of ABE) was produced in
288 h with a butanol recovery ratio of 97.36%. The overall
yield and productivity of butanol were 0.23 g/g and 0.64 g/L/
h, respectively. This study demonstrated the feasibility of
using FAFIGS under non-strict anaerobic conditions with
TSH06. This work is helpful in characterizing the butanol
anabolism performance of TSH06 and provides a simple and
efficient scheme for butanol production.
Keywords Fed-batch ABE fermentation . TSH06 . Gas
stripping . Recovery rate regulation . Non-strict anaerobic
fermentation
Introduction
Butanol production by acetone-butanol-ethanol (ABE) fer-
mentation is now receiving increasing global attention due
to the gradual exhaustion of fossil fuels and the rising con-
sciousness of environmental protection (Qureshi et al. 2008a,
b; Nigam and Singh 2011). Compared with ethanol, butanol
has many advantages as a fuel, including higher energy den-
sity, lower volatility, more similar performance to gasoline,
and less corrosion to current automobile engines and transpor-
tation pipelines (Dürre 2007). However, product toxicity, es-
pecially that of butanol, is one of the main problems restricting
the industrialization of ABE fermentation. Butanol can inhibit
cell growth, which results in low butanol production, produc-
tivity, and yield (Setlhaku et al. 2013; Ezeji et al. 2010). In
general, the total ABE production is no more than 20 g/L, of
which butanol is only approximately 13 g/L (Ezeji et al.

2004), making the recovery of butanol an energy-intensive
process (Mariano et al. 2011; Kraemer et al. 2011).
The solutions to overcoming the issue of butanol toxicity
have been studied during the last few decades. One approach
is to utilize genetic techniques, such as mutagenesis and met-
abolic engineering, to improve butanol tolerance of strains;
butanol production reaches 20 g/L in mutants (Ezeji et al.
2004; Chen and Blaschek 1999). However, only 20 g/L of
butanol was obtained when such skills were applied, which
was far lower than the concentration of bio-ethanol that has
been obtained (120–150 g/L). In comparison, the integration
of ABE fermentation with product recovery techniques has
improved butanol production and productivity significantly.
Online recovery of ABE via gas stripping, extraction, adsorp-
tion, or pervaporation could not only reduce the cytotoxicity
of butanol remarkably but also obtain high concentrations of
ABE products at the same time (Staggs and Nielsen 2015).
Among these technologies, gas stripping has been demonstrat-
ed to cause no harm to cells, be environmental friendly, oper-
ate easily, and be more economical, leading to it being a fa-
vorable choice (Ezeji et al. 2013; Xue et al. 2012). And nu-
merous studies of integrated ABE fermentation with online
gas stripping have shown huge increases in production and
productivity (Ezeji et al. 2007; Ezeji et al. 2013; Xue et al.
2012; Cai et al. 2015). However, all previous studies on ABE
fermentation integrated with online gas stripping were con-
ducted under anaerobic conditions and without concern for
the adjustment of the recovery rate.
Traditional butanol fermentation has to be operated under
strict anaerobic conditions, and the fermentation medium
needs to be flushed with nitrogen gas to remove the dissolved
oxygen, which makes the butanol fermentation process com-
plex and of high cost. The symbiotic system TSH06, which
includes Clostridium acetobutylicum TSH1 and Bacillus
cereus TSH2, was obtained in our previous work and exhib-
ited excellent butanol production under non-strict anaerobic
condition (Wu et al. 2016a). Compared with strict anaerobes,
the culture of TSH06 does not need special equipment and
complicated operations to eliminate the oxygen in the sub-
strate. In batch fermentation, butanol production reached
11.9 g/L under non-strict anaerobic condition (Wu et al.
2016a, b). To further improve butanol productivity, fed-
batch ABE fermentation integrated with gas stripping
(FAFIGS) for butanol production using TSH06 under non-
strict anaerobic condition was conducted in this study. In this
process, FAFIGS was carried out without nitrogen gas flush-
ing and oxygen removal, which could make the process eco-
nomical and easy to operate.
In the integrated fermentation process, the butanol concen-
tration should be maintained at a relatively high concentration
(> 8 g/L) to reach a high product recovery rate (Xue et al.
2012). But a too high butanol concentration has a negative
effect on the strain or could even stop the fermentation. It is
Appl Microbiol Biotechnol
necessary to develop a kinetic model to control the butanol
concentration during the fermentation. Considering that the
butanol productivities were different at different fermentation
periods, in the process of FAFIGS, the butanol recovery rate
should be adjusted based on the changing butanol productiv-
ities in order to control the butanol concentration.
The objective of this study is to investigate the feasibility of
FAFIGS under non-strict anaerobic conditions and in a more
stable state for high-concentration butanol production with the
non-anaerobic butanol-producing symbiotic system TSH06.
The butanol tolerance of TSH06 was investigated first in this
study. And a kinetic model was developed to illustrate the
relationship between gas flow rate and the gas-stripping effi-
ciency. The developed model was further used for keeping a
dynamic equilibrium of butanol concentration during the in-
tegrated ABE fermentation process. This study is helpful for
the deep understanding on the butanol synthesis capability of
TSH06 and could promote the industrialization of butanol
fermentation.
Materials and methods
Strains
The symbiotic system TSH06 included C. acetobutylicum
TSH1 and B. cereus TSH2, which was isolated from corn
powder in the agricultural areas of Beijing.
C. acetobutylicum TSH1 and B. cereus TSH2 were deposited
in the China General Microbiological Culture Collection
Center (CGMCC) as CGMCC 8071 and CGMCC 8072,
respectively.
Medium
The bacterial enrichment LB medium (pH 7.5) contained
yeast extract (3.0 g/L), peptone (10.0 g/L), and NaCl (5.0 g/
L). The hemi-synthesis P2 medium contained solution I (glu-
cose (65 g/L) and yeast extract (1 g/L)), buffer (ammonium
acetate (220 g/L), KH2PO4 (50 g/L), and K2HPO4 (50 g/L)),
and vitamins and mineral salts (biotin (0.001 g/L), thiamin
(0.1 g/L), para-amino-benzoic acid (0.1 g/L), NaCl (1 g/L),
MnSO4·H2O (1 g/L), FeSO4·7H2O (1 g/L), and MgSO4·7H2O
(20 g/L)). Solution I was autoclaved at 115 °C for 15 min.
Buffer and vitamins and mineral solutions were sterilized by
filtration. Buffer and vitamins and mineral solutions were
added to solution I at a ratio of 1% before inoculation. The
feeding medium contained glucose (500 g/L) and yeast extract
(5 g/L). The corn meal medium contained 6% corn, was
boiled at 100 °C for 40 min, and then was autoclaved at
121 °C for 20 min.
Appl Microbiol Biotechnol

Gas stripping for butanol separation in a simulated system

The separation of ABE by gas stripping was studied with a 1-

L reactor containing 0.5 L of ABE-simulated solution at 37 °C
with an ethanol-acetone-butanol ratio of 2:5:8 (Fig. 1). A peri-
staltic pump was used to cycle gas between the bioreactor and
the condenser system. The recycled gas was passed through
the ABE solution and condenser at 1–6 vvm. The condensate
was collected in a three-neck flask immersed in a low-
temperature water bath with a temperature of − 5 °C. A double
gas outlet was used in the gas circulation system. The recovery
rate Rs (g/L/h) was defined as the rate of solvent removal from
the ABE solution. The relative recovery rate (/h) was calcu-
lated as β = Rs/Cs, where Cs was the solvent concentration in
the ABE solution.

Culture conditions Fig. 2 Schematic diagram for ABE fermentation with in situ product
removal. 1 fermentor, 2 gas distributor, 3 gas-circulating pump, 4
Batch fermentation condenser, 5 collection bottle, 6 cooling water tank, 7 gas flow meter, 8
peristaltic pump, 9 concentrated substrate tank

The bacteria activation was performed in corn powder medi-

um (6.5% corn mash). The P2 medium was used for seed
cultures and fermentation cultures. The batch fermentation
was cultured in a 2-L bioreactor with a liquid volume of 1 L
and was statically incubated at 37 °C. In the symbiotic system
TSH06, the inoculum volume of TSH1 was 5% and that of
TSH2 was 0.5%, which was the optimal inoculation propor-
tion in our previous study (Wu et al. 2016a, b). The initial pH
was adjusted to 6.0, and the pH was natural during the fer-
mentation process. All the experiments were done in triplicate.
Fermentation system coupled with in situ butanol removal
A gas-stripping system including a gas-circulating pump, a
condenser, a collection bottle, and a cooling water tank was
connected to the fermentation reactor, which is illustrated in
Fig. 2. The fed-batch fermentation was conducted at 37 °C
with a 1-L bioreactor containing 500 mL P2 medium. Batch
fermentation proceeded in the first 36 h followed by gas strip-
ping with the fermentation gasses (CO2 and H2) as carrier
gasses. The gas produced by TSH06 was circulated through
the bioreactor and the condenser system with a peristaltic
pump, which was turned on when the butanol concentration
in the fermentation broth reached 8 g/L. The temperature of
the condenser was maintained at − 5 °C. The fermentation
broth was fed with feeding medium when the glucose concen-
tration was lower than 10 g/L, and 2.5 mL each of the buffer
and mineral and vitamin solutions was added to the broth at
the same time. A controlled-pH strategy for integration pro-
cess was proposed as follows: the initial pH was adjusted to
6.0, and pH was natural during the first 36 h, then, the pH was
shifted to 5.0 and maintained at 5.0 for the remaining process
by addition of 4 M NaOH.

Analytic methods

A UV-visible spectrophotometer (UV-2802PC; Unico, China)

Fig. 1 Schematic diagram of gas-stripping device
was used to determine the cell density by measuring the
OD600. During the fermentation period, samples were with-
drawn on time and centrifuged at 12000 rpm for 5 min. The
supernatant was used for ABE, organic acid, and residual
sugar concentration analyses. The concentrations of ABE,
acetic acid, butyric acid, and glucose were detected by a
high-performance liquid chromatograph (HPLC) (LC-20AT,
Shimadzu, Japan) equipped with a refractive index detector.
An HPX-87H column (Bio-Rad, USA) was used at 35 °C with
5 mM sulfuric acid as mobile phase (0.60 mL/min).
 Appl Microbiol Biotechnol

Results A gradual decrease in the cell growth rate was observed

Effect of butanol on cell growth and butanol accumulation
of TSH06
The high concentration of products presented in the broth
severely inhibits the fermentation performance of TSH06,
limiting the production of butanol. Among the products
formed during ABE fermentation, butanol exhibited the dom-
inant inhibitory effect on cell growth. Therefore, it is neces-
sary to study the inhibitory effect of butanol on TSH06 to
assess the critical concentrations of butanol, above which cells
cease to grow.
To measure the inhibition induced by butanol, batch
fermentation with different concentrations of added buta-
nol (0–12 g/L) was conducted. Figure 3 shows the impacts
of the different initial concentrations of butanol on cell
growth and butanol synthesis.
with increasing butanol concentration. For initial butanol con-
centrations lower than 8 g/L, the strain can enter the exponen-
tial growth stage in the first 24 h with an OD value of higher
than 1.77, and at the end of the fermentation, about 11 g/L of
butanol was observed, which was comparable to the control
groups. This indicated that TSH06 was able to grow and pro-
duce solvents when the butanol concentration ranged from 0
to 8 g/L. However, at higher butanol concentrations of 10–
12 g/L, the inhibition was markedly more severe. For butanol
concentration above 10 g/L, the strain stopped growing and
reproducing.
High butanol concentrations inhibited the fermentation ca-
pacity as well. In the early stages of fermentation, the presence
of butanol results in an obvious lag phase of glucose con-
sumption and butanol synthesis. The higher the butanol con-
centration is, the longer the lag phase lasts. When the initial
butanol concentration was 8 g/L, the strain still had the ability

Control
Butanol(6g/L) 14
4
Butanol(8g/L)
Butanol(10g/L) 12

Butanol(12g/L)
3 10
Butanol (g/L)

8
OD

2
6
Control
4 Butanol(6g/L)
1 Butanol(8g/L)
2 Butanol(10g/L)
Butanol(12g/L)
0
0
0 15 30 45 60 75 90
0 15 30 45 60 75 90
Time (h)
Time (h)

70

60

50
Glucose (g/L)

40

30
Control
20 Butanol(6g/L)
Butanol(8g/L)
10 Butanol(10g/L)
Butanol(12g/L)
0
0 20 40 60 80
Time (h)
Fig. 3 Effects of butanol on cell growth and butanol accumulation by TSH06 (E ethanol, A acetone, B butanol)
Appl Microbiol Biotechnol

to synthesize butanol and started to produce butanol after 36 h, approximately 0.035/h, indicating that the butanol recovery
and the final butanol concentration reached 10.53 g/L. The ratio per unit time was not affected by the butanol concentra-
strain could not synthesize butanol anymore if the initial bu- tion when the gas flow rate was constant.
tanol concentration was above 10 g/L. There was a sensitive correlation between the butanol con-
The results indicate that a high concentration (> 10 g/L) of centration in the model ABE solution and the butanol concen-
butanol can strongly suppress the cell growth and fermenta- tration in the condensate. The butanol solubility is 7.7% (w/w,
tion capability of TSH06. When ABE fermentation is integrat- at 20 °C) in water, and a phase separation appears when its
ed with gas stripping to improve butanol production, the bu- concentration is above 7.7%. When the butanol concentration
tanol concentration should be controlled at values lower than in the ABE solution increased from 6 to 12 g/L, the butanol
8 g/L. concentration in the condensate increased from 71.14 to
135.64 g/L. The butanol concentration in the condensate
Simulated gas-stripping experiments exceeded 77 g/L when the butanol concentration in the feed
solution was above 8 g/L, and thus, the condensate separated
The simulation of the gas-stripping process was studied in a into aqueous and organic phases. More than 600 g/L of buta-
mixture solvent system with an ethanol-acetone-butanol ratio nol could be obtained in the organic phase of the condensate,
of 2:5:8. The solvent that was removed by gas stripping from which made the process of butanol purification and
the simulated solutions was condensed at − 5 °C. The effects dewatering more energy-efficient.
of the initial butanol concentration and gas flow rate on gas For the FAFIGS system, butanol, as the compound being
stripping efficiency were detected. stripped, is both a desired product and an inhibitor to the
microorganism producing it. If gas stripping was conducted
Effect of butanol concentration on gas-stripping efficiency at relatively low butanol concentrations (< 5 g/L), the high gas
circulation rate would not only increase the tubes wastage but
The butanol concentration in the model ABE solution greatly also cause sporulation and induce culture degeneration (Xue
affected the recovery rate and butanol concentration in the et al. 2012). The results showed that in order to capture highly
condensate. The results are shown in Fig. 4. concentrated butanol in the condensate and keep the high ef-
The butanol recovery rate increased with increasing buta- ficiency of gas stripping, it is necessary to conduct gas strip-
nol concentration. When the butanol concentration in the ping at butanol concentrations as high as possible in the ABE
model ABE solution increased from 6 to 12 g/L with a con- solution. In consideration of the butanol tolerance of TSH06
stant gas flow rate of 1 vvm, the recovery rate of butanol and the effect of butanol concentration on the gas-stripping
increased from 0.20 to 0.41 g/L/h. The relative recovery rate efficiency, the concentration of butanol in the model solutions
(/h) represented the proportion of recovery amount of butanol should be maintained at 8 g/L.
versus the total amount of butanol in unit time, which elimi-
nated the concentration interference in the analysis of gas- Effects of the gas flow rate on gas-stripping efficiency
stripping efficiency. As can be seen in Fig. 4, no obvious
difference was observed in the relative butanol recovery rate. The impacts of the gas flow rate on the butanol concentration
The relative butanol recovery rate in each experiment was in the condensate and its recovery rate were investigated. The

Fig. 4 Effects of butanol 0.06 0.50

Butanol concentration in condensate
Butanol concentration in condensate (g/L)
concentration on gas-stripping
140
Relative butanol recovery rate (h-1)

efficiency 0.45 Relative recovery rate

0.05
Butanol recovery rate
Butanol recovery rate (g/L/h)

0.40
0.04 120

0.35
0.03
0.30 100

0.02
0.25
80
0.01
0.20

0.00 0.15 60
6 7 8 9 10 11 12

Butanol concentration in ABE solution (g/L)


butanol concentration was kept at approximately 8 g/L by
continuously adding butanol during the gas-stripping process.
The results of gas stripping with different gas flow rates are
shown in Fig. 5. The empirical relationship between the buta-
nol extraction rate and gas flow rate for the regression models
was obtained and expressed as follows:
y ¼ −0:0298x2 þ 0:3421x
where y and x represent the response (recovery rate) and the
independent variable (gas flow rate), respectively.
The value of the coefficient of determination (R2) was
0.9948, which indicated that a very high proportion of the
experimental variability could be explained by the quadratic
model. As can be seen in Fig. 5, gas flow rates in the range of
1–6 vvm had a significant effect on gas stripping. When the
gas flow rate increased from 1 to 5 vvm, the butanol extraction
rate increased from 0.28 to 0.97 g/L/h. According to the pre-
diction of model, the highest extraction rate (0.98 g/L/h)
would be obtained when the gas flow rate is 5.74 vvm.
However, the relationship between the butanol extraction rate
and the gas flow rate was not linear. The higher the gas flow
rate was, the less the specific rate of gas stripping efficiency
was increased. When the gas flow rate was 6 vvm, the butanol
extraction rate decreased to 0.96 g/L/h. This phenomenon was
observed most likely because as the gas flow rate increased,
some butanol was not immediately condensed and bubbled
into the solution again.
The objective of this kinetic study is to develop a model for
describing the relationship between the butanol-stripping rate
and the gas flow rate; the model should be able to be further
applied to adjust the recovery rate on the basis of the changing
butanol productivity. Therefore, we used the developed model
to regulate the gas flow rate in the process of FAFIGS.
1.2
Butanol
Ethanol
1.0
Acetone
Recovery rate (g/L/h)
0.8
0.6
0.4
0.2
0.0
1 2 3 4 5 6
Gas flow rate (vvm)
Fig. 5 The effect of gas flow rate on recovery rate
Appl Microbiol Biotechnol
Glucose
70 Acetic acide 14
Ethanol
Butanol
60 Butyric acid 12
Solvents concentration (g/L)
Glucose concentration(g/L)
Acetone
Acid concentration (g/L)
OD
50 10
40 8
OD
30 6
ð1Þ
20 4
10 2
0 0
0 20 40 60 80 100
Time (h)
Fig. 6 Glucose consumption and solvent production by TSH06
Fed-batch fermentation integrated with in situ butanol
recovery
First, batch fermentation without product recovery was con-
ducted in a 2-L bioreactor. With the fermentation medium
containing 60 g/L of glucose, as shown in Fig. 6, 11.79 g/L
of butanol and 17.29 g/L of total ABE were produced in 72 h.
At the end of the fermentation, 8.30 g/L of glucose remained
in the fermentation broth. The yield and productivity were
0.22 g/g and 0.16 g/L/h, respectively.
In contrast, FAFIGS was conducted in a 2-L bioreactor
containing 800 mL of medium with the pH controlled at 5.0.
The results of the FAFIGS experiment are shown in Table 1.
In this process, the consumption of 839.76 g/L of glucose
resulted in the production of 185.65 g/L of butanol, 56.49 g/
L of acetone, and 25.01 g/L of ethanol (total ABE 267.15 g/L).
The overall product yields were 0.23 g/g for butanol and
0.33 g/g for ABE. The productivities of butanol and ABE
were 0.64 and 0.93 g/L/h, respectively. The recovery ratios
of butanol, acetone, and ethanol reached 97.36, 93.33, and
92.41%, respectively. The composition of the condensate in
the collection bottle was determined, and the ABE solution
separated into two phases: 624.07 g/L butanol, 13.15 g/L eth-
anol, and 27.73 g/L acetone were obtained in the upper, or-
ganic phase, while 84.64 g/L butanol, 14.20 g/L ethanol, and
32.61 g/L acetone were obtained in the lower, aqueous phase.
Fermentation intermediates, such as butyric acid and acetic
acid, were not detected in the condensate during gas stripping,
suggesting that either gas stripping did not separate the acids
from the bioreactor or their concentration levels (in the con-
densate) were too low to be detected by LC.
The kinetics of the FAFIGS during the 288-h experiment is
shown in Fig. 7. The integrated ABE fermentation system was
initiated with a glucose concentration of 65.42 g/L. Following
36 h of fermentation, 8.56 g/L of butanol, 3.44 g/L of acetone,
and 1.51 g/L of ethanol were measured in the fermentation
broth. At this stage, product separation by gas stripping was
Appl Microbiol Biotechnol

Table 1 Fed-batch ABE

fermentation integrated with gas Butanol Ethanol Acetone ABE
stripping
Titer (g/L) 185.65 25.01 56.49 267.15
Yield (g/g) 0.23 0.03 0.07 0.33
Productivity(g/L/h) 0.64 0.09 0.20 0.93
ABE ratio 6.9 0.9 2.2 –
Residual ABE concentration in broth (g/L) 4.91 1.90 3.77 9.46
Initial glucose (g/L) 65.42
Final glucose (g/L) 20.18
Glucose utilized (g/L) 839.76
Glucose utilization rate (g/L/h) 2.92
Recovery ratio(%) 97.36 92.41 93.33 –
ABE concentration in collector (g/L) Organic phase 624.07 13.15 27.73 –
Aqueous phase 84.64 14.20 32.61 –

started. The glucose concentrations of 10–50 g/L were main- buffer and 2 mL of vitamin and mineral salt solution were
tained in the bioreactor by feeding it with a medium contain- added to the fermentation broth. The addition of feeding me-
ing 500 g/L glucose and 5 g/L yeast; at the same time, 4 mL of dium and the rate of gas circulation which varied with time

A B OD
18 Butanol 12 pH 20
Butyric acid
70
16
Ethanol Acetic acid Glucose 18
Acetone 10
60 16
ABE
Acetic acid;Butyric acid(g/L)

14
ABE concentration(g/L)

14
12 8 50
12
Glucose (g/L)

OD; pH
10 40
6 10
8
30 8

6 4 6
20
4 4
2 10
2 2

0 0
0 0 0 50 100 150 200 250 300
0 25 50 75 100 125 150 175 200 225 250 275
Time (h)
Time (h)
C D ABE Gas flow rate 8
300 1.75
ABE Butanol
275 Butanol Acetone
1.50
250 Acetone Ethanol
Solvent productivity (g/L/h)

6
225 Ethanol
Solvent production (g/L)

1.25
Gas flow rate (vvm)

200
175 1.00
150 4

125 0.75

100
0.50
75 2

50
0.25
25
0 0.00 0
0 25 50 75 100 125 150 175 200 225 250 275 300 325 0 25 50 75 100 125 150 175 200 225 250 275 300
Time (h) Time (h)
Fig. 7 The time course profiles of fed-batch ABE fermentation with gas stripping. a Product concentration in the fermentation broth. b Glucose
concentration, OD, and pH profiles. c Cumulative ABE production. d Productivities of the solvents and the variation of gas flow rate

were dependent on the rates of sugar utilization and ABE
production.
ABE productivities were calculated at different stages;
along with the cell growth, ABE productivity increased rap-
idly in the first 48 h. In addition, the highest ABE productivity
(1.22 g/L/h) was observed at 96 h. During the period from 36
to 276 h, an average ABE productivity of 1.06 g/L/h was
achieved. A relatively stable butanol concentration of
7 ~ 9 g/L was maintained over the period, indicating a dynam-
ic balance between butanol production and separation. The
results demonstrate that the developed model for controlling
the recovery rate of butanol fits the process of FAFIGS and the
model can be used to adjust the gas flow rate based on the
changing butanol productivity.
Comparing the ABE concentration in the collected conden-
sate with the ABE concentration in the fermentation broth, it
was apparent that gas stripping was an effective method in
separating butanol during the fermentation process, maintain-
ing its values at approximately 8 g/L throughout the process
and thus alleviating the cytotoxicity of butanol and allowing
cell growth and ABE synthesis in a long period.
Four different physiological stages of C. acetobutylicum
cells have been reported to occur in the fed-batch fermentation
system (Ezeji et al. 2013). These cell stages include actively
growing, ABE-producing, sporulating, and dead cells. Due to
long-time fermentation, dead or inactive cells were present
with the active solvent-producing cells in the bioreactor
(Chen et al. 2014). Both the solvent productivity and glucose
consumption rates dropped significantly after 276 h, indicat-
ing decreased cell viability and fermenting performance.
Compared with batch fermentation without gas stripping,
the biomass was increased significantly. The highest cell OD
of 11.88 was obtained at 252 h, compared with 3.85 in batch
fermentation, indicating a higher cell concentration in the
broth. The improved cell growth in the bioreactor resulted in
faster ABE production, leading to a butanol productivity that
was four times higher than that of batch fermentation without
gas stripping. The butanol production was 15 times than that
produced by the batch fermentation, which suggests that bu-
tanol separation by gas stripping could effectively relieve bu-
tanol toxicity to TSH06 and thus increase fermentation
production.
Discussion
In this study, ABE fermentation-integrated on-line product
recovery by TSH06 was conducted under non-strict anaerobic
condition. TSH06 included two strains, butanol-producing,
strict anaerobic C. acetobutylicum TSH1 and facultative
B. cereus TSH2 without butanol synthesis capability.
Facultative TSH2 can provide anaerobic microcosms for
TSH1 in the medium by consuming oxygen, which makes
Appl Microbiol Biotechnol
anaerobic TSH1 grow and produce butanol (Wang et al.
2015). The culture of TSH06 does not need to operate in a
special anaerobic equipment or remove the oxygen in the me-
dium, which make this process simple and economical. These
characteristics make TSH06 a potential application for indus-
trialization of ABE fermentation.
ABE fermentation integrated with butanol recovery by gas
stripping has been widely studied as an effective technique to
alleviate the cytotoxicity of butanol and improve butanol pro-
duction. Recent studies on FAFIGS are summarized and com-
pared in Table 2. Different from FAFIGS, in this study, all of
the ABE fermentations coupled with gas stripping in previous
studies were conducted under strict anaerobic condition, as all
of the strains used were anaerobic bacteria.
The effects of factors such as butanol concentration in feed,
temperature, gas flow rate, and condensational condition on
gas-stripping efficiency had been analyzed in some studies
(Ezeji et al. 2005; Xue et al. 2012; Xue et al. 2014).
However, no methods were proposed for controlling the prod-
uct recovery rate efficiency during the fermentation process.
FAFIGS without product recovery rate efficiency regulation
resulted in fluctuations in ABE concentrations. ABE fermen-
tation coupled with two-stage gas stripping in a fibrous bed
bioreactor, at a gas flow rate of 1.6 L/min, was investigated by
Xue et al. (2014); a butanol fluctuation from 7.2 to 12.7 g/L
was observed in the fermentation broth during the integrated
process. Ezeji et al. (2013) studied ABE fermentation in an
integrated continuous system with a gas recycling adjustment,
but the amount of gas recycles was regulated only based on
the ABE concentrations; the butanol concentrations varied
between 1 and 6 g/L. An integrated fermentation conducted
at a relatively low butanol concentration, such as 2 ~ 5 g/L,
could result in low recovery efficiency and high energy con-
sumption in the subsequent distillation process (Xue et al.
2012). But when butanol concentration was higher than 8 g/
L, the cell growth and fermentation of TSH06 could be
inhibited. It is necessary to operate the integrated fermentation
with product recovery rate efficiency regulation. In the present
work, we developed a kinetic model to describe the relation-
ship between gas flow rate and butanol recovery efficiency.
And the developed model was further used to keep the dy-
namic equilibrium of butanol concentration during the process
of FAFIGS by adjusting butanol recovery rate efficiency. As
the gas recycling adjustment in this study was based on the
changing butanol productivity during the fermentation pro-
cess, the butanol concentration, which varied between 7 and
9 g/L, was relatively stable. Different productivities of buta-
nol, acetone, and ethanol were produced due to the different
fermentation activities, which may be attributed to the accu-
mulation of active cells in the bioreactor. The greatest produc-
tivity of ABE (1.14 g/L/h) in the bioreactor was obtained from
96 to 120 h, and lower ABE productivities were observed in
the first and last 24 h.
Appl Microbiol Biotechnol

Table 2 ABE fermentations with gas stripping

Process Condition Strain Butanol in Production Productivity Yield (g/g) Reference

fermentor with/ (g/L) (g/L)
without gas
stripping (g/L)
Butanol ABE Butanol ABE Butanol ABE

Batch/free cell Strict C. beijerinckii 1.9–8.8/11.9 46.4 75.9 0.42 0.61 0.28 0.40 Ezeji et al.
anaerobic BA101 (2003)
Batch/free cell Strict C. beijerinckii 2–6/7.4 – 21.42 – 0.31 – 0.41 Qureshi
anaerobic P260 et al.
(2008a)
Batch/immobilizition Strict C.acetobutylicum < 15/16.2 19.8 31.8 0.41 0.66 0.25 0.40 Xue et al.
anaerobic JB200 (2013)
Continuous Strict C. beijerinckii 1–6/11.9 – 461.3 – 0.92 – 0.41 Ezeji et al.
anaerobic BA101 (2013)
Fed-batch/immobilizition Strict C.acetobutylicum 8–10/19.4 113.3 172 0.35 0.53 0.24 0.36 Xue et al.
anaerobic JB200 (2012)
Fed-batch/immobilizition Strict C. acetobutylicum 6–12/19.3 48.5 73.3 0.24 0.36 0.27 0.40 Xue et al.
anaerobic JB200 (2014)
Fed-batch/immobilizition Strict C. acetobutylicum 6–15/20.4 76.4 108.1 0.32 0.45 0.23 0.32 Lu et al.
anaerobic JB200 (2013)
Fed-batch/free cell Strict C. beijerinckii 2–5.8/7.4 – 192 – 0.36 – 0.44 Qureshi
anaerobic P260 et al.
(2008a, b)
Fed-batch/immobilizition Strict C. acetobutylicum 7–10/12 66.1 106.27 0.38 0.61 0.23 0.36 Chen et al.
anaerobic B3 (2014)
Fed-batch/immobilizition Strict C. acetobutylicum 9–13/14.3 101.3 170.6 0.52 0.88 0.22 0.37 Cai et al.
anaerobic ABE 1401 (2015)
Fed-batch/free cell Strict C. beijerinckii 2–9.6/11.9 151.7 232.8 0.75 1.16 0.30 0.47 Ezeji et al.
anaerobic BA101 (2004)
Fed-batch/free cell Strict C. beijerinckii 2–5.5/18.6 – 81.3 – 0.59 – 0.36 Ezeji et al.
anaerobic BA101 (2007)
Fed-batch/free cell Non-strict TSH06 7–9/11.8 185.7 267.2 0.64 0.93 0.23 0.33 This study
anaerobic

Butanol fermentation using a fibrous bed reactor, integrated
with gas stripping, has been reported in recent years.
However, once the immobilized materials were fixed in the
bioreactor, the gas diffusion could be blocked by the
immobilized materials; on the other hand, the bubbles across
the carrier surface could decrease the adsorption between the
carrier and the cells. Considering the poor mass transfer in the
fibrous bed bioreactor and the problem of carrier adsorptivity,
gas stripping was often conducted in an additional stripper that
was connected to the bioreactor (Chen et al. 2014; Xue et al.
2013; Lu et al. 2013). As a result, there must be a circula-
tion system to transport fermentation broth between the
stripper and the bioreactor, which needs more devices,
making the integration process become complicated. In
this study, cell growth, fermentation, and product recov-
ery were performed in the same reactor. A high butanol
recovery ratio of 97.36% was obtained in the single-stage
integrated fermentation system.
In this study, the ABE fermentation ceased suddenly. The
productivity of ABE decreased obviously after 264 h, and the
OD value of the culture dropped from a high of ~ 12 to ~ 10
during this time (Fig. 7b). The presence of inactive cells, accu-
mulation of salts, culture degeneration, and possible changes in
cell metabolic pathways toward acid production could be con-
sidered as possible reasons for the cessation of ABE production
(Mariano et al. 2011; Ezeji et al. 2007; Chen et al. 2014).
Maddox et al. (2000) reported that the biomass and ABE
production of C. acetobutylicum decreased with an increase of
the NaCl concentration (0 ~ 25 g/L) in the medium. During the
process of FAFIGS, plenty of water in the fermentation broth
was removed by gas stripping as well, thus causing the accu-
mulation of salt, which could inhibit cell growth and solvent
production (Gapes et al. 1996).
Culture degeneration, as a physiological phenomenon, is
usually associated with a metabolic shift and occurs over a
long period of fermentation. Woolley and Morris (1990)

studied C. acetobutylicum degeneration during continuous
fermentation and found that non-active C. acetobutylicum
coexisted with the solvent-producing C. acetobutylicum for
some time, before gradually taking over. At the end of the
fermentation, sporulated and dead cells were observed in the
broth, which could be an inducement of culture degeneration
to stop ABE fermentation.
The time-course profiles of ABE fermentation (Fig. 7a) show
that a dramatic increase of butyric acid (from 1.64 to 2.55 g/L)
and acetic acid (from 1.97 to 4.67 g/L) concentrations was ob-
served in the last 36 h of the fermentation, which shows a
change in the cellular metabolism. The high concentration of
acetic acid and butyric acid caused an inhibitory effect on cell
growth, and the acid inhibition on ABE fermentation was pH-
dependent. Wu et al. (2016b) found that no fermentation was
observed when 7.5 g/L acetic acid or 5.5 g/L butyric acid was
added to the medium without pH control, while
C. acetobutylicum could tolerate an acetic acid concentration
up to 11.7 g/L when the medium’s initial pH was adjusted to
6.5 (Cho et al. 2012). The butyric acid concentration of 2.55 g/L
and acetic acid concentration of 4.67 g/L at pH 5.0 resulted in
1.01 g/L undissociated butyric acid and 1.70 g/L undissociated
acetic acid contained in the culture broth, which could be a
factor for the termination of ABE fermentation.
In this study, the butanol tolerance of TSH06 and the effect of
butanol concentration and gas flow rate on the gas-stripping
efficiency were investigated. In consideration of the butanol
tolerance of TSH06 and the gas-stripping efficiency, the optimal
butanol concentration for the FAFIGS was 8 g/L. The integrated
in situ gas-stripping process with product separation efficiency
adjustments under non-strict anaerobic conditions has been
shown to be an effective method for improving ABE produc-
tion. With the non-strict anaerobic butanol-producing TSH06,
267.15 g/L of ABE (185.65 g/L of butanol) was produced from
839.76 g/L of glucose, with a yield and productivity of 0.33 g/g
and 0.93 g/L/h, respectively. A stable concentration of bu-
tanol was maintained during the FAFIGS process, which
made the method attractive for the industrial production
of bio-butanol. This study provided a simple, efficient,
and stable in situ separation strategy for integrated fed-
batch ABE fermentation, and it helped to promote the
industrial development of ABE fermentation.
Funding information This work was supported by the National Key
R&D Program of China (No. 2016YFE0131300), the Boeing Company
(No. 2015-156), and the Foundation of Key Laboratory for Industrial
Biocatalysis (Tsinghua Univesity), Ministry of Education (No. 2015302).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Appl Microbiol Biotechnol
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