Biochemical Engineering Journal 91 (2014) 220230

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Biochemical Engineering Journal

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

Regular Article

Characterization and kinetics of bio-butanol production with

Clostridium acetobutylicum ATCC824 using mixed sugar medium
simulating microalgae-based carbohydrates
Yue Wang a , Wan-Qian Guo a , Yung-Chung Lo b , Jo-Shu Chang a,b,c,d, , Nan-Qi Ren a,
a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China
b
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
c
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
d
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan

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

Article history: Our recent work showed that Chlorella vulgaris JSC-6 can accumulate over 50% carbohydrates per dry
Received 25 May 2014 weight, which mainly contain glucose and xylose as the monosaccharides. In this study, synthetic mixed
Received in revised form 12 July 2014 sugars simulating the hydrolyzed biomass of C. vulgaris JSC-6 (primarily containing glucose and xylose
Accepted 4 August 2014
at a ratio of 5:16.5:1) was used as the carbon source for bio-butanol production with Clostridium aceto-
Available online 13 August 2014
butylicum ATCC824. The growth and product formation kinetics based on glucose, xylose, and mixtures
of the two sugars were determined using Monod-type and MichaelisMenten models, respectively. C.
Keywords:
acetobutylicum ATCC824 can grow faster and produce butanol more efciently on glucose, while the per-
Butanol
Fermentation
formance was markedly poorer when using xylose. The butanol production kinetics were quite similar
Carbohydrates when using glucose alone or using mixed sugars (glucose to xylose ratio = 5:1 or 6.5:1), resulting in a
Growth kinetics maximum butanol production rate of 0.890.93 g/h/L. This work demonstrated the feasibility of using
Production kinetics the microalgae-based carbohydrates as the feedstock for biobutanol production with C. acetobutylicum
Microalgae ATCC824.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Today about 80% of global energy demand is produced from
fossil fuels [1]. However, rising concerns over energy security
and climate change make biofuels (such as biodiesel, ethanol,
and butanol) attract increasing amounts of attention as potential
sources of clean, renewable energy. Among these, biobutanol is
especially promising, since it is less corrosive, less hydroscopic,
and can potentially replace ethanol as a gasoline additive due to
several advantages, such as low vapor pressure (reduced emis-
sions), high energy density (greater miles per gallon), and blending
options (increased concentration) [2]. The current production scale
of biobutanol is only second to that of bioethanol, although the
butanol that is now produced is mainly used as an industrial sol-
vent, rather than an alternative energy, due to the high production
Corresponding author at: Department of Chemical Engineering, National Cheng
Kung University, Tainan, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146.
Corresponding author.
E-mail addresses: changjs@mail.ncku.edu.tw (J.-S. Chang), rnq@hit.edu.cn
(N.-Q. Ren).
cost [3]. A cheaper way to produce butanol is thus required before
its widespread commercial utilization.
The use of Clostridial species (such as Clostridium acetobutylicum,
Clostridium beijerinckii and Clostridium saccharoperbutylaceton-
icum) via ABE fermentation is the most commonly used process
for butanol production [4]. Clostridium sp. are known to have the
capability of utilizing simple and complex sugars, including pen-
tose and hexose, as well as CO2 , H2 and CO [58]. Butanol has
traditionally been produced by ABE fermentation using carbon sub-
strates obtained from corn, sugar beets and sugar cane, potatoes,
tapioca and millet [5,9,10]. However, in order to obtain enough
fermentable carbon substrates without getting into the competi-
tion with food supplies, other efcient feedstocks are now being
considered, such as cellulosic materials or microalgal biomass [11].
Moreover, butanol production using food wastes or plants is ben-
ecial only when the biomass is produced in a sustainable way,
without having adverse impacts on the environment. The concept
of photonol has recently has been proposed to address this issue,
based on the direct conversion of solar energy in the production
of fermentation end products, such as biofuels, by photosynthetic
microalgae [5]. In this approach, H2 O, CO2 and solar energy are used
as inputs for the production of fermentation products [5]. This is

http://dx.doi.org/10.1016/j.bej.2014.08.007
1369-703X/ 2014 Elsevier B.V. All rights reserved.
 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230
referred to as photofermentation, and is a process that is likely
to make biofuel production more economically attractive when
its photosynthetic efciency is further improved [12,13]. The use
of microalgal biomass as a feedstock for biobutanol production is
thus one of the most promising approaches to the production of
biobutanol, although few studies have examined this issue. More
information is thus needed on the characterization and kinetics
of microalgae-based biobutanol production, in order to aid in the
design of bioreactors, scale up the process, and eventually achieve
sustainable commercialization of this renewable fuel.
Our previous works showed that isolated Chlorella vulgaris
strains can accumulate over 50% carbohydrates per dry weight
under autotrophic condition, and have been successfully used as a
feedstock to produce bioethanol and biohydrogen [1418]. The car-
bohydrates produced from the microalga grown under autotrophic
conditions consist mainly of glucose and xylose, which account
for 44.9% and 6.14% of dry cell weight, respectively. Mixotorophic
cultivation of microalage can greatly shorten cultivation time for
the biomass growth and carbohydrate accumulation. The current
work was thus undertaken to evaluate the feasibility of using
this carbohydrate-rich microalga cultivated under mixotrophic
condition as feedstock for biobutanol production. Before using
hydrolysate of real microalgal biomass for biobutanol fermenta-
tion with a C. acetobutylicum strain, it is useful to obtain clearer cell
growth and butanol production properties with pure and mixed
mono sugars (i.e., glucose and xylose) as the carbon source. There-
fore, in this study, a simulated hydrolysate of the microalgae-based
carbohydrates (i.e., an appropriate ratio of glucose and xylose) was
used as the carbon source to characterize the kinetic properties of
bio-butanol fermentation.
2. Materials and methods
2.1. Microalgal strain and its cultivation conditions
The microalga used in this study was C. vulgaris JSC-6, which is
a UV-mutated strain of C. vulgaris ESP6 isolated from fresh water
in Southern Taiwan [1416]. The basal medium used to grow the
microalga consisted of (g/L): KNO3 , 1.25; KH2 PO4 , 1.25; MgSO4 , 1.0;
CaCl2 , 0.111; FeSO4 , 0.0498; EDTA 2 Na, 0.5. Mixotrophic cultiva-
tion was used, in which carbon dioxide (2.5%) was continuously
supplied to the microalgal culture at a ow rate of 0.2 vvm, and
0.3 g/L acetate and 0.8 g/L butyrate was added to the basal medium,
as mentioned above. The organic acids were used as the carbon
source for the heterotrophic growth of C. vulgaris, since they are the
predominant soluble metabolites in the dark fermentation culture
associated with Clostridium sp. [14]. The microalga was grown in a
1-L glass photobioreactor illuminated by an external light source
(TL5 uorescent lamp) mounted on both sides of the photobioreac-
tor at a light intensity of 150 mol/m2 /s. The initial optical density
(measured at 685 nm) and pH used for the microalga cultivation
were 2.0 and 7.0, respectively. The liquid sample was collected from
the sealed glass vessel to determine the microalgae cell concentra-
tion, carbohydrate content in the microalga, pH, and residual nitrate
concentration over time.
2.2. Fermentative bacterium and its growth condition
C. acetobutylicum ATCC824 was used for butanol fermentation.
The inoculum was prepared from spore suspension with a 2 min
heat-shock at 70 C for germination induction, and then incuba-
tion for 24 h at 37 C. The medium used to grow C. acetobutylicum
ATCC824 consisted of (g/L): yeast extract, 5; KH2 PO4 , 0.75; K2 HPO4 ,
2; NaCl, 1; MgSO4 , 0.2; MnSO4 , 0.01; and FeSO4 , 0.01; l-cysteine,
0.5, adjusted to pH 6.0.
221
2.3. ABE fermentation using glucose and xylose as sole or dual
carbon sources
The butanol fermentation was conducted at a controlled tem-
perature of 37 C. The initial pH of the culture was 6.0, and this
was then maintained at 4.5 with NaOH (1 N). The batch cultiva-
tion of C. acetobutylicum ATCC824 was carried out in a 250 mL glass
vessel and the working volume was 100 mL. Glucose alone, xylose
alone, and the mixture of the two with a glucose to xylose ratio of
5:1 or 6.5:1 (simulating the sugar composition of the hydrolyzed
C. vulgaris JCS-6), were used as the carbon source for biobutanol
fermentation. The concentrations of 20, 40, 60, and 80 g/L were
examined for the batch butanol fermentation.
2.4. Analytical methods
The carbohydrate content and prole in the microalgal biomass
were determined using a modied quantitative saccharication
(QS) method (Huang) originally reported by the National Renew-
able Energy Laboratory (NREL), USA [19]. In general, after grown
under mixotrophic conditions for nearly 11 days, the broth of
C. vulgaris JSC-6 culture was centrifuged at 6000 rpm for 3 min,
washed three times, and then lyophilized. The lyophilized microal-
gal biomass was dissolved into 3 mL 72% (w/w) sulfuric acid in a
water bath at 30 C for about 60 min to completely dissolve the
biomass. The liquid samples were then mixed with 87 mL water to
reach an acid concentration of 4% (w/w), and the acidmicroalga
mixture was autoclaved at 121 C for 20 min for hydrolysis of car-
bohydrates to obtain sugars. The supernatant of the hydrolyzed
microalgal biomass was neutralized with solid CaCO3 and analyzed
using a high-performance liquid chromatograph (HPLC) equipped
with a refraction index detector (RID-10A, Waters, USA) for the
assay of monosaccharides (glucose, xylose). The column used was
ICSep 156ICE-COREGEL 87H3 (Transgenomic, USA) [20]. The solu-
ble metabolites produced during the ABE fermentation were also
detected with the same HPLC device used for sugar analysis men-
tioned above following the procedures described in our earlier
work [21]. The mobile phase for sugar and other soluble metabo-
lites production was 0.008 N H2 SO4 with a ow rate of 0.4 mL/min,
the injection sample volume was 2.0 mL and the column tempera-
ture was controlled at 70 C. The retention time for glucose, xylose,
acetate, butyrate, ethanol and butanol were 13.8, 14.8, 22.6, 32.1,
32.6, 55.2 min, respectively.
The gas products (mainly H2 and CO2 ) were measured by a
gas meter and analyzed by gas chromatography (Model 9800,
China Chromatography, Taipei, Taiwan) with a thermal conductiv-
ity detector [22].
Dry cell concentrations were obtained based on the optical den-
sity (OD) values measured at 685 nm with a spectrophotometer
(model U-2001, Hitachi, Tokyo, Japan) via appropriate calibration.
3. Results and discussions
3.1. Carbohydrate composition of the biomass of C. vulgaris JSC-6
The microalga C. vulgaris JSC-6 is feasible for use as a feedstock
because of its fast growth rate and high carbohydrate accumulation
ability [15]. The carbohydrate composition of C. vulgaris JSC-6 under
mixotrophic growth was analyzed after cultivation for nearly 11
days, a carbohydrate content of 51.2% per dry weight of biomass
was detected. The major sugar composition in the carbohydrates
was glucose and xylose. The glucose to xylose ratio in the microalgal
carbohydrates was typically in the range of 5:16.5:1 (based on a
glucose and xylose content (cell dry weight basis) of 28.0243.6%
and 5.416.5%, respectively). Herein, the different ratio of glucose
222 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230
to xylose represents different cultivation period of C. vulgaris JSC-
6 as carbohydrate content will accumulate after nitrogen source
exhausted [17].
3.2. Butanol fermentation performance at different sugar
concentrations
Carbohydrates are the main carbon source used in the culture
medium for ABE fermentation using Clostridial species [23]. Butanol
production with a theoretical yield of about 90% was obtained when
glucose was used as the sole carbon source. Since the objective
of this work is to use microalgae-based carbohydrates (containing
mainly glucose and xylose after hydrolysis) as the carbon source for
butanol production, glucose and xylose were employed as sole and
dual carbon sources, based on the results of the composition anal-
ysis of C. vulgaris biomass, to investigate the kinetics of butanol
production when using the major monosaccharides originating
from microalgal carbohydrates as the carbon source. Moreover, the
concentration of the carbon source usually plays a crucial role in the
efciency of anaerobic butanol fermentation [24]. Therefore, differ-
ent concentrations of carbon sources (glucose, xylose and mixed
sugars at a total concentration of 20 g/L, 40 g/L, 60 g/L and 80 g/L)
were used to examine the effects of this concentration on the per-
formance of ABE fermentation.
The process of butanol production in ABE fermentation can
be divided into the acid production phase (acidogenesis) and the
solvent production phase (solventogenesis) [4]. In the acid produc-
tion phase, the growth of C. acetobutylicum is accompanied by the
production of organic acids (mainly acetate and butyrate) and H2
(Fig. 1). In the solvent production phase, the organic acids gener-
ated in the earlier phase are converted to produce acetone, ethanol
and butanol (Fig. 1). Since glucose accounts for over 80% of total
microalgal sugars, according to the composition analysis (Section
3.1), the butanol fermentation was rst carried out at different
concentrations of glucose (Fig. 1).
At a glucose concentration of 20 g/L, a higher nal butyric
acid concentration and lower solvent concentration were detected,
suggesting that butanol fermentation should be carried out at
a relatively higher carbon source concentration to achieve bet-
ter butanol producing performance. The concentration of butanol
increased from 1.85 to 13.03 g/L as the glucose concentration was
increased from 20 to 80 g/L. The butanol yield increased as the
glucose concentration increased from 20 to 60 g/L, reaching a max-
imum butanol yield and productivity of 0.45 mol/mol glucose and
0.33 g/L/h, respectively, with nearly complete glucose utilization
(Table 1). A further increase in glucose concentration to 80 g/L
resulted in an increase in maximum butanol production and a sim-
ilar butanol yield, but the glucose utilization efciency became
much lower (87.6%) (Table 1).
Based on the metabolism of Clostridial species, the fermenta-
tion process always produces both liquid (ABE) and gaseous (H2 )
biofuels [24,26]. As indicated in Table 1, a maximum H2 accumu-
lation (10,021 mL/L) and yield (1.76 mol/mol glucose) were also
obtained at a glucose concentration of 60 g/L. A further increase
in glucose concentration from 60 to 80 g/L led a decrease in both
H2 production and yield. These results suggest that the best glucose
concentration for butanol fermentation would be 60 g/L.
Butanol fermentation was also carried out using different con-
centrations of xylose as the carbon source, with the results shown
in Fig. 2. It can be see that butanol was successfully produced using
xylose, but a long lag phase (1626 h) occurred when the xylose
concentration was increased from 20 to 80 g/L. This lag time is much
longer than that observed when using glucose as a carbon source
(Figs. 1 and 2). The concentration of butanol increased from 1.34
to 8.89 g/L when the xylose concentration was increased from 20
to 80 g/L (Table 2). The butanol production performance was quite
poor when the xylose concentration was lower than 20 g/L. How-
ever, when the xylose concentration was increased to 4080 g/L,
the performance was better, giving a similar butanol yield and
productivity of 0.320.38 mol/mol xylose and 0.120.14 g/L/h,
respectively (Table 2). During ABE fermentation with C. aceto-
butylicum ATCC824, a maximum H2 production of 73087352 mL/L
was obtained at xylose concentrations of 6080 g/L (Table 2). More-
over, as the xylose concentration increased from 20 to 80 g/L, the
xylose utilization efciency decreased from 99 to 70% (Table 2). As
shown in Tables 1 and 2, the butanol production performance when
using glucose as the carbon source is signicantly better than when
using xylose.
Since the results of the composition analysis show that the ratio
of glucose and xylose content in the microalgal biomass ranged
from 5:1 to 6.5:1 (data not shown), a mixed sugar with a glucose-
to-xylose ratio of 5:1 was then used as the carbon source for
butanol fermentation. As shown in Fig. 3, the concentration of
butanol increased from 1.36 to 11.5 g/L as the total sugar con-
centration increased from 20 to 80 g/L. Although a higher total
sugar concentration resulted in a higher nal butanol concentra-
tion, as well as a slightly higher butanol productivity, a similar
maximum butanol yield of 0.420.47 mol/mol sugar was observed
when the total sugar concentration was 4080 g/L (Table 3). On the
other hand, H2 production increased as the sugar concentration
increased. However, the maximum H2 accumulation (7756 mL/L)
and yield (2.13 mol/mol sugar) were obtained at a total sugar con-
centration of 60 g/L and 40 g/L, respectively (Table 3). Moreover,
when the mixed sugar was used as the carbon source, the C. aceto-
butylicum ATCC824 strain preferred to utilize glucose over xylose.
The glucose utilization efciency was 99100% for the xylose con-
centration of 2060 g/L, while a lower glucose utilization of 96.6%
was observed when the xylose concentration was increased to
80 g/L. In contrast, the xylose utilization was only 2044% (Table 3).
To determine the effect of the glucose to xylose ratio in the
mixed sugars on the ABE fermentation performance, another
glucose-to-xylose ratio (i.e., 6.5:1) was also used to carry out ABE
fermentation (Fig. 4). This increased glucose-to-xylose ratio did not
affect the trends of solvent generation and H2 accumulation, as
indicated in Table 4. These results also show that the butanol pro-
duction performance using mixed sugar with a glucose-to-xylose
ratio of 5:1 and 6.5:1 is quite similar to that obtained from using
glucose alone as the carbon source. Based on the butanol produc-
tion results and taking the sugar utilization efciency into account,
the preferable total sugar concentration for butanol fermentation
is thus 60 g/L.
3.3. Kinetics of cell growth and product (H2 , solvent, and butanol)
formation using different compositions of carbon sources
To date, little research has been done to describe the kinetics of
cell growth and product generation in relation to the current study,
and thus two kinetic models were used to describe the ABE fer-
mentation system in this work. The dependence of the cell growth
rate of C. acetobutylicum ATCC824 on glucose, xylose and mix sugar
concentrations is described by the following Monod-type kinetic
model (Eq. (1)) [27]:
max S
= (1)
KS + S
where  denotes the specic growth rate (h1 ), S denotes the
carbon substrate concentration (g/L), max denotes the maximum
specic growth rate (h1 ), and KS denotes the half-saturation con-
stant (g/L).
 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 223

12000
(a) 20 g/L 70 6 12000
(b) 40 g/L 70 6

H2 content in biogas (%)

H2 content in biogas (%)

H2 accumulation (ml/L)

H2 accumulation (ml/L)
10000 60 5 10000 60 5
50 50

Biomass (g/L)

Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 10 1

0 0 0 0 0 0
80 14 80 14

Solvent production (g/L)

Solvent production (g/L)

Glucose conc. (g/L)

Glucose conc. (g/L)

12 12
60 60
10 10
8 8
40 40
6 6

20 4 20 4
2 2
0 0 0 0
5 5

Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH
5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (h) Time (h)

12000
(c) 60 g/L 60 6 (d) 80 g/L
12000 60 6
H2 content in biogas (%)
H2 accumulation (ml/L)

10000 50 5

H2 content in biogas (%)

H2 accumulation (ml/L)

10000 50 5
Biomass (g/L)

8000 40 4

Biomass (g/L)
8000 40 4
6000 30 3
6000 30 3
4000 20 2
4000 20 2
2000 10 1
2000 10 1
0 0 0
80 14 0 0 0
80
Solvent production (g/L)

14

Solvent production (g/L)

Glucose conc. (g/L)

12
Glucose conc. (g/L)

12
60
10 60
10
8
40 8
6 40
6
20 4
20 4
2
2
0 0
0 0
5
Organic acid conc. (g/L)

6.0 5
Organic acid conc. (g/L)

6.0
4
4
5.5
3 5.5
pH

3
pH

5.0
2 5.0
2
4.5 1 4.5 1
4.0 0
4.0 0
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Time (h)
Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)

Fig. 1. BioH2 and ABE fermentation performance at different glucose concentrations (2080 g/L). (Operating condition: temperature: 37 C; initial pH: 6.0; controlled nal
pH: 4.5; agitation rate: 200 rpm.)
224 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230

(a) 20 g/L 70 7
(b) 40 g/L 70 7
8000 8000

H2 content in biogas (%)

H2 content in biogas (%)

H2 accumulation (ml/L)

H2 accumulation (ml/L)
60 6 60 6

6000 50 5 6000 50 5

Biomass (g/L)

Biomass (g/L)
40 4 40 4
4000 4000
30 3 30 3

20 2 20 2
2000 2000
10 1 10 1

0 0 0 0 0 0
80 14 80 14

Solvent production (g/L)

Solvent production (g/L)

12 12
Xylose conc. (g/L)

Xylose conc. (g/L)

60 60
10 10
8 8
40 40
6 6

20 4 20 4
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH
5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
70 7 70
8000 8000
H2 content in biogas (%)

H2 content in biogas (%)

H2 accumulation (ml/L)

H2 accumulation (ml/L)

60 6 60 6

6000 50 5 50
Biomass (g/L)

6000

Biomass (g/L)
40 4 40 4
4000 4000
30 3 30
20 2 20 2
2000 2000
10 1 10
0 0 0 0 0 0
80 14 80 14
Solvent production (g/L)

Solvent production (g/L)

12 12
Xylose conc. (g/L)

Xylose conc. (g/L)

60 60
10 10
8 8
40 40
6 6

20 4 4
20
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH

5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
H2 accumulation (ml/L) Xylose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)

Fig. 2. BioH2 and ABE production performance at different xylose concentrations (2080 g/L). (Operating conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH:
4.5; agitation rate: 200 rpm.)
 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 225

(a) 20 g/L 70 6
(b) 40 g/L 70 6

H2 content in biogas (%)

8000

H2 content in biogas (%)

8000
H2 accumulation (ml/L)

H2 accumulation (ml/L)
60 5 60 5
50 50

Biomass (g/L)

Biomass (g/L)
6000 4 6000 4
40 40
3 3
4000 30 4000 30
2 2
20 20
2000 2000
10 1 10 1

0 0 0 0 0 0
14 14

Solvent production (g/L)

Solvent production (g/L)

60 12 60 12
Sugar conc. (g/L)

Sugar conc. (g/L)

10 10
40 8 40 8
6 6
20 4 20 4
2 2
0 0 0 0
5 5

Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH
5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
70 6 70 6
8000
H2 content in biogas (%)

8000

H2 content in biogas (%)

H2 accumulation (ml/L)

H2 accumulation (ml/L)

60 5 60 5
50 50
Biomass (g/L)

Biomass (g/L)
6000 4 6000 4
40 40
3 3
4000 30 4000 30
2 2
20 20
2000 2000
10 1 10 1

0 0 0 0 0 0
14 14
Solvent production (g/L)

Solvent production (g/L)

60 12 60 12
Sugar conc. (g/L)

Sugar conc. (g/L)

10 10
40 8 40 8
6 6
20 4 20 4
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH

5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Xylose (g/L)

Fig. 3. BioH2 and ABE fermentation performance at different mixed sugar concentrations (2080 g/L). The ratio of the glucose and xylose concentration is 5:1. (Operating
conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH: 4.5; agitation rate: 200 rpm.)
226 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230

12000
(a) 20 g/L 70 6
(b) 40 g/L
12000 70 6

H2 content in biogas (%)

H2 content in biogas (%)

H2 accumulation (ml/L)

60

H2 accumulation (ml/L)
10000 5 10000 60 5
50

Biomass (g/L)
50

Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 1
10
0 0 0 0 0 0
70 14 70 14

Solvent production (g/L)

Solvent production (g/L)

60 12 60 12
Sugar conc. (g/L)

Sugar conc. (g/L)

50 10 50 10
40 8 40 8
30 6 30 6
20 4 20 4
10 2 10 2
0 0 0 0
5 5
Organic acid conc. (g/L)

Organic acid conc. (g/L)

6.0 6.0
4 4
5.5 5.5
3 3
pH

pH
5.0 5.0
2 2

4.5 1 4.5 1

4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
12000 70 6 12000 70 6
H2 content in biogas (%)

H2 content in biogas (%)

H2 accumulation (ml/L)

60
H2 accumulation (ml/L)

10000 5 10000 60 5
50
Biomass (g/L)

50

Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 1
10
0 0 0 0 0 0
70 14 70 14
Solvent production (g/L)

Solvent production (g/L)

60 12 60 12
Suagr conc. (g/L)

Sugar conc. (g/L)

50 10 50 10
40 8 40 8
30 6
30 6
20 4
20 4
10 2
10 2
0 0
0 0
5
Organic acid conc. (g/L)

5
Organic acid conc. (g/L)

6.0
6.0
4
4
5.5
5.5
3
pH

3
pH

5.0
2 5.0
2
4.5 1 4.5 1
4.0 0
4.0 0
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Time (h)
Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Xylose (g/L)

Fig. 4. BioH2 and ABE fermentation performance at different mixed sugar concentrations (2080 g/L). The ratio of the glucose and xylose concentration was 6.5:1. (Operating
conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH: 4.5; agitation rate: 200 rpm.)
 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 227

Table 1
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different glucose concentrations.

Glucose Glucose H2 production Butanol production

conc. (g/L) utilization (%)

H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(mL/L) glucose) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)

20 99.8 3293.3 1.34 2.78 (4.6:11.8:1) 1.85 0.23 0.12

40 99.8 6068.8 1.50 10.33 (6.5:16.5:1) 7.05 0.43 0.26
60 99.6 10,021.2 1.76 16.71 (8.0:16.7:1) 10.55 0.45 0.33
80 87.6 9912.2 1.46 20.30 (6.1:13.2:1) 13.03 0.45 0.27

Table 2
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different xylose concentrations.

Xylose Xylose H2 production Butanol production

conc. (g/L) utilization (%)

H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(mL/L) xylose) (acetone:butanol:ethanol) production (g/L) xylose) productivity (g/L/h)

20 99 4366.2 1.42 1.98 (7.3:13.7:1) 1.34 0.13 0.04

40 98 5592.5 0.99 10.9 (5.3:12.8:1) 7.40 0.38 0.12
60 83 7351.7 1.08 11.42 (5:13.7:1) 7.86 0.33 0.14
80 70.4 7308.3 0.88 13.29 (4.9:12.5:1) 8.89 0.32 0.13

Table 3
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different total mixed sugar concentrations with a glucose-to-xylose ratio of 5:1.

Total sugar Sugar utilization H2 production Butanol production

concentration (g/L)

Glucose Xylose H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(%) (%) (mL/L) sugar) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)

20 99.0 19.8 2423.7 0.021 2.09 (3.7:9.3:1) 1.36 0.19 0.07

40 99.9 43.8 5984.9 2.13 10.24 (7.0:14.7:1) 6.34 0.47 0.21
60 99.9 32.7 7756.3 1.34 13.70 (6.3:15.2:1) 9.09 0.44 0.33
80 96.6 29.7 8518.8 1.05 17.95 (6.3:13.5:1) 11.5 0.42 0.36

In addition, the dependences of gas product (bio-H2 ) and solvent
product (bio-butanol and total solvent production) were evaluated
by the MichaelisMenten (MM) model (Eq. (2)) [24]:
vmax S
v=
Km + S
where v represents the specic product production rate (L/h/L for
H2 , and g/h/L for liquid production), vmax represents the maximum
specic product production rate (L/h/L, g/h/L for butanol or g/h/L
for solvent), and Km represents the Michaelis constant (g/L).
The dependence of cell growth and product production rates
on glucose concentration is shown in Figs. 5 and 6, respectively.
The estimated parameters for the Monod-type model (max and
KS ) are 0.35 h1 and 7.43 g/L, respectively (Table 5). Furthermore,
the MM type model ts the experimental results for the kinetics
of H2 , solvent, and butanol with an R2 value of 0.95 (Fig. 6a), 0.97
(Fig. 6b) and 0.99 (Fig. 6c). The vmax and Km values for H2 production
are 1.25 L/h/L and 23.12 g/L, for solvent production 1.22 g/h/L and
58.91 g/L, and for butanol production 0.93 g/h/g VSS and 65.28 g/L,
respectively.
The effects of various xylose concentrations on cell growth
and product formation rates were also investigated, with the
model simulation results shown in Figs. 5 and 6, respectively.
The Monod-type model tted the cell growth data with an R2
value of 0.96, while the MM model also ts the data well
(2)
(R2 = 0.920.97). The parameters (max and KS ) were 0.18 h1 and
4.21 g/L, respectively (Table 5). The vmax and Km values for H2
production were 0.45 L/h/L and 6.29 g/L, for solvent production
0.77 g/h/L and 45.43 g/L, and for butanol production 0.63 g/h/L and
57.73 g/L, respectively (Table 5).
The growth kinetics of C. acetobutylicum ATCC824 on mixed sug-
ars at a glucose-to-xylose ratios of 5:1 and 6.5:1 are shown in
Fig. 5, while simulations with the Monod-type model gave max
values of 0.31 h1 and 0.34 h1 , respectively, which are quite simi-
lar (Table 5). Kinetic modeling with the MM model also shows that
the bio-H2 and ABE production performances with the two glucose
to xylose ratios were also very similar in terms of the maximum
production rate (i.e., vmax ) and the dynamic trends (as represented
by the Km value) (Table 5). Nevertheless, the vmax is slightly higher
when a higher glucose to xylose ratio is used. These results indicate

Table 4
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different total xylose concentrations with a glucose-to-xylose ratio of 6.5:1.

Total sugar Sugar utilization H2 production Butanol production

conc. (g/L)

Glucose Xylose H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(%) (%) (mL/L) sugar) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)

20 99.7 12.3 3174.1 1.32 1.79 (4.4:13.2:1) 1.27 0.12 0.07

40 99.8 51.8 5094.7 1.87 8.82 (6.5:17.0:1) 6.12 0.42 0.24
60 99.8 31.9 7574.0 1.24 13.14 (6.0:15.2:1) 8.99 0.40 0.32
80 96.3 29.2 7718.8 1.09 18.82 (5.5:11.2:1) 11.07 0.40 0.26
228 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230

Table 5
Kinetics of cell growth, bioH2 production, and butanol production with Clostridium acetobutylicum ATCC824 using different carbon sources.

Carbon source Monod type model MichaelisMenten model for MichaelisMenten model for MichaelisMenten model for
H2 production butanol production solvent production

max (h1 ) KS (g/L) R2 max (L/h/L) Km (g/L) R2 max (g/h/L) Km (g/L) R2 max (g/h/L) Km (g/L) R2

Glucose 0.35 7.43 0.96 1.25 23.12 0.95 0.93 65.28 0.99 1.22 58.91 0.97
Xylose 0.18 4.21 0.96 0.45 6.29 0.97 0.63 57.73 0.94 0.77 45.43 0.96
Mixed (G/X = 5.0) 0.31 4.54 0.99 0.83 13.38 0.98 0.89 59.25 0.99 0.97 66.76 0.96
Mixed (G/X = 6.5) 0.34 4.67 0.99 0.94 16.11 0.96 0.90 62.16 0.99 1.00 66.33 0.97

3.4. Effects of different carbon sources on the cell growth and

Glucose
0.5 Xylose product formation kinetics
Specific cell gorwth rate (h )
-1

Mixed sugar (Glucose:Xylose = 5:1)

Mixed sugar (Glucose:Xylose = 6.5:1) The type and concentration of carbon substrate are critical fac-
0.4
tors affecting the fermentation kinetics. The results presented in
Figs. 5 and 6 and Table 5 clearly show that glucose is a better carbon
0.3 source than xylose for cell growth as well as butanol and H2 produc-
tion. The maximum growth rate (max ) and production formation
rate (vmax ) obtained when using glucose as the carbon source
0.2
appear to be much higher than that obtained from using xylose
(Table 5). For the case of mixed sugars, since glucose was the pre-
0.1 dominant carbon source over xylose (glucose-to-xylose ratio = 5:1
or 6.5:1), the kinetics with regard to the cell growth and product
formation was still similar to that of using glucose alone (Table 5).
0.0
However, since xylose still accounts for a considerable portion in
0 20 40 60 80 100
the microalgae-based carbohydrates, it still needs to be included in
Sugar concentration (g/L) the carbon substrate in this work to simulate the performance of
using microalgal carbohydrates for ABE fermentation.
Fig. 5. Cell growth kinetics of Clostridium acetobutylicum ATCC824 using glucose
Details of the kinetic parameters for the cell growth and for-
alone (), xylose alone (), mixed sugar (glucose-to-xylose ratio = 5:1) (), and
mixed sugar (glucose-to-xylose ratio = 6.5:1) () as the carbon source. (Symbols: mation of the target products are vital for the effective design of
experimental data; curves: model prediction.) bioreactors and scaling up the butanol fermentation process [28].
However, little information is known concerning the growth and
butanol production kinetics with different carbon sources, with
very few reports revealing the kinetics of cell growth and butanol
that in the mixed sugar containing different ratios of the two carbon production with C. acetobutylicum using sugars originating from the
sources, glucose is still the most important carbon source, dominat- hydrolyzed biomass of C. vulgaris JSC-6 (i.e., mixture of glucose and
ing the kinetic behaviors of cell growth and product formation of xylose) [29]. Moreover, the information obtained from this study
C. acetobutylicum ATCC824, while xylose is used less efciently by also indicates that using mixture of glucose and xylose at differ-
the microalgal strain. ent ratios based on the carbohydrates composition resulting from

Table 6
Electron mass balances (as mg COD) according to the results using glucose, xylose and mixed sugars.

Carbon source Substrate CODin CODres CODSMP Cell yield (g cell/g CODBio CODH2 CODsum Missing electron
conc. (g/L) (mg COD)a (mg COD)b (mg COD)c substrate) (mg COD)d (mg COD)e (mg COD)f equivalents (%)g

Glucose 20 2133 4 1327 0.16 512 215 2058 3.5

40 4267 9 2919 0.09 585 396 3909 8.4
60 6400 26 4365 0.08 776 654 5821 9.0
80 8533 1058 5201 0.07 791 647 7697 9.8

Xylose 20 2133 21 792 0.27 859 285 1958 8.2

40 4267 85 2801 0.15 963 365 4214 1.2
60 6400 1088 3579 0.12 989 480 6137 4.1
80 8533 2526 3512 0.11 975 477 7490 12.2

Mixed sugar (G:X = 5.0) 20 2133 303 1114 0.15 489 158 2065 3.2
40 4267 403 2801 0.09 628 391 4223 1.0
60 6400 723 3797 0.07 724 507 5751 10.1
80 8533 1242 4682 0.07 875 556 7355 13.8

Mixed sugar (G:X = 6.5) 20 2133 255 1075 0.15 489 207 2027 5.0
40 4267 282 2753 0.10 681 333 4049 5.1
60 6400 592 3707 0.08 828 495 5622 12.2
80 8533 1079 4525 0.07 857 504 6966 18.4
a
CODin : mg COD of initial sugar, calculated with sugar concentration (mg COD/L) 0.1 L.
b
CODres : mg COD of residual sugar, calculated with CODin,glucose (1 glucose conversion) + CODin,xylose (1 xylose conversion).
c
CODSMP : mg COD of soluble microbial products (SMP).
d
CODBio : mg COD of biomass in the reactor, calculated with mg cell 1.63 mg COD/mg cell; assuming that cell formula is C5 H7 O2 N.
e
CODH2 : mg COD of H2 evolved, calculated with mol H2 16 g COD/mol H2 1000 mg/g.
f
CODsum : mg COD of residual sugar, SMP, biomass, and H2 .
g
Calculated with (1 CODsum /CODin ) 100%.
 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 229

Average H2 production rate (L/h/L) different cultivation stage of C. vulgaris JSC-6 (i.e., different status
1.4 Glucose
a
Xylose of nitrogen deciency) is all suitable for bio-butanol fermentation
1.2
Mixed sugar (Glucose:Xylose = 5:1) with C. acetobutylicum. The kinetic parameters determined in this
Mixed sugar (Glucose:Xylose = 6.5:1) study thus represent new information that is of great importance in
1.0 understanding the kinetic properties of producing butanol and H2
from microalgae-based carbohydrates. Nevertheless, future stud-
0.8 ies should also use the real hydrolysate of the microalgal biomass
as a carbon source for ABE fermentation, in order to conrm the
0.6 observations obtained in the current work by using a simulated
synthetic mixed sugar carbon source mimicking the microalgal car-
0.4
bohydrates.
0.2
3.5. Electron mass balance of the substrate and products
0.0
0 20 40 60 80 100 The electron mass balances (based on the level of chemical oxy-
Sugar concentration (g/L) gen demand (COD)) of the substrates and products were obtained
for all the experiments to verify whether the quantitative analyses
of the gaseous and liquid metabolites from ABE fermentation were
reasonable. The missing electron equivalent (MEE) is an indicator
Average solvent production rate (g/h/L)

of the deviation between the measured values and the theoretical

Glucose ones. As indicated in Table 6, most of the MEE values are lower
1.0
Xylose
b
than 10%, while only 4 out of 16 experiments have an MEE greater
Mixed sugar (Glucose:Xylose = 5:1)
Mixed sugar (Glucose:Xylose = 6.5:1) than 10% (i.e., 1018%). The electron mass balances conrm that the
0.8 analyses of the gaseous and liquid products were quite accurate,
and thus the technical quality of this work is fairly reliable.
0.6
4. Conclusion

0.4
This work demonstrated the feasibility of using the simulated
hydrolysate of C. vulgaris JSC-6 (primarily containing glucose and
0.2 xylose at a ratio of 5:16.5:1) as the carbon source to produce biobu-
tanol with C. acetobutylicum ATCC824. Kinetic studies show that the
maximum butanol production rate (vmax ) on glucose and xylose
0.0
alone was 0.93 and 0.63 g/h/L, respectively, indicating that glucose
0 20 40 60 80 100
is a more effective carbon source than xylose for ABE fermentation.
Sugar concentration (g/L) In the mixed sugar systems, the bio-butanol production was pre-
dominantly affected by glucose, while the utilization efciency of
xylose by C. acetobutylicum ATCC824 was quite poor (ranging from
20 to 50%).
Average butanol production rate (g/h/L)

Glucose
c
Xylose Acknowledgements
0.6 Mixed sugar (Glucose:Xylose = 5:1)
Mixed sugar (Glucose:Xylose = 6.5:1)
This work was supported by Open Project of State Key Labora-
tory of Urban Water Resource and Environment, Harbin Institute of
Technology (HCK201205). This work is also supported by National
0.4 Natural Science Foundation of China (grant nos. 51008105 and
51121062) and Academician Workstation Construction in Guang-
dong Province (2012B090500018). The authors also gratefully
acknowledged the support by Taiwans Ministry of Science and
0.2
Education (MOST) under grant numbers of 103-2221-E-006-190-
MY3 and 103-3113-E-006-006.

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