Bioresource Technology 129 (2013) 680–685

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Bioresource Technology
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Short Communication

Continuous butanol fermentation from inexpensive sugar-based

feedstocks by Clostridium saccharobutylicum DSM 13864
Ye Ni ⇑, Ziyi Xia, Yun Wang, Zhihao Sun
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., Wuxi 214122, China

h i g h l i g h t s

" Butanol can be produced from cane molasses and corn stover hydrolysate by C. saccharobutylicum.
" A four-stage continuous butanol fermentation using CSH was steadily operated for 220 h.
" A high solvent productivity of 0.429 g/L/h and solvent titer of 11.43 g/L were attained.

a r t i c l e i n f o a b s t r a c t

Article history: Corn stover hydrolysate (CSH) and cane molasses were studied for butanol fermentation by Clostridium
Received 30 July 2012 saccharobutylicum DSM 13864 in continuous fermentation. Using cane molasses as substrate, solvent
Received in revised form 21 November 2012 of 13.75 g/L (butanol 8.65 g/L) and productivity of 0.439 g/L/h were achieved in a four-stage continuous
Accepted 23 November 2012
fermentation at a gradient dilution mode of 0.15–0.15–0.125–0.1 h 1. In continuous fermentation using
Available online 8 December 2012
CSH as substrate, total solvent titer of 11.43 g/L (butanol 7.81 g/L) and productivity of 0.429 g/L/h were
reached at a dilution rate of 0.15 h 1, and the steady process was continuously operated for 220 h with-
Keywords:
out compromise in solvent titer.
Butanol
Continuous fermentation
Ó 2012 Elsevier Ltd. All rights reserved.
Corn stover hydrolysate (CSH)
Cane molasses
Dilution rate

1. Introduction of the largest industrial fermentation processes in the early 20th

With an ever-growing global depletion of natural oil and gas re-
sources, and various environmental issues resulting from the rapid
consumption of petroleum fuels, the development of alternative fuel
resources has been getting significant attentions for decades
(Bankar et al., 2012). Butanol, an important C4 platform compounds,
is considered as one of the most promising biofuels. Compared with
traditional biofuel ethanol, butanol provides many advantages such
as higher energy content, less hygroscopy, less volatile, less hazard-
ous to handle, and lower vapor pressure (Qureshi and Ezeji, 2008). In
addition, this chemical is also an excellent fuel extender as it con-
tains 22% oxygen (Qureshi et al., 2010). Importantly, butanol can
be used directly or blended with gasoline or diesel at any ratio with-
out retrofit of vehicle engines, and can be supplied and stored
through the existing gasoline pipeline (Ni and Sun, 2009).
Butanol is a main product of acetone-butanol-ethanol (ABE) fer-
mentation by Clostridia under anaerobic conditions, which was one
⇑ Corresponding author. Tel./fax: +86 510 85329265.
E-mail address: yni@jiangnan.edu.cn (Y. Ni).
century. Compared with continuous fermentation, the commercial
development of batch ABE fermentation has been hampered by a
number of deficiencies including lower productivity, and stronger
end-product inhibition, etc. The productivity of a traditional ABE
batch fermentation, usually less than 0.5 g/L/h, is obviously unde-
sirable for industrial production (Qureshi and Ezeji, 2008). While in
continuous fermentation, the solvent productivity could be en-
hanced to 0.5–1.0 g/L/h (Maddox, 1989) . An average overall sol-
vent concentration of 15 g/L and an overall solvent productivity
of 0.27 g/L/h were achieved using a two-stage continuous cultiva-
tion with Clostridium beijerinckii NRRL B592 (Mutschlechner et al.,
2000). Furthermore, higher solvent productivity and concentration
could also be achieved in single-stage continuous process. In a sin-
gle-stage continuous fermentation with wood pulp as a cell hold-
ing material, maximum solvent productivity of 13.66 g/L/h was
obtained at a dilution rate of 1.9 h 1 (Survase et al., 2012). Re-
cently, using a cell recycling continuous fermentation with glycerol
as substrate, a solvent productivity of 8.3 g/L/h was reached at a
dilution rate of 0.9 h 1 (Malaviya et al., 2012). Although a number
of studies on continuous ABE fermentation have been carried out,

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.11.142
 Y. Ni et al. / Bioresource Technology 129 (2013) 680–685
glucose or corn starch were mostly utilized as major feedstock. As a
matter of fact, the cost of fermentation substrate, which account
for 60–70% of total production cost, is the most influential factor
in the butanol fermentation (Qureshi and Blaschek, 2000). It is
therefore necessary to explore cheaper feedstocks for making
ABE fermentation economically more attractive. Cane molasses, a
by-product of sugar industry, contains approximately 45–50%
(w/w) total sugars (sucrose, glucose and fructose), vitamins, and
nitrogenous compounds, etc. (Najafpour and Shan, 2003). It has
therefore been regarded as an inexpensive and appropriate
substrate for ABE fermentation (Ni et al., 2012). In the last decade,
cellulosic hydrolysates of various agricultural residues and other
biomasses have been investigated for the biofuel production,
including degermed corn, corn stover (CS), wheat straw (WS), bar-
ley straw (BS), and energy crops like switchgrass (SG) and miscan-
thus (Qureshi and Ezeji, 2008).
In China, the annual production of cane molasses and agricul-
tural residues were about three million tons and one billion tons,
respectively. To date, there is no Chinese solvent plant using corn
stover hydrolysate (CSH) and cane molasses as sole substrate to
produce butanol, especially in a continuous fermentation. In this
work, the effect of dilution rate on solvent production in continu-
ous ABE fermentation using corn stover hydrolysate (CSH) and
cane molasses as substrates were investigated.
2. Methods
2.1. Microorganism, maintenance and inoculum preparation
Clostridium saccharobutylicum DSM 13864 used in this study
was purchased from DSMZ (German Collection of Microorganisms
and Cell Cultures). It was cultivated in Reinforced Clostridia Med-
ium (RCM) at 37 °C for 7 days to induce sporulation, followed by
storage at room temperature. A 1.2 mL spore culture was inocu-
lated into a glass tube (£1.5 cm  15 cm) containing 12 mL sterile
RCM. It was then placed in a desiccator pumped to a vacuum level
of 0.065 MPa for providing an anaerobic condition. Afterwards, the
culture was maintained at 37 °C for 12–16 h and supplied as the
seed medium. An inoculum size of 8% (v/v) was used in all fermen-
tation in this study.
2.2. Fermentation medium
Cane molasses was purchased from Xinyue chemical industry
Co. Ltd., (Changshu, China). The fermentation medium reported
by Ni et al. (2012) was used, which contains (per liter): cane molas-
ses, 60 g; CaCO3, 3.2 g; (NH4)2SO4, 2 g; K2HPO4, 0.5 g; MnSO4
H2O,
0.01 g; corn steep liquor, 10 g. The pH of medium was adjusted to
6.1 with 2 M NaOH or 2 M HCl prior to sterilization, and then the
medium was autoclaved at 121 °C for 20 min. Cane molasses
mainly consists of 270.4 g/l sucrose, 58.2 g/l glucose and 78.6 g/l
fructose, with a total sugar of 407.20 g/L.
The corn stover pretreatment method reported by Lin et al.
(2011) was used with slight modification. The dried corn stover
was pulverized and passed through a 40 mesh sieve. For pretreat-
ment, 112 g of corn stover was added into 1.6 L of 1% (w/w) sodium
hydroxide in a 3-L erlenmeyer flask, and autoclaved at 120 °C for
2 h. After cooling to room temperature, the pH of pretreated corn
stover was adjusted to 4.8 using 250 g/L H2SO4 solution. Then cel-
lulase solution of 11.2 mL (filter paper enzyme activity:
51.70 FPIU/mL), which was purchased from Zesheng bioengineer-
ing technology Co. Ltd., (Shandong, China), was added followed
by incubation at 50 °C and 150 rpm for 40 h. Afterwards, the pre-
treated corn stover mixture was centrifuged at 8000 rpm for
10 min to obtain corn stover hydrolysate. The sugar composition
681
(g/L) of CSH includes: glucose 29.48, xylose 11.51, arabinose 2.44,
and cellobiose 4.19. The fermentation medium contains the follow-
ing components (per liter CSH): CaCO3, 2.0 g; (NH4)2SO4, 1.0 g;
K2HPO4, 0.5 g; MnSO4H2O, 0.01 g; corn steep liquor, 15 g (based
on our previous optimization study, unpublished data). Prior to
autoclaving, glucose was supplemented into CSH to raise the total
sugar to 60 g/L. The medium was adjusted to pH6.1, and was then
autoclaved at 115 °C for 20 min. After sterilization, the medium
was sparged with carbon dioxide for 5–10 min to remove dissolved
oxygen.
2.3. Continuous fermentation
Continuous fermentations using cane molasses as substrate
were also performed in 3-L stirred tank bioreactors. The experi-
mental setup consists of four stirred tank bioreactors with working
volume of 1-L. Four tanks were arranged sequentially and desig-
nated as stage one (R1), stage two (R2), stage three (R3) and stage
four (R4), respectively. In order to investigate the effect of dilution
rate on fermentation performance, continuous fermentations were
conducted without pH control.
For cane molasses substrate, to achieve a better sugar utiliza-
tion in the later two stages of continuous fermentation, R3 and
R4 were inoculated first and then incubated for 10–20 h, after
which R1 and R2 were inoculated, and the continuous fermenta-
tion was started when R1 arrived at a desired stage (3–4 g/L total
solvent). The dilution rates of R1, R2, R3 and R4 were controlled
at 0.1–0.1–0.1–0.1 h 1 and 0.15–0.15–0.125–0.1 h 1, respectively.
Sterilized fresh fermentation medium was stored in a feeding tank
with a total volume of 20 L. The culture volume in each tanks was
kept constant by using a peristaltic pump. First, R3 and R4 were
started by inoculating 8% (v/v) seed culture, following by incuba-
tion for 14 h. Then R1 and R2 were inoculated and fermented for
16 h. Thereafter, continuous fermentations were started by feeding
fresh fermentation medium at certain flow rates. It is assumed that
the continuous fermentation process enters into a steady phase
when a stable solvent titer was observed during the following per-
iod (for example: 100–200 h). Samples of 2 mL were taken at a reg-
ular time interval to monitor the progress of fermentation.
Continuous fermentations using CSH as substrate were carried
out in four 500 mL serum bottles with a working volume of
300 mL. The four bioreactors were inoculated with 8% (v/v) of seed
culture simultaneously and proceed in batch fermentation for 16 h,
after which continuous fermentation was started by feeding fresh
fermentation medium using a peristaltic pump. The dilution rates
were controlled at 0.1 and 0.15 h 1, separately.
2.4. Analytical procedure
The cell concentration was measured at 660 nm (OD660) using an
ultraviolet spectrophotometer (INESA, China). Sterilized fresh cane
molasses or corn steep liquor medium was diluted 5–10 times with
2% HCl and used as blank control to eliminate the influence of their
color in the detection. The total sugar in cane molasses fermentation
medium was determined by 3,5-dinitrosalicylic acid (DNS) method
(Tesun and Dhese, 1970). The sugar compositions of CSH were ana-
lyzed by HPLC (Waters 1525, USA) equipped with a refractive index
detector and an Aminex HPX-87H column. The temperatures of col-
umn and detector were controlled at 55 °C and 35 °C, respectively.
The injection volume was 10 lL. The concentrations of solvent
and organic acids (acetic and butyric acid) were analyzed by GC
(Varian cp 3900, USA) equipped with the flame ionization detector
(FID) and an capillary column PEG-20 M (30 m0.32 mm0.5 lm,
JK, China) using nitrogen as the carrier gas. The oven temperature
was maintained at 60 °C for 0.5 min and then programmed with
the increment of 10 °C to 120 °C, held for 0.5 min, and subsequently
682 Y. Ni et al. / Bioresource Technology 129 (2013) 680–685

increased to 190 °C with the increment of 15 °C with 1 min final
hold. The temperature of the injector and detector was held at
180 °C and 210 °C, respectively. The injection volume was 1 lL.
3. Results and Discussion
3.1. Continuous fermentation with 6% cane molasses
Continuous fermentation is usually adopted in industrial ABE
fermentation to improve solvent productivity, relieve end-product
inhibition, and reduce downtime, and the main feedstocks used
were simple sugars and starchy materials. (Maddox, 1989). Re-
cently, various substrates such as wheat bran (Liu et al., 2010), bar-
ley straw hydrolysate (Qureshi et al., 2010), sugar maple wood
(Sun and Liu, 2012), and cassava bagasse hydrolysate (Lu et al.,
2012) have been used in the ABE fermentation. Based on our pre-
liminary studies on batch and semi-continuous fermentation (Ni
et al., 2012), we achieved 17.88 g/L total solvent at 36 h, including
acetone 4.60 g/L, butanol 11.86 g/L, ethanol 1.42 g/L, and the pro-
ductivity and yield were 0.50 g/L/h and 0.33 g/g, respectively. In
this study, four-stage continuous fermentations using 6% cane
molasses at two sets of dilution rates (constant mode: 0.1–0.1–
0.1–0.1 h 1 and gradient mode: 0.15–0.15–0.125–0.1 h 1) were
carried out. The gradient mode was attempted to increase the
retention time of R3 and R4, and also improve the sugar consump-
tion. The concentrations of residual sugar, solvents, organic acids
and cell density (OD660) were shown in Fig. 1A–F. Under the
constant mode when the dilution rate in all four stages were main-

Fig. 1. Time courses of continuous ABE fermentation by C. saccharobutylicum DSM 13864 using 6% cane molasses.(A) OD600 and residual sugar, (B) butanol and total solvent,
(C) organic acids at constant dilution rates of 0.1–0.1–0.1–0.1 h 1. (D) OD600 and residual sugar, (E) butanol and total solvent, (F) organic acids at gradient dilution rates of
0.15–0.15–0.125–0.1 h 1. R1, R3 and R4 represent the first-stage, third-stage and fourth-stage bioreactor tanks, respectively.
 Y. Ni et al. / Bioresource Technology 129 (2013) 680–685 683

Table 1
Steady state parameters of four-stage ABE continuous fermentation using cane molasses and corn stover hydrolysate (CSH) as substrates.

Cane molasses CSH

Parameters D = 0.1–0.1–0.1–0.1 D = 0.15–0.15–0.125–0.1 D = 0.1 D = 0.15
R1 R3 R4 R1 R3 R4 R4 R4
OD660 4.57 3.12 3.06 4.72 4.56 4.17 1.37 3.79
ABE/(g/L) 7.71 11.37 11.74 7.15 12.24 13.75 13.44 11.43
Butanol/(g/L) 4.68 6.93 7.18 4.04 7.52 8.26 9.29 7.81
Acetone/(g/L) 2.26 3.16 3.32 1.86 3.68 3.99 3.85 3.32
Ethanol/(g/L) 0.77 1.28 1.25 1.26 1.03 1.49 0.30 0.30
Acetic acid/(g/L) 2.17 1.77 1.78 3.00 2.67 2.55 1.24 0.46
Butyric acid/(g/L) 1.33 0.72 0.70 2.32 2.13 2.10 1.01 1.80
Initial sugar/(g/L) 56.62 58.22 57.06 59.81 59.09 58.07 51.31 51.58
Residual sugar/(g/L) 33.69 24.77 20.27 35.14 18.26 15.78 14.98 23.63
Consumed sugar/(g/L) 22.93 33.45 36.79 24.67 40.83 42.29 36.33 27.95
Yield of Butanol a/(g/g) 0.204 0.207 0.195 0.164 0.184 0.195 0.256 0.279
Yield of ABE a/(g/g) 0.336 0.340 0.319 0.290 0.300 0.325 0.370 0.409
Productivity of Butanol b/(g/L/h) 0.468 0.231 0.180 0.606 0.353 0.264 0.232 0.293
Productivity of ABE b/(g/L/h) 0.771 0.379 0.294 1.072 0.574 0.439 0.336 0.429
a
Yield of ABE or butanol: concentration of ABE or butanol divided by concentration of sugar consumed.
b
Productivity of ABE or butanol: concentration of ABE or butanol divided by fermentation time.

tained at 0.1 h 1, continuous fermentation could be stably oper-
ated during 30–130 h of fermentation where the average total sol-
vent of R1, R3 and R4 were 7.71, 11.37 and 11.74 g/L, respectively
(Table 1). Consequently, the solvent productivity in R1 was calcu-
lated to be 0.771 g/L/h, representing a 54.2% increase compared
with that of batch fermentation. While productivities in R3
(0.379 g/L/h) and R4 (0.294 g/L/h) were slightly reduced due to
the extended retention time. In continuous fermentation under
gradient dilution mode, where the dilution rates in each stage were
increased to 0.15, 0.15, 0.125 and 0.1 h 1, the average ABE titers of
7.15, 12.24 and 13.75 g/L were obtained for R1, R3, and R4, respec-
tively (Fig. 1E). As a result, the solvent productivities of R1, R3 and
R4 were 1.072, 0.574 and 0.439 g/L/h, respectively. Our results are
comparable with those reported by Liew and coworkers, in which
total solvent of 9.10 g/L and overall productivity of 0.46 g/L/h were
attained using a single-stage continuous fermentation by Clostrid-
ium saccharobutylicum DSM 13864 at a dilution rate of 0.05 h 1
(Liew et al., 2006). Clearly, the butanol toxicity to cell culture could
be alleviated by continuous fermentation, thereby enhancing sol-
vent productivity in this study. Unexpectedly, solvent production
began to reduce after 140 h of continuous fermentation, likely
due to a drop in pH to around 4.5 (data not shown) which resulted
in strain degeneration.
As shown in Fig. 1A and D, average residual sugars in two R4
tanks were maintained at 20.27 and 15.78 g/L respectively, which
would lead to a compromised solvent yield. Under the gradient
mode, the accumulation of acetic acid and butyric acid in R4 were
2.55 and 2.10 g/L, respectively, which were slightly higher than
1.78 and 0.70 g/L at constant dilution rates. It is known that the
production of organic acids is often accompanied by the ATP regen-
eration system driven by several enzymes including acetate kinase
(AcK) in ABE fermentation (Jiang et al., 2009). Dilution rate is one
major reason for the different cell density observed under gradient
mode (4.17 OD660) and constant mode (3.06 OD660). Also, ATP
regeneration rate could somewhat affect the cell growth. As dem-
onstrated above, a continuous fermentation at a gradient dilution
rate of 0.15–0.15–0.125–0.1 h 1 was advantageous to improve sol-
vent productivity.
3.2. Continuous fermentation with CSH
To exploit the utilization of sustainable sugar-based feedstock,
ABE fermentation from various lignocellulosic hydrolysates of
agricultural residues has been extensively investigated during the
last few years (Qureshi et al., 2010; Liu et al., 2010; Lin et al.,
2011). Inhibitors including furfural, hydroxymethyl furfurl, ferulic,
glucuronic, and phenolic compounds, are usually generated during
the pretreatment of cellulosic materials, and are deleterious for cell
growth and solvent production (Qureshi et al., 2010). A number of
approaches have been developed to remove these inhibitors,
including overliming, adsorbent resin, dilution of hydrolysate, and
development of inhibitor-tolerant strains, etc. (Qureshi et al.,
2008, 2010; Liu et al., 2010). Based on our previous work (unpub-
lished data), 1% NaOH was used for the pretreatment of corn stover,
which was autoclaved at 120 °C for 2 h to remove the inhibitors and
followed by enzymatic hydrolysis. CSH was then obtained and used
in four-stage continuous ABE fermentation in this study.
A control batch fermentation with CSH as substrate was con-
ducted, maximum total solvent of 16.09 g/L (10.59 g/L butanol,
4.04 g/L acetone, and 1.46 g/L ethanol) was attained at 40 h, and
the ABE yield and productivity were 0.334 g/g and 0.402 g/L/h,
respectively (data not show). The four-stage continuous fermenta-
tion was operated at two constant dilution rates of 0.1 and 0.15 h 1,
respectively. The concentrations of residual sugar, solvent, organic
acids and OD660 are shown in Table 1. Our results indicate that
CSH is well suited to solvent production. At both dilution rates, con-
tinuous fermentation reached a steady state after 60 h of fermenta-
tion (Fig. 2A–B), where cell densities of R4 tanks were maintained at
1.37 and 3.79 OD660, respectively. The steady fermentation process
could be continuously operated for 220 h at 0.15 h 1. At dilution
rate of 0.1 h 1, the average total solvent concentration of 13.44 g/
L (9.29 g/L butanol) and total solvent productivity of 0.336 g/L/h
were achieved. For 0.15 h 1, a relatively lower average total solvent
titer of 11.43 g/L (7.81 g/L butanol) was obtained, accompanied by a
higher total solvent productivity of 0.429 g/L/h. The accumulation
of total organic acid (2.25 g/L) was almost equivalent at two
dilution rates. The glucose was completely consumed at the dilu-
tion rate of 0.1 h 1, and the residual sugars consisted of xylose
7.71 g/L, arabinose 4.84 g/L, and cellobiose 2.43 g/L. Higher total
residual sugars (23.63 g/L) was however detected at 0.15 h 1 dilu-
tion rate, which contained glucose 12.77 g/L, xylose 5.04 g/L,
arabinose 4.44 g/L, and cellobiose 1.38 g/L, indicating dilution rate
of 0.15 h 1 is inappropriate for efficient sugar utilization.
It has been reported that higher dilution rate was beneficial to
cell growth whereas lower dilution rate favored solvent production
by C. saccharobutylicum DSM 13864 in a single-stage continuous cul-
ture using gelatinized sago starch as substrate. (Liew et al., 2006),
and our results are in accordance with it. In comparison with other
684 Y. Ni et al. / Bioresource Technology 129 (2013) 680–685

Fig. 2. Time courses of ABE fermentation in the final stage of four-stage continuous fermentation by C. saccharobutylicum DSM 13864 using CSH as substrate. (A) Dilution rate
of 0.1 h 1. (B) Dilution rate of 0.15 h 1. OD660 (open upright triangle), residual sugar (filled square), butanol (filled inverted triangle), total solvent (filled circle), acetic acid
(open inverted triangle), butyric acid (open circle).

Table 2
Comparison of ABE continuous fermentation parameters in the literatures and this study.
1
Operation mode Strain Substrate Dilution rate (h ) Productivity Total solvent Reference
(g/L/h) (g/L)
Immobilized cell C. acetobutylicum DSM 792 Glucose 1.9 13.66 7.19 Survase et al. 2012
Membrane cell bioreactor C. pasteurianum ATCC 6103 Glycerol 0.9 8.3 9.2 Malaviya et al. (2012)
C. acetobutylicum ATCC 824 Glucose 0.017 0.37 124.4 Wouter et al. (2012)
Free cell C. saccharobutylicum DSM 13864 Gelatinised sago starch 0.05 0.46 9.10 Liew et al. (2006)
C. beijerinckiiNRRL B592 Glucose 0.022 (R2) 0.27 15.00 Mutschlechner et al. (2000)
C. saccharobutylicum DSM 13864 Corn stover hydrolysate 0.15 0.429 11.43 This study

literatures (Table 2), total solvent concentration in this study is the
highest although the productivity is not greatly improved.
Therefore, further efforts should be dedicated to enhance the
productivity while maintaining high solvent titer, e.g.: continuous
fermentation using immobilized cells, integrated solvent removal
strategy. It was presumed that the cost of solvent recovery would
be reduced by half if the butanol titer could be enhanced from 1.2
to 2.0%.
4. Conclusion
Butanol can be produced from cane molasses and CSH by
C. saccharobutylicum DSM 13864 in continuous fermentation. In a
four-stage continuous fermentation using cane molasses, a steady
solvent production of 13.75 g/L and productivity of 0.439 g/L/h
were obtained under a gradient dilution mode. In continuous fer-
mentation using CSH, a high solvent productivity of 0.429 g/L/h
was maintained for an extended period of 220 h. Therefore, the
continuous fermentation using inexpensive sugar-based feed-
stocks could potentially help to drive butanol fermentation to-
wards an economically viable process.
Acknowledgements
The research was financially supported by the National High
Technology Research and Development Program of China
(2011CB710800), New Century Excellent Talents in University
(NCET-11-0658), Natural Science Foundation of Jiangsu Province
(BK2011150), the Program of Introducing Talents of Discipline to
Universities No. 111-2-06, the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions.
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