i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6

Available online at www.sciencedirect.com

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

Bioproduction of hydrogen by simultaneous saccharification

and fermentation of cassava starch with 2-deoxyglucose-
resistant mutant strains of Clostridium tyrobutyricum

Ling Jiang a, Liying Zhu c, Xian Xu b, Ming Lin b, Yanping Li c, Xiaotong Li b, Huaiyan Cui b,
He Huang b,*
a
College of Food Science and Light Industry, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of
China
b
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009,
People’s Republic of China
c
College of Sciences, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China

article info abstract

Article history: Laboratory mutagenesis of microorganisms offers the possibility of relating acquired mu-
Received 15 October 2012 tations to improve the quality of microbial cultures. In the present study, a mutant strain,
Received in revised form Clostridium tyrobutyricum ATCC 25755 DG-8, with significantly elevated a-amylase activity
11 February 2013 as well as resistant to the non-metabolizable and toxic glucose analog 2-deoxyglucose
Accepted 23 February 2013 (2-DG) was obtained by implanting the low-energy nitrogen ion beam. DG-8 was further
Available online 6 April 2013 developed to produce hydrogen by simultaneous saccharification and fermentation (SSF)
directly form cassava starch in batch fermentation mode, which to our knowledge is at the
Keywords: first attempt in genus Clostridium. Our results demonstrated that the increased activity of
Clostridium tyrobutyricum a-amylase would be attributed to the hydrogen over-producing. Higher hydrogen yield
Hydrogen (3.2 mol/mol glucose) was achieved with the volumetric productivity of 0.41 L/h/L when the
Cassava starch initial total sugar concentration of cassava starch rise up to 100 g/L. The present work will
Simultaneous saccharification and help to decrease the cost of hydrogen fermentation process and stimulate its industrial
fermentation application in the near future.
2-Deoxyglucose-resistant mutant Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

1. Introduction Among the known biochemical routes, hydrogen can be

Hydrogen, the most abundant and lightest element in the
universe, has much potential as an attractive further energy
source [1]. Because of the upward trend in the price of oil and
public concerns about the environmental pollution caused by
the petrochemical industry, production of hydrogen by mi-
croorganisms, therefore, has been recognized as a promising
approach and attracted great research interest in recent years.
mainly produced via two distinct pathways: photosynthesis
and fermentation. Compared with photosynthetic processes,
fermentative hydrogen production generally yields two orders
of magnitude higher rates, does not rely on the availability of
light, utilizes a variety of renewable sources, requires less
energy, and is technically much simpler and more stable [2,3].
In addition, fermentative microorganisms have rapid growth
and are not affected by oxygen as the main process is

* Corresponding author. Tel./fax: þ86 25 83172094.

E-mail address: biotech@njut.edu.cn (H. Huang).
0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2013.02.109
6350 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6
anaerobic. Therefore, fermentative hydrogen production is
more advantageous than the photosynthetic hydrogen pro-
duction and appears to have more potential for practical ap-
plications [4].
The genus Clostridium, one of the largest genera of pro-
karyotes, is often reported used to produce hydrogen, such as
Clostridium butyricum [5], Clostriduim acetobutylicum [6,7], Clos-
tridium saccharoperbutylacetonicum [8], Clostridium pasteurianum
[9,10]. Among them, Clostridium tyrobutyricum has many ad-
vantages over other species, including simple medium for cell
growth and relatively high product purity and yield [11,12].
Previous studies regarding this anaerobe were extensively
focused on acid production, while an increasing attention has
been paid on the fermentation processes for biohydrogen
production from glucose, xylose, sucrose, and so on [13e15].
However, the high cost of carbon source come to be one of the
greatest obstacles restraining the industrialization of
hydrogen fermentation with C. tyrobutyricum. Therefore, the
exploitation of cheap, renewable carbohydrate feedstock has
been strongly stimulated.
Among the possible candidates, starch, the second abun-
dant organic resource on earth, is well suited to serve as a
cost-effective substrate instead of expensive refined sugars
for commercialization of hydrogen fermentation. However,
the rate-limiting step for bioconversion of starch is the
effective hydrolysis of the macromolecule into reducing
sugars [16]. The a-amylase was found to be facilitated efficient
starch degradation, which was conducted to enhance the
feasibility of using starch feedstock for hydrogen production
by Chen et al. [17]. Results from our laboratory also demon-
strated incomplete hydrolysis of corn starch by C. tyrobutyr-
icum ATCC 25755 (unpublished results), which suggested that
C. tyrobutyricum can secrete a-amylase at a low basal level,
while a similar observation has been previously reported for
other strains of genus Clostridium [18e20]. It is of scientific
interest and technological importance to utilize this indige-
nous bacterium for hydrolytic pretreatment of raw starch
materials to facilitate the efficiency of biohydrogen produc-
tion. If the activity of the a-amylase in C. tyrobutyricum could
be further improved, the simultaneous saccharification and
fermentation (SSF) process for hydrogen fermentation from
starch feedstock without the addition of commercial
a-amylase might be established, which will have remarkable
advantages in terms of operational cost and space.
Investigators from various laboratories have suggested
that a-amylase enzyme biosynthesis in genus Clostridium is
subject to catabolite repression by glucose or metabolizable
compounds [21,22]. The most effective random mutation
methods for obtaining variants with enhanced a-amylase
production are those combining mutagenesis and enrich-
ment, one of which is the use of the anti-metabolite
2-deoxyglucose (2-DG), a glucose analog, which, when incor-
porated into a suitable medium containing starch, causes
catabolite repression of nonmutant strains, allowing only
mutated strains resistant to this form of suppression to form
colonies [23]. However, to the best of our knowledge, the
foregoing has not been applied into the isolation of C. tyrobu-
tyricum mutant with the capability to produce a high yield of
a-amylase. The objective of this research was undertaken to
evolve glucose-derepressed and/or hyperamylase strains of
C. tyrobutyricum and to further develop an SSF process for
hydrogen production from starch materials without com-
mercial a-amylase supplementation.
2. Materials and methods
2.1. Chemicals
Cassava starch of food-grade (as per the specifications of the
Chinese Edible Cassava Standard NY/T875-2012) was pur-
chased from Wuming Cassava Starch Co. Ltd. (Guangxi,
China), dried to constant weight and passed through an
80-mesh screen. The starch content was measured more than
99.5% by the starch-iodide method [24]. The 2-DG was pur-
chased from Aladdin Reagent Co. Ltd. (Shanghai, China). All
other chemicals were of analytical grade and commercially
available.
2.2. Microorganism and medium
C. tyrobutyricum ATCC 25755 (purchased from Guangdong
culture collection center, collection number: GIM 1.262) was
used as the present strain, and stored in reinforced clostridial
medium [25]. Unless otherwise indicated, all experiments
were performed under anaerobic conditions as previously
described [26]. The cultivation medium was composed of 5 g/L
yeast extract, 5 g/L peptone, 3 g/L (NH4)2SO4, 1.5 g/L K2HPO4,
0.6 g/L MgSO4$7H2O, 0.03 g/L FeSO4$7H2O. Carbon source was
added separately in the seed medium of 20 g/L glucose, and in
the fermentation medium of 60e120 g/L cassava starch. All
the media were sterilized by autoclaving at 121  C, 15 psig, for
20 min.
2.3. SSF process
The batch SSF cultivations were performed in a laboratory-
scale 5-L stirred-tank fermentor (B. Braun, B. Braun Biotech
International, Melsungen, Germany) with a working volume
of 2 L operated at 37  C, agitated at 150 rpm, and pH controlled
at 6.0 by adding 5 N NaOH facilitated by an on-line sensing and
dosing system. Anaerobiosis was maintained by sparging the
medium with N2 (10 mL/min) for 60 min. Fermentation broth
and biogas in the bioreactor were taken at regular intervals for
the analyses of cell growth, extracellular metabolite concen-
trations, including carbon sources, a-amylase, lactic acid,
butyric acid, acetic acid, H2 and CO2 [27].
2.4. Mutagenesis and selection of 2-DG-resistent
mutants
The implantation sources were produced using an ion beam
implantation instrument (LZD-900, Southwestern Institute of
Physics, China). A late exponential phase of C. tyrobutyricum
was centrifuged at 12,000 g for 20 min at 4  C, washed once
with sodium potassium phosphate buffer (pH 7.2), and diluted
in sterilized 2% glucose solution. About 200 mL suspension was
spread to form a single-cell layer on a sterilized Petri dish
(75 mm) and dried in the hood. The dishes were put into the
sample holder and implanted by a nitrogen ion beam with
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6
energy of 10 keV using pulse-type implantation [28]. The
implanted samples were then diluted with 1 mL sterilized
water and plated on the growth medium containing 5 g/L of
starch and 1 g/L of 2-DG. After cultivation for 2 days, the
growing colonies were picked at random and replica plated
onto agar medium containing 20 g/L of glucose. After another
2 days, the master plates were overlaid with an agar solution
containing 20 g/L of starch (pH 5), incubated for 3 h, and then
stained with iodine vapor. The iodine vapor was generated by
heating iodine crystals in a water bath at 80  C. The colonies
exhibiting large clear zones on the iodine-stained plates were
picked for further analysis.
2.5. Analytical procedures
Samples were collected during the exponential phase to
measure the cells, substrates and products for extracellular
metabolite analysis. Cell concentration was analyzed by
measuring the optical density of the cell suspension at OD600
with a spectrophotometer (Ultrospec 3300 pro, Amersham
Bioscience). Dry cell weight (DCW) was determined by
centrifugation of the fermentation broth (10 mL) at 10,000 g for
10 min, washing the sediment twice with distilled water, and
drying at 80  C for 24 h to a constant weight [28].
The concentrations of glucose were measured by SBA-80C
biosensor analyzer (Institute of Biology, Shandong Academy
of Sciences, China). Total sugar concentration was estimated
by the phenol-sulfuric acid method with D-(þ)-glucose (Sigma,
G8270-100G, EC 200-075-1) as the standard [17]. The H2 and
CO2 concentrations in the exhaust gas were determined using
the MultiRAE IR gas monitor PGM 54 (RAE system Inc., San
Jose, USA). Quantitative analysis of butyric acid and acetic
acid was performed by GC (Agilent 6820 GE, Agilent Technol-
ogies) as previously reported [29].
The preparation of the crude extract was described previ-
ously [13]. Protein concentration was determined by the
method of Bradford with bovine serum albumin as the stan-
dard (Bio-Rad protein assay). The activity of a-amylase was
determined as previously described by Annous and Blaschek
[30]. Glucoamylase activity was measured according to the
method of Deng et al. [28]. One unit of a-amylase and glu-
coamylase activity was defined as the amount of enzyme
producing 1 mmol of reducing sugar per min under the
Table 1 e Effect of glucose addition to starch-based medium on the localization of a-amylase activity in C. tyrobutyricum
ATCC 25755.
Carbon sourcea Total activity (U/mg protein)b
2.0%S 2.7  0.4
1.5%S þ 0.5%G 1.1  0.2
1.0%S þ 1.0%G 0.8  0.1
a S: Sucrose; G: Glucose.
b Total activity (U/mg protein) equals the sum of the total activities of all cell fractions: [cell-adsorbed activity (U/mg protein/
mL)  50 mL] þ [intracellular activity (U/mg protein/mL)  10 mL] þ [extracellular activity (U/mg protein/mL)  200 mL]. It was measured at the
highest activity produced.
c Cell fraction activity (U/mg protein)  100/total activity.
6351
reaction conditions specified. All results were obtained from
the means of triplicate determinations.
3. Results and discussion
3.1. Mutagenesis, enrichment, and isolation of mutants
Low-energy nitrogen ion (Nþ) implantation was used as the
mutagenizing agent to improve the wild-type strain C. tyro-
butyricum ATCC 25755. Cells were found to be very sensitive to
low-energy Nþ implantation, which finally resulted in 98.7%
loss of cell viability at a fixed energy of 10 keV. Then, a total of
261 colonies of the 1.3  105 survivors were picked at random.
Nine of these, termed DG-1, DG-2-DG-9, respectively, were
further examined based on the production of large clear zones
on starch-overlaid glucose plates. The mutants were firstly
transferred to the growth medium containing 1.0 g/L 2-DG to
investigate their hereditary stability for 10 generations, and
the results showed that all of them grew normally.
3.2. Effect of carbon source on amylolytic activity
Previous results suggested that the production of amylolytic
enzymes, including a-amylase and glucoamylase, in genus
Clostridia are subject to the influence by different carbon
sources [30]. The activity of a-amylase is wherein a decisive
factor with regard to the conversion efficiency from starch to
reducing sugars, while the low utilization of starch is usually
due to the low activity of the a-amylase produced by Clostridia
[31]. Therefore, the effect of various combinations of glucose
and cassava starch in the growth medium on the localization
of a-amylase activity in C. tyrobutyricum was first investigated
and the results shown in Table 1 were obtained. An increase in
the concentration of glucose in the growth medium was
accompanied by a corresponding increase in the cell-adsorbed
and the intracellular a-amylase activity while a decrease in
the extracellular and the total a-amylase activity. The activity
of the cell-adsorbed fraction suggests that this enzyme is
rather loosely attached to the surface of the cell, which is in
accordance with the findings of Annous and Blaschek [30].
The effect of carbon sources on the formation of extracel-
lular a-amylase and glucoamylase by Clostridium tyrobutylicum
% Of activity in cell fractionc
Cell-adsorbed Intracellular Extracellular
0.0 1.8 94.6
0.4 44.8 32.2
0.7 52.8 21.6
6352 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6

Table 2 e Total a-amylase and glucoamylase activity produced by C. tyrobutyricum wide-type and mutant strains grown in
growth medium containing 20 g/L of carbon source.
Strain Total a-amylase Total glusoamylase activitya Repression ratio for
activitya (U/mg protein) (U/mg protein) of strain grown on a-amylase/glucoamylaseb
of strain grown on

Starch Glucose Starch Glucose

Wide-type 2.6  0.3 0.4  0.1 7.2  0.9 3.0  0.3 6.5/2.4
DG-1 2.8  0.3 0.5  0.1 8.0  0.5 3.8  0.5 5.6/2.1
DG-2 1.5  0.2 0.3  0.1 5.4  0.7 2.7  0.5 5.0/2.0
DG-3 1.3  0.2 0.2  0.1 4.6  0.6 1.9  0.4 6.5/2.4
DG-4 1.4  0.2 0.1  0.0 5.0  0.6 0.8  0.1 14/6.0
DG-5 2.7  0.4 0.6  0.1 7.4  0.9 5.3  0.5 4.5/1.4
DG-6 3.2  0.4 0.8  0.1 8.4  1.1 4.9  0.4 4.0/1.7
DG-7 2.4  0.4 0.3  0.1 6.8  0.8 2.1  0.2 8.0/3.2
DG-8 5.4  0.5 1.9  0.2 15.6  1.2 14.0  0.9 2.8/1.1
DG-9 3.8  0.3 0.7  0.1 9.6  0.5 4.4  0.4 5.4/2.2

a It was measured at the highest activity produced.

b Specific activity produced when the culture was grown on starch divided by the specific activity produced by a culture grown on glucose.

ATCC 25755 and mutants were shown in Table 2. As can be
seen in Table 2, there was a dramatic increase in the total
amylolytic activity (6.5-fold of a-amylase, 2.4-fold of glucoa-
mylase) of C. tyrobutylicum wild-type for cell outgrowth from
starch over that seen when this strain was grown on glucose,
which was correspondence with a lengthened lag phase in
starch-based medium. Both a-amylase and glucoamylase ac-
tivities of the 9 mutants when grown in the medium con-
taining 20 g/L of glucose or cassava starch demonstrated
on the total a-amylase activity. Therefore, the production of
a-amylase enzymes by C. tyrobutyricum wild-type and mutant
strain were proved to be subject to induction by starch and
catabolite repression by glucose, which was consistent with
the observations previously reported in genus Clostridia [21].
The variability in a-amylase activity values and the corre-
sponding repression ratios of the mutant DG-8 isolated in this
10 6

considerable variability (Table 2), but with the similar change (A)

Total amylase activity (U/mg protein)

trend, which indicated that the production of extracellular a-
amylase and glucoamylase was under similar biosynthetic 1 4
control [18]. Among them, DG-8 demonstrated a 2.1-fold in-
Biomass (g/L)

crease in total a-amylase activity relative to those of the wild-

type strain grown in starch-based medium (5.4 vs. 2.6 U/mg
0.1 2
protein). This finding, together with the corresponding
repression ratio of 2.8 (the lowest value of the tested strains),
suggests that a-amylase enzyme production in the DG-8
mutant strain is not significantly catabolite repressed, which 0.01 0
0 20 40 60
was picked out for further examination. Time (h)
10 6
3.3. Regulation of a-amylase enzymes biosynthesis (B)
Total amylase activity (U/mg protein)

The effect of the addition of 1% glucose to 2% starch-based

1 4
medium at time zero (0 h) and during the early exponential
Biomass (g/L)

phase (12 h) on the growth response and the total a-amylase

activities of C. tyrobutyricum wild-type were shown in Fig. 1.
The addition of glucose at time zero (Fig. 1A) reduced the lag 0.1 2

phase of wild-type by ca. 10 h and the total a-amylase activity

by 60%. A similar addition at 12 h (Fig. 1B) had no effect on the
growth response of wild-type, but reduced the total a-amylase 0.01 0
0 20 40 60
activity by 14% relative to the values obtained without added
Time (h)
glucose.
Fig. 2 shows the effect of the addition of 1% glucose to 2%
starch-based medium at 0 and 12 h on the growth response Fig. 1 e Effect of addition of 1% glucose to 2% starch-based
and the total a-amylase activity of C. tyrobutyricum DG-8. The medium at time zero (A) and during the early exponential
addition of glucose at time zero (Fig. 2A) reduced the lag phase phase (B) on the growth response (open symbols) and the
by approximately 6 h, the specific growth rate by 40%, and the total a-amylase activities (closed symbols) of C. tyrobutylicum
total a-amylase activity by 26%. The addition of glucose at 12 h wide-type. Symbols: 2% starch (,, -); 2% starch D 1%
(Fig. 2B) had no effect on the growth response and little effect glucose (6, :).
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6 6353

10 6 3.5
(A)

Total amylase activity (U/mg protein)

3.0

Hydrogen yield (mol / mol)

1 2.5
4
Biomass (g/L)

2.0

0.1
1.5
2

1.0
0.01
0.5
0
0 20 40 60
Time (h) 0.0
WT DG-1 DG-2 DG-3 DG-4 DG-5 DG-6 DG-7 DG-8 DG-9

10
Strain number
6
(B)
Fig. 3 e Hydrogen production of C. tyrobutyricum wide-type

Total amylase activity (U/mg protein)

(WT) and its nine mutants (DG-1-DG-9). The data presented
here were the averages of three separate experiments.
1
4
Biomass (g/L)

0.1
2

(A) 100 50 4

Production of acid and biomass (g/L); gas (L)

0.01
0 80 Total sugar 40
Total sugar concentration (g/L)

0 20 40 60 amylase 3

Amylase (U/mg protein)

Time (h) H2
CO2
60 30
Butyric acid
Fig. 2 e Effect of addition of 1% glucose to 2% starch-based Acetic acid 2
medium at time zero (A) and during the early exponential Lactic acid
40 Biomass 20
phase (B) on the growth response (open symbols) and the
total a-amylase activities (closed symbols) of C. tyrobutylicum 1
20 10
DG-8. Symbols: 2% starch (,, -); 2% starch D 1% glucose
(6, :).
0 0 0
0 20 40 60 80 100
Time (h)

study suggest an alteration in carbohydrate metabolism. (B) 100 80 Production of acid and biomass (g/L); gas (L) 6

Recent work by Annous and Blaschek suggested that the

Total sugar
a-amylase in C. acetobutylicum ATCC 824 is subject to catabo- 80
Total sugar concentration (g/L)

amylase 60
Amylase (U/mg protein)

lite repression by glucose at the level of transcription [30]. H2

4
CO2
This type of regulation of enzyme synthesis in Clostridia has 60
Butyric acid
been shown to be mediated by the phosphoenolpyruvate- Acetic acid 40
Lactic acid
dependent sugar phosphotransferase system, which has 40 Biomass
been proved to be a widespread mechanism for carbohydrate 2
20
uptake by bacteria, including C. tyrobutyricum [13,26,32,33]. 20

3.4. Improvement of hydrogen fermentation ability 0 0 0

0 20 40 60 80 100
Time (h)
To determine the feasibility of using starch materials as a low
cost substrate for fermentative hydrogen production, the Fig. 4 e Time courses of fermentative production by C.
batch SSF of cassava starch (80 g/L total sugar) was converted tyrobutylicum wide-type (A) and the mutant DG-8 (B) with
to hydrogen using C. tyrobutyricum wild-type and mutants. As cassava starch as carbon source (100 g/L total sugar). For
can be seen in Fig. 3, five DG-resistant mutants (DG-1, DG-5, clarity, the three acid products, CO2 as well as the biomass
DG-6, DG-8 and DG-9) with higher a-amylase activity were shown in grey lines. The total sugar, amylase and H2
showed enhanced hydrogen production from cassava starch. were shown in black lines. The data presented are the
Among the five mutants, mutant strain DG-8, with the highest averages of three separate experiments.
6354 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6
Table 3 e Effect of initial sugar concentration (cassava starch) on hydrogen production of C. tyrobutyricum DG-8 in batch SSF.
Total sugar Total residual Total
con. (g/L) sugar con. (g/L) amount (L)
60 0.8  0.1 49.1  2.1
80 1.5  0.2 65.1  3.2
100 10.2  0.4 79.4  3.8
120 21.4  0.9 54.5  5.0
a Yield of 1 mol hydrogen/mol glucose is equal to 1.11 mol hydrogen/mol starch on the basis of the stoichiometric equation.
a-amylase activity previously mentioned, had the best per-
formance for hydrogen production.
The fermentative performance based on the cassava starch
was shown in Fig. 4 to present more clearly the change in
mutant DG-8 compared with the wild-type. Production of acid
products (butyric acid, acetic acid and lactic acid) and gas (H2
and CO2) of both strains were low at the beginning but
increased afterwards and continued in the stationary phase,
while the a-amylase activity peak for both strains appeared at
40 h. The mutant DG-8 could utilize cassava starch for cell
metabolism more efficiently than the wild-type strain. The
hydrogen and a-amylase production levels of mutant DG-8
were higher than those of wild-type during the whole cultiva-
tion period. The maximum amount of hydrogen produced by
DG-8 was 3.0 mol/mol glucose, 1.55 times higher than that of
the wild-type strain (vs. 1.94 mol/mol glucose). However, when
compared with that of C. tyrobutyricum wild-type, the specific
growth rate of C. tyrobutyricum mutant DG-8 appears to be
affected by the low-energy nitrogen ion treatment. This may
facilitate both the hydrogen generation and a-amylase pro-
duction to follow a parallel pattern to that of cell growth,
especially before the stationary phase. It has already been
proved that hydrogen is growth associated product in bacteria
[34]. In this work, screening of 2-DG mutant strains through
mutagenesis contributed to both enzyme productivity and
fermentation potential. The mutants showed higher hydrogen
production in batch SSF of cassava starch, which was in
accordance with that of similar reports. Lin and Blaschek re-
ported that higher starch utilization by C. acetobutylicum SA-1, a
butanol-tolerant strain, correlated with a higher a-amylase
activity and butanol production. They also suggested that
further amplification of the amylase enzyme production in
Clostridium acetobutyricum SA-1 may increase the final butanol
concentration in the batch fermentation process [35]. Altintas
et al. [36] reported that a recombinant Saccharomyces cerevisiae
YPB-G expressing a-amylase produced 12.1 g/L ethanol in 76 h
by SSF of starch. The parent strain had no detectable activity
and no ethanol was detected. These findings all indicated the
effectiveness of improving the a-amylase related to the
degradation of starch to increase the output of the end-product.
3.5. Effect of initial sugar concentration on hydrogen
production in SSF
Bacterial growth and production become low in the higher
initial sugar concentration in the medium due to the substrate
inhibition, which is an universal phenomenon in practical
applications. A relevant characteristic of the medium formu-
lated with high initial sugar concentration was the intrinsic
Fermentation Yield Volumetric
time (h) (mol/mol glucose)a productivity (L/h/L)
64 3.0  0.1 0.38  0.08
88 3.0  0.1 0.37  0.10
96 3.2  0.1 0.41  0.07
126 2.0  0.1 0.22  0.05
high permeability due to the elevated amount of medium
components. For example, osmolality of cultures at initial
glucose concentration as high as 120 g/L was very high,
reaching values of closed to 1.8 osm/kg in the most concen-
trated medium, which have been reported to inhibit growth of
saccharolytic Clostridia [37], and can therefore be a cause for
the decreased growth rate of the bacteria. In the present work,
60e120 g/L (initial total sugar) cassava starch was used in
batch SSF mode with mutant DG-8 (Table 3). When the initial
total sugar concentration was set at a level of 100 g/L, the
highest hydrogen yield of 3.2 mol/mol glucose and volumetric
productivity of 0.41 L/h/L were achieved. So far, the maximum
hydrogen yield with genus Clostridium previously produced in
fermentation was 2.81 mol/mol glucose by Clostridium beijer-
inckii L9 in batch fermentation mode [38]. Liu also reported
that 2.61 mol of H2 per mole of glucose was produced by
immobilized cells of C. tyrobutyricum PAK-Em mutant in fed-
batch fermentation [14]. The further addition of starch did
not improve hydrogen production, but decreased the yield and
volumetric productivity due to the exhaustion of other
nutrient components [39]. Under the same experimental
conditions, hydrogen production and productivity in separate
saccharification and fermentation (TSF) were much lower
than the results of the mode of fermentation presented in the
current study (data not shown). The most probable reason for
this finding is that the high concentrations of the starch seg-
ments of cassava starch were gradually degraded to
fermentable sugar in SSF, and the inhibition by high substrate
concentrations was overcome [40]. Hence, SSF has absolute
competitive advantage in terms of high substrate concentra-
tion in lower reactor volume and low fermentation cost.
4. Conclusions
Starch is one of the most abundant resources on earth and is
suited to serve as a cost-effective feedstock for biological
hydrogen production. However, producing hydrogen from
direct fermentation of starch is usually inefficient, as the
starch hydrolysis is often the rate-limiting step. The present
work describes the isolation of 2-DG resistant mutants of
C. tyrobutyricum for the improvement of a-amylase, and con-
sequencely, for the enhancement of hydrogen production by
SSF of cassava starch without the additional commercial
enzyme supplementation. We also found that the a-amylase
enzymes produced by C. tyrobutyricum appear to be subject to
induction by starch and catabolite repression by glucose.
About 3.2 mol hydrogen per mol glucose was successfully
obtained form cassava starch (100 g/L total sugar) using the
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6
mutant DG-8, which is 1.65-fold as much as that of the wild-
type strain. This mutant with higher a-amylase applied in
SSF opens a new avenue for the cost-effective fermentation of
hydrogen from raw starchy materials. In order to fully un-
derstand the mutation and its effects on gene regulation and
the metabolism, a series of genetic experiments should be
performed in further trials.
Acknowledgments
This work was obtained from the National Science Foundation
for Distinguished Young Scholars of China (No. 21225626), the
National Basic Research Program of China (2012CB725204), the
National Natural Science Foundation of China for Young
Scholars (No. 21106064), the Key Program of the National
Natural Science Foundation of China (No. 20936002), the Na-
tional High Technology Research and Development Program
of China (No. 2012AA021700) and the Program for New Cen-
tury Excellent Talents in University from the Ministry of Ed-
ucation of China (NCET-09-0157).
references
[1] Vardar-Schara G, Maeda T, Wood TK. Metabolically
engineered bacteria for producing hydrogen via
fermentation. Microb Biotechnol 2008;1:107e25.
[2] Chen X, Sun Y, Xiu Z, Li X, Zhang D. Stoichiometric analysis
of biological hydrogen production by fermentative bacteria.
Int J Hydrogen Energy 2006;31:539e49.
[3] Ust’ak S, Havrland B, Muñoz JOJ, Fernández EC, Lachman J.
Experimental verification of various methods for biological
hydrogen production. Int J Hydrogen Energy
2007;32:1736e41.
[4] Das D, Veziroglu TN. Hydrogen production by biological
processes: a survey of literature. Int J Hydrogen Energy
2001;26:13e28.
[5] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative
hydrogen production with Clostridium butyricum GCS5
isolated from anaerobic sewage sludge. Int J Hydrogen
Energy 2005;30:1063e70.
[6] Chin HL, Chen ZS, Chou CP. Fed-batch operation using
Clostriduim acetobutylicum suspension culture as biocatalyst
for enhancing hydrogen production. Biotechnol Prog
2003;19:383e8.
[7] Pan CM, Fan YT, Zhao P, Hou HW. Fermentative hydrogen
production by the newly isolated Clostridium beijerinckii
Fanp3. Int J Hydrogen Energy 2008;33:5383e91.
[8] Ferchichi M, Crabbe E, Gil GH, Hintz W, Almadidy A.
Influence of initial pH on hydrogen production from cheese
whey. J Biotechnol 2005;120:402e9.
[9] Liu G, Shen J. Effects of culture medium and medium
conditions on hydrogen production from starch using
anaerobic bacteria. J Biosci Bioeng 2004;98:251e6.
[10] Lin CY, Lay CH. Carbon/nitrogen-ratio effect on fermentative
hydrogen production by mixed microflora. Int J Hydrogen
Energy 2004;29:41e5.
[11] Michel-Savin D, Marchal R, Vandecasteele JP. Control of the
selectivity of butyric acid production and improvement of
fermentation performance with Clostridium tyrobutyricum.
Appl Microb Biotechnol 1990;32:387e92.
6355
[12] Jiang L, Wang JF, Liang SZ, Cai J, Xu ZN, Cen PL, et al.
Enhanced butyric acid tolerance and bioproduction by
Clostridium tyrobutyricum immobilized in a fibrous bed
bioreactor. Biotechnol Bioeng 2011;108(1):31e40.
[13] Jiang L, Li S, Hu Y, Xu Q, Huang H. Adaptive evolution for fast
growth on glucose and the effects on the regulation of
glucose transport system in Clostridium tyrobutyricum.
Biotechnol Bioeng 2012;109(3):708e18.
[14] Liu XG, Zhu Y, Yang ST. Butyric acid and hydrogen
production by Clostridium tyrobutyricum ATCC 25755 and
mutants. Enzyme Microb Technol 2006;38:521e8.
[15] Jo HJ, Lee DS, Park D, Park JM. Biological hydrogen production
by immobilized cells of Clostridium tyrobutyricum JM1 isolated
from food waste treatment process. Bioresour Technol
2008;99:6666e72.
[16] Watson SD, Pletschke BI. The effect of sulfide on a-
glucosidases: Implications for starch degradation in
anaerobic bioreactors. Chemosphere 2006;65:159e64.
[17] Chen SD, Sheu DS, Chen WM, Lo YC, Huang TI, Lin CY, et al.
Dark hydrogen fermentation from hydrolyzed starch treated
with recombinant amylase originating from
Caldimonastaiwanensis On1. Biotechnol Prog 2007;23:1312e20.
[18] Ensley B, McHugh JJ, Barton LL. Effect of carbon sources on
formation of alpha-amylase and glucoamylase by Clostridium
acetobutylicum. J Gen Appl Microbiol 1975;21:51e9.
[19] Madihah MS, Ariff AB, Khalil MS, Suraini AA, Karim MIK.
Partial purification and some properties of a-amylase and
glucoamylase obtained as by-product from direct
fermentation of Sago starch to solvent by Clostridium
acetobutylicum. Pak J Biol Sci 2000;3:744e9.
[20] Hyun HH, Zeikus JG. Simultaneous and enhanced production
of thermostable amylases and ethanol from starch by
cocultures of C. thermosulfurogenes and C.
thermohydrosulfuroicum. Appl Environ Microbiol
1985;49:1174e81.
[21] Chojecki A, Blaschek HP. Effect of carbohydrate source on
alpha-amylase and glucoamylase formation by Clostridium
acetobutylicum SA-1. J Ind Microbiol 1986;1:63e7.
[22] Melasniemi H. Effect of carbon source on production of
thermostable a-amylase, pullulanase and a-glucosidase by
Clostridium thermo-hydrosulfuricum. J Gen Microbiol
1987;133:883e90.
[23] Dillon AJP, Zorgi C, Camassola M, Henriques JAP. Use of
2-deoxyglucose in liquid media for the selection of mutant
strains of Penicillium echinulatum producing increased
cellulase and b-glucosidase activities. Appl Microbiol
Biotechnol 2006;70:740e6.
[24] Yoo YJ, Hong J, Hatch RT. Comparison of a-amylase activities
from different assay methods. Biotechnol Bioeng
1987;30:147e51.
[25] Jiang L, Wang JF, Liang SZ, Wang XN, Cen PL, Xu ZN.
Production of butyric acid from glucose and xylose with
immobilized cells of Clostridium tyrobutyricum in a fibrous-
bed bioreactor. Appl Biochem Biotechnol 2010;160:350e9.
[26] Jiang L, Wang JF, Liang SZ, Xu ZN, Yang ST.
Phosphoenolpyruvate-dependent phosphorylation of
sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for
the phosphotransferase transport system. Bioresour Technol
2010;101:304e9.
[27] Jiang L, Wang JF, Liang SZ, Cai J, Xu ZN. Control and
optimization of Clostridium tyrobutyricum ATCC 25755
adhesion into fibrous matrix in a fibrous bed bioreactor. Appl
Biochem Biotech 2011;165:98e108.
[28] Deng YF, Li S, Xu Q, Gao M, Huang H. Production of fumaric
acid by simultaneous saccharification and fermentation of
starchy materials with 2-deoxyglucose-resistant mutant
strains of Rhizopus oryzae. Bioresour Technol
2012;107:363e7.
6356 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 6 3 4 9 e6 3 5 6
[29] Jiang L, Wang JF, Liang SZ, Wang XN, Cen PL, Xu ZN. Butyric
acid fermentation in a fibrous bed bioreactor with
immobilized Clostridium tyrobutyricum from cane molasses.
Bioresour Technol 2009;100:3403e9.
[30] Annous BA, Blaschek HP. Regulation and localization of
amylolytic enzymes in Clostridium acetobutylicum ATCC 824.
Appl Environ Microbiol 1990;56:2559e61.
[31] Tashiro Y, Takeda K, Kobayashi G, Sonomoto K, Ishizaki A,
Yoshino S. High butanol production by Clostridium
saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-
stat continuous butyric acid and glucose feeding method. J
Biosci Bioeng 2004;98:263e8.
[32] Haseltine C, Rolfsmeier M, Blum P. The glucose effect and
regulation of alpha-amylase synthesis in the
hyperthermophilic archaeon Sulfolobus solfataricus. J Bacteriol
1996;78:945e50.
[33] Priest FG. Effect of glucose and cyclic nucleotides on the
transcription of a-amylase mRNA in Bacillus subtilis. Biochem
Biophys Res Commun 1975;63:606e10.
[34] Kumar N, Das D. Production and purification of a-amylase
from hydrogen producing Enterobacter cloacae IIT-BT 08.
Biopreocess Eng 2000;23:205e8.
[35] Lin YL, Blaschek HP. Butanol production by a butanol-
tolerant strain of Clostridium acetobutylicum in extruded corn
broth. Appl Environ Microbiol 1983;45:966e73.
[36] Altintas MM, Kirdar M, Önsan ZI, Ülgen KÖ. Cybernetic
modeling of growth and ethanol production in a recombinant
Saccharomyces cerevisiae strain secreting a bifunctional fusion
protein. Process Biochem 2002;37:1439e45.
[37] Walter RP, Morris JG, Kell DB. The roles of osmotic stress and
water activity in the inhibition of the growth, glycolysis and
glucose phosphotransferase system of Clostridium
pasteurianum. J Gen Microbiol 1987;133:259e66.
[38] Lin PY, Whang LM, Wu YR, Ren WJ, Hsiao CJ, Li SL, et al.
Biological hydrogen production of the genus Clostridium
metabolic study and mathematical model simulation. Int J
Hydrogen Energy 2007;32:1728e35.
[39] Kim DY, Yim SC, Lee PC, Lee WG, Lee SY, Chang HN. Batch
and continuous fermentation of succinic acid from wood
hydrolysate by Mannheimia succiniciproducens MBEL55 E.
Enzym Microb Technol 2004;35:648e65.
[40] John RP, Anisha GS, Nampoothiri KM, Pandey A. Direct lactic
acid fermentation: focus on simultaneous saccharification
and lactic acid production. Biotechnol Adv 2009;27:145e52.