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

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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

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

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

E-mail address: [email protected] (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 C. tyrobutyricum and to further develop an SSF process for
more advantageous than the photosynthetic hydrogen pro- hydrogen production from starch materials without com-
duction and appears to have more potential for practical ap- mercial a-amylase supplementation.
plications [4].
The genus Clostridium, one of the largest genera of pro-
karyotes, is often reported used to produce hydrogen, such as 2. Materials and methods
Clostridium butyricum [5], Clostriduim acetobutylicum [6,7], Clos-
tridium saccharoperbutylacetonicum [8], Clostridium pasteurianum 2.1. Chemicals
[9,10]. Among them, Clostridium tyrobutyricum has many ad-
vantages over other species, including simple medium for cell Cassava starch of food-grade (as per the specifications of the
growth and relatively high product purity and yield [11,12]. Chinese Edible Cassava Standard NY/T875-2012) was pur-
Previous studies regarding this anaerobe were extensively chased from Wuming Cassava Starch Co. Ltd. (Guangxi,
focused on acid production, while an increasing attention has China), dried to constant weight and passed through an
been paid on the fermentation processes for biohydrogen 80-mesh screen. The starch content was measured more than
production from glucose, xylose, sucrose, and so on [13e15]. 99.5% by the starch-iodide method [24]. The 2-DG was pur-
However, the high cost of carbon source come to be one of the chased from Aladdin Reagent Co. Ltd. (Shanghai, China). All
greatest obstacles restraining the industrialization of other chemicals were of analytical grade and commercially
hydrogen fermentation with C. tyrobutyricum. Therefore, the available.
exploitation of cheap, renewable carbohydrate feedstock has
been strongly stimulated. 2.2. Microorganism and medium
Among the possible candidates, starch, the second abun-
dant organic resource on earth, is well suited to serve as a C. tyrobutyricum ATCC 25755 (purchased from Guangdong
cost-effective substrate instead of expensive refined sugars culture collection center, collection number: GIM 1.262) was
for commercialization of hydrogen fermentation. However, used as the present strain, and stored in reinforced clostridial
the rate-limiting step for bioconversion of starch is the medium [25]. Unless otherwise indicated, all experiments
effective hydrolysis of the macromolecule into reducing were performed under anaerobic conditions as previously
sugars [16]. The a-amylase was found to be facilitated efficient described [26]. The cultivation medium was composed of 5 g/L
starch degradation, which was conducted to enhance the yeast extract, 5 g/L peptone, 3 g/L (NH4)2SO4, 1.5 g/L K2HPO4,
feasibility of using starch feedstock for hydrogen production 0.6 g/L MgSO4$7H2O, 0.03 g/L FeSO4$7H2O. Carbon source was
by Chen et al. [17]. Results from our laboratory also demon- added separately in the seed medium of 20 g/L glucose, and in
strated incomplete hydrolysis of corn starch by C. tyrobutyr- the fermentation medium of 60e120 g/L cassava starch. All
icum ATCC 25755 (unpublished results), which suggested that the media were sterilized by autoclaving at 121  C, 15 psig, for
C. tyrobutyricum can secrete a-amylase at a low basal level, 20 min.
while a similar observation has been previously reported for
other strains of genus Clostridium [18e20]. It is of scientific 2.3. SSF process
interest and technological importance to utilize this indige-
nous bacterium for hydrolytic pretreatment of raw starch The batch SSF cultivations were performed in a laboratory-
materials to facilitate the efficiency of biohydrogen produc- scale 5-L stirred-tank fermentor (B. Braun, B. Braun Biotech
tion. If the activity of the a-amylase in C. tyrobutyricum could International, Melsungen, Germany) with a working volume
be further improved, the simultaneous saccharification and of 2 L operated at 37  C, agitated at 150 rpm, and pH controlled
fermentation (SSF) process for hydrogen fermentation from at 6.0 by adding 5 N NaOH facilitated by an on-line sensing and
starch feedstock without the addition of commercial dosing system. Anaerobiosis was maintained by sparging the
a-amylase might be established, which will have remarkable medium with N2 (10 mL/min) for 60 min. Fermentation broth
advantages in terms of operational cost and space. and biogas in the bioreactor were taken at regular intervals for
Investigators from various laboratories have suggested the analyses of cell growth, extracellular metabolite concen-
that a-amylase enzyme biosynthesis in genus Clostridium is trations, including carbon sources, a-amylase, lactic acid,
subject to catabolite repression by glucose or metabolizable butyric acid, acetic acid, H2 and CO2 [27].
compounds [21,22]. The most effective random mutation
methods for obtaining variants with enhanced a-amylase 2.4. Mutagenesis and selection of 2-DG-resistent
production are those combining mutagenesis and enrich- mutants
ment, one of which is the use of the anti-metabolite
2-deoxyglucose (2-DG), a glucose analog, which, when incor- The implantation sources were produced using an ion beam
porated into a suitable medium containing starch, causes implantation instrument (LZD-900, Southwestern Institute of
catabolite repression of nonmutant strains, allowing only Physics, China). A late exponential phase of C. tyrobutyricum
mutated strains resistant to this form of suppression to form was centrifuged at 12,000 g for 20 min at 4  C, washed once
colonies [23]. However, to the best of our knowledge, the with sodium potassium phosphate buffer (pH 7.2), and diluted
foregoing has not been applied into the isolation of C. tyrobu- in sterilized 2% glucose solution. About 200 mL suspension was
tyricum mutant with the capability to produce a high yield of spread to form a single-cell layer on a sterilized Petri dish
a-amylase. The objective of this research was undertaken to (75 mm) and dried in the hood. The dishes were put into the
evolve glucose-derepressed and/or hyperamylase strains of 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 6351

energy of 10 keV using pulse-type implantation [28]. The reaction conditions specified. All results were obtained from
implanted samples were then diluted with 1 mL sterilized the means of triplicate determinations.
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 3. Results and discussion
onto agar medium containing 20 g/L of glucose. After another
2 days, the master plates were overlaid with an agar solution 3.1. Mutagenesis, enrichment, and isolation of mutants
containing 20 g/L of starch (pH 5), incubated for 3 h, and then
stained with iodine vapor. The iodine vapor was generated by Low-energy nitrogen ion (Nþ) implantation was used as the
heating iodine crystals in a water bath at 80  C. The colonies mutagenizing agent to improve the wild-type strain C. tyro-
exhibiting large clear zones on the iodine-stained plates were butyricum ATCC 25755. Cells were found to be very sensitive to
picked for further analysis. 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
2.5. Analytical procedures 261 colonies of the 1.3  105 survivors were picked at random.
Nine of these, termed DG-1, DG-2-DG-9, respectively, were
Samples were collected during the exponential phase to further examined based on the production of large clear zones
measure the cells, substrates and products for extracellular on starch-overlaid glucose plates. The mutants were firstly
metabolite analysis. Cell concentration was analyzed by transferred to the growth medium containing 1.0 g/L 2-DG to
measuring the optical density of the cell suspension at OD600 investigate their hereditary stability for 10 generations, and
with a spectrophotometer (Ultrospec 3300 pro, Amersham the results showed that all of them grew normally.
Bioscience). Dry cell weight (DCW) was determined by
centrifugation of the fermentation broth (10 mL) at 10,000 g for 3.2. Effect of carbon source on amylolytic activity
10 min, washing the sediment twice with distilled water, and
drying at 80  C for 24 h to a constant weight [28]. Previous results suggested that the production of amylolytic
The concentrations of glucose were measured by SBA-80C enzymes, including a-amylase and glucoamylase, in genus
biosensor analyzer (Institute of Biology, Shandong Academy Clostridia are subject to the influence by different carbon
of Sciences, China). Total sugar concentration was estimated sources [30]. The activity of a-amylase is wherein a decisive
by the phenol-sulfuric acid method with D-(þ)-glucose (Sigma, factor with regard to the conversion efficiency from starch to
G8270-100G, EC 200-075-1) as the standard [17]. The H2 and reducing sugars, while the low utilization of starch is usually
CO2 concentrations in the exhaust gas were determined using due to the low activity of the a-amylase produced by Clostridia
the MultiRAE IR gas monitor PGM 54 (RAE system Inc., San [31]. Therefore, the effect of various combinations of glucose
Jose, USA). Quantitative analysis of butyric acid and acetic and cassava starch in the growth medium on the localization
acid was performed by GC (Agilent 6820 GE, Agilent Technol- of a-amylase activity in C. tyrobutyricum was first investigated
ogies) as previously reported [29]. and the results shown in Table 1 were obtained. An increase in
The preparation of the crude extract was described previ- the concentration of glucose in the growth medium was
ously [13]. Protein concentration was determined by the accompanied by a corresponding increase in the cell-adsorbed
method of Bradford with bovine serum albumin as the stan- and the intracellular a-amylase activity while a decrease in
dard (Bio-Rad protein assay). The activity of a-amylase was the extracellular and the total a-amylase activity. The activity
determined as previously described by Annous and Blaschek of the cell-adsorbed fraction suggests that this enzyme is
[30]. Glucoamylase activity was measured according to the rather loosely attached to the surface of the cell, which is in
method of Deng et al. [28]. One unit of a-amylase and glu- accordance with the findings of Annous and Blaschek [30].
coamylase activity was defined as the amount of enzyme The effect of carbon sources on the formation of extracel-
producing 1 mmol of reducing sugar per min under the lular a-amylase and glucoamylase by Clostridium tyrobutylicum

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 % Of activity in cell fractionc

Cell-adsorbed Intracellular Extracellular

2.0%S 2.7  0.4 0.0 1.8 94.6

1.5%S þ 0.5%G 1.1  0.2 0.4 44.8 32.2
1.0%S þ 1.0%G 0.8  0.1 0.7 52.8 21.6

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

60 0.8  0.1 49.1  2.1 64 3.0  0.1 0.38  0.08

80 1.5  0.2 65.1  3.2 88 3.0  0.1 0.37  0.10
100 10.2  0.4 79.4  3.8 96 3.2  0.1 0.41  0.07
120 21.4  0.9 54.5  5.0 126 2.0  0.1 0.22  0.05

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- high permeability due to the elevated amount of medium
formance for hydrogen production. components. For example, osmolality of cultures at initial
The fermentative performance based on the cassava starch glucose concentration as high as 120 g/L was very high,
was shown in Fig. 4 to present more clearly the change in reaching values of closed to 1.8 osm/kg in the most concen-
mutant DG-8 compared with the wild-type. Production of acid trated medium, which have been reported to inhibit growth of
products (butyric acid, acetic acid and lactic acid) and gas (H2 saccharolytic Clostridia [37], and can therefore be a cause for
and CO2) of both strains were low at the beginning but the decreased growth rate of the bacteria. In the present work,
increased afterwards and continued in the stationary phase, 60e120 g/L (initial total sugar) cassava starch was used in
while the a-amylase activity peak for both strains appeared at batch SSF mode with mutant DG-8 (Table 3). When the initial
40 h. The mutant DG-8 could utilize cassava starch for cell total sugar concentration was set at a level of 100 g/L, the
metabolism more efficiently than the wild-type strain. The highest hydrogen yield of 3.2 mol/mol glucose and volumetric
hydrogen and a-amylase production levels of mutant DG-8 productivity of 0.41 L/h/L were achieved. So far, the maximum
were higher than those of wild-type during the whole cultiva- hydrogen yield with genus Clostridium previously produced in
tion period. The maximum amount of hydrogen produced by fermentation was 2.81 mol/mol glucose by Clostridium beijer-
DG-8 was 3.0 mol/mol glucose, 1.55 times higher than that of inckii L9 in batch fermentation mode [38]. Liu also reported
the wild-type strain (vs. 1.94 mol/mol glucose). However, when that 2.61 mol of H2 per mole of glucose was produced by
compared with that of C. tyrobutyricum wild-type, the specific immobilized cells of C. tyrobutyricum PAK-Em mutant in fed-
growth rate of C. tyrobutyricum mutant DG-8 appears to be batch fermentation [14]. The further addition of starch did
affected by the low-energy nitrogen ion treatment. This may not improve hydrogen production, but decreased the yield and
facilitate both the hydrogen generation and a-amylase pro- volumetric productivity due to the exhaustion of other
duction to follow a parallel pattern to that of cell growth, nutrient components [39]. Under the same experimental
especially before the stationary phase. It has already been conditions, hydrogen production and productivity in separate
proved that hydrogen is growth associated product in bacteria saccharification and fermentation (TSF) were much lower
[34]. In this work, screening of 2-DG mutant strains through than the results of the mode of fermentation presented in the
mutagenesis contributed to both enzyme productivity and current study (data not shown). The most probable reason for
fermentation potential. The mutants showed higher hydrogen this finding is that the high concentrations of the starch seg-
production in batch SSF of cassava starch, which was in ments of cassava starch were gradually degraded to
accordance with that of similar reports. Lin and Blaschek re- fermentable sugar in SSF, and the inhibition by high substrate
ported that higher starch utilization by C. acetobutylicum SA-1, a concentrations was overcome [40]. Hence, SSF has absolute
butanol-tolerant strain, correlated with a higher a-amylase competitive advantage in terms of high substrate concentra-
activity and butanol production. They also suggested that tion in lower reactor volume and low fermentation cost.
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 4. Conclusions
et al. [36] reported that a recombinant Saccharomyces cerevisiae
YPB-G expressing a-amylase produced 12.1 g/L ethanol in 76 h Starch is one of the most abundant resources on earth and is
by SSF of starch. The parent strain had no detectable activity suited to serve as a cost-effective feedstock for biological
and no ethanol was detected. These findings all indicated the hydrogen production. However, producing hydrogen from
effectiveness of improving the a-amylase related to the direct fermentation of starch is usually inefficient, as the
degradation of starch to increase the output of the end-product. starch hydrolysis is often the rate-limiting step. The present
work describes the isolation of 2-DG resistant mutants of
3.5. Effect of initial sugar concentration on hydrogen C. tyrobutyricum for the improvement of a-amylase, and con-
production in SSF sequencely, for the enhancement of hydrogen production by
SSF of cassava starch without the additional commercial
Bacterial growth and production become low in the higher enzyme supplementation. We also found that the a-amylase
initial sugar concentration in the medium due to the substrate enzymes produced by C. tyrobutyricum appear to be subject to
inhibition, which is an universal phenomenon in practical induction by starch and catabolite repression by glucose.
applications. A relevant characteristic of the medium formu- About 3.2 mol hydrogen per mol glucose was successfully
lated with high initial sugar concentration was the intrinsic 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 6355

mutant DG-8, which is 1.65-fold as much as that of the wild- [12] Jiang L, Wang JF, Liang SZ, Cai J, Xu ZN, Cen PL, et al.
type strain. This mutant with higher a-amylase applied in Enhanced butyric acid tolerance and bioproduction by
SSF opens a new avenue for the cost-effective fermentation of Clostridium tyrobutyricum immobilized in a fibrous bed
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This work was obtained from the National Science Foundation 2008;99:6666e72.
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National Basic Research Program of China (2012CB725204), the glucosidases: Implications for starch degradation in
National Natural Science Foundation of China for Young anaerobic bioreactors. Chemosphere 2006;65:159e64.
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Natural Science Foundation of China (No. 20936002), the Na-
with recombinant amylase originating from
tional High Technology Research and Development Program
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