i n t e r n a t i o n a l j o u r n al of hy d r o g en e n e r g y 33

(2008) 51615168

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Hydrogen production by the hyperthermophilic

eubacterium, Thermotoga neapolitana,
using cellulose pretreated by ionic liquid
a

Tam-Anh D. Nguyen , Se Jong Han , Jun Pyo Kim ,

b
b
a,
Mi Sun Kim , You Kwan Oh , Sang Jun Sim *
a

Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea

Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-343, South Korea

a r t i c l e i n
f o

a b s t r a c t
The application of thermophilic bacteria to the production of hydrogen (H 2) from a cellu-

Article history:
Received 13 May 2008
Accepted 26 May 2008
Available online 9 August 2008

losic

biomass

has

captured

attention

of

engineering

researchers.

Thermotoga

neapolitana is considered as a potential hydrogen producer because of its ability to be

used for the di- rect H 2 fermentation from raw cellulose, but its H 2 yield is not high. In this
study, three chemical pretreatment methods with an ionic liquid, acid and alkali,
respectively, were tested on cellulose to enhance the cellulose conversion of this strain into

Keywords:

H2. Batch cultiva- tions were carried out to investigate the influence of the chemical

Hydrogen production

pretreatment methods on H 2 production under non-sterile conditions. The highest H 2

Ionic liquid pretreatment

Thermotoga neapolitana

36.1% (v/v) with ethanol as an anti-solvent. This was clearly higher than that from the

N2 sparging

concentration in the head- space obtained from the ionic liquid-pretreated cellulose was
acid- and alkali-pretreated cellu- loses (2224%). Cultivation with cellulose pretreated with
the ionic liquid at a concentration of 10% combining with N 2 sparging

showed

the

maximum cumulative H2 yield of 1280 mL H 2/L culture. This value is approximately 10fold higher than that of raw cellulose (122 mL H2/L culture). The H2 yield obtained from
fermentation with ionic liquid-pretreated cellulose was 2.2 mol H 2/mol glucose
equivalents. The results showed that the pretreat- ment of cellulose using an ionic liquid
has considerable potential for improving the direct conversion of cellulosic substrates into
H2 by bacteria.
2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All
rights
reserved.

1.

Introduction

Hydrogen (H2) is considered as an ideal alternative to fossil

fuels in the energy and chemical industries in the future, because the energy derived from hydrogen via combustion or
via fuel cells is non-polluting [6]. Another major advantage
of H2 is that it can be directly produced by thermochemical
(gasification) or biological processes (fermentation) from

cellulose, thus representing a renewable supply of hydrogen

from a biomass. Although cellulose has been recognized as
the feedstock source having the most potential for bioenergy
production because of its sustainability, availability and lowcost, the very low cellulose digestibility of the microbial
cellulose system represents a serious obstacle to the
biological conversion of cellulose to bioenergy products and
H2. Cellulose is a polymer of glucose which is waterinsoluble

516
2

internatio nal j ournal of hydr ogen e ne r g y 3 3 ( 2008)

51615168

* Corresponding author. Tel.: 82 31 290 7341; fax: 82 31 290

7272. E-mail address: simsj@skku.edu (S.J. Sim).
0360-3199/$ see front matter 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved. doi:10.1016/j.ijhydene.2008.05.019

and recalcitrant to hydrolysis into its individual glucose

subunits, because of the tightly packed, highly crystalline
structures in its polymer chains [5]. Pretreatment methods
are essential to overcome this highly ordered structure and
make the hydrolysis process viable for producing
fermentable sugars.
Pretreatment is an important tool for practical cellulosic
bioconversion processes, because it has great potential for
im- proving the efficiency and lowering of cost. Many
pretreatment methods have been studied for improving the
rate of enzy- matic hydrolysis and increasing yields of
fermentable sugars from cellulose. They can be physical,
chemical or a combina- tion of these two processes. Physical
methods include commi- nution, steam explosion and
hydrothermolysis [18]. Chemical methods are techniques
using acids and bases as pretreatment agents. The most
commonly used acid and base are H2SO4 and NaOH,
respectively. Another chemical method is pretreatment of
cellulose by some specific solvents. These solvents can dissolve cellulose, disrupt its crystalline structures and enhance
its hydrolysis [8,9]. Among these, ionic liquids (ILs) have recently attracted much attention as new novel chemical agents
for cellulose pretreatment because of their high dissolubility
of cellulose up to 39% without derivatization [8,25]. The
ability of ILs to dissolve cellulose depends on their halide
anions, which have been shown to be strongly disruptive to
hydrogen bonding, having the effect of breaking up the
extensive hydro- gen-bonding network of the polysaccharide
and promote its dissolution [21]. Moreover, cellulose
pretreatment by ionic liquid is a green process that
prevents pollution and waste production and is economical
because of its 100% reusability. The enhancement of the
cellulose saccharification kinetics using an ionic liquid
pretreatment step was investigated in the study of Dadi et al.
using [C4mim]Cl [7]. The initial enzy- matic hydrolysis rates
of cellulase, from Trichoderma reesei (ATCC# 26799) were
approximately 50-fold higher for the regenerated cellulose as
compared to untreated cellulose.
Thermotoga neapolitana is a member of a group of
extremely thermophilic, rod-shaped, non-sporeforming
eubacteria [3,11]. This strain demonstrates heterotrophic
growth with acetic acid, carbon dioxide (CO 2) and H2 as the
main products from strictly anaerobic fermentation using a
wide range of dif- ferent carbon source [3,11,24]. Recently, the
biotechnological applications of hyperthermophiles such as
T. neapolitana have been widely studied because of their
prominent proper- ties. The use of extreme temperatures
makes fermentation processes less sensitive to contamination
in increasing the rates of thermodynamic reactions. The
specific enzyme sys- tems of hyperthermophiles utilize
biomass sources as an effi- cient feedstock [10].
In this study, we enhanced the H2 production of T.
neapoli- tana from cellulose by various chemical
pretreatment methods. This process is called simultaneous
saccharification and fermentation (SSF) which is thought to
be the best route for biological conversion of cellulosic
biomass to biofuels and valuable products. The sugars
produced in pretreatment solution were immediately
converted into H2 via anaerobic fermentation by the same
microbial system at
the
same
time. Three different
pretreatment agents including NaOH, H 2SO4 and 1-butyl-3-

methylimidazolium chloride ([C4mim]Cl), a non-derivatizing

ionic liquid [19], were used to pretreat

cellulose which was utilized as a carbon source for H2

fermen- tation. The growth, H2 production and substrate
utilization on the untreated and different kinds of
pretreated cellulose were compared and analyzed. These
pretreatment methods resulted in an increase of cellulose
degradability and H2 fermentation in comparison with the
untreated cellulose. The method using the ionic liquid,
[C4mim]Cl, as the pretreat- ment agent showed very
impressive results in terms of the improvement of the H 2
production from cellulose.

ensure an anaerobic atmosphere. The growth conditions for

this strain were agitation at 150 rpm and 80 C.

2.

Materials and methods

2.1.

Strain and cultivation conditions

atmospheric pressure (1 atm). The pretreatment agents

(H2SO4 and NaOH) were re- moved from the pretreated
cellulose by washing with water or by neutralization (pH
7.5). In the case where the pretreated cellulose subjected to
the neutralization step was used as the substrate, NaCl was
not added to the medium for fermenta- tion. In the
pretreatment method using the ionic liquid, cellu- lose was
dissolved in intact [C4mim]Cl at ratios of 5, 10 and 15%
(w/w) at 80 C within 6 h (completely dissolved). After dissolution, the cellulose was regenerated by using three differ4
ent anti-solvents (H2O, methanol and ethanol) to precipitate
the cellulose from [C4mim]Cl. The regenerated cellulose was
separated from mixtures of [C4mim]Cl and anti-solvent with
a filter under reduced pressure and washed with distilled
water before use for fermentation. The [C 4mim]Cl in the antisolvents was easily recovered for subsequent reutiliza- tions
by means of a rotary evaporator (BUCHI Rotavapor R- 200,
Switzerland). After the cellulose is precipitated, the antisolvents used for the cellulose precipitation such as water,
ethanol, methanol are removed from the mixture of IL and
anti-solvent via evaporation based on the property

T. neapolitana (DSM 4359) was obtained from the

Deutsche
Sammlung
von
Mikroorganismen
und
Zellkulturen (DSM) (Braunchweig, Germany). This strain
was grown on an opti- mized Thermotoga maritima basal
(TMB) medium [12] consist- ing of (amounts are in grams
per liter of deionized water) 1.5 g KH 2PO4, 4.2 g
Na2HPO4$12H2O (22 mM PO

), 0.5 g NH4Cl, 0.2 g

MgCl2$6H2O (1 mM), 20.0 g NaCl, 2.0 g yeast ex- tract, 15.0

mL trace element solution 141 [1], and 1 mg resa- zurin.
The medium was reduced with 0.1% (w/v) cysteine$HCl.
The initial pH for growth was adjusted to 7.5 with 5 N
NaOH. Batch cultivation of T. neapolitana was performed in 120-mL serum bottles containing 40 mL medium
and 0.5% (w/v) cellulose as a carbon source under nonsterile conditions. The bottles were sparged with 100% N2 to

2.2.

Methods of substrate pretreatment

Microcrystalline cellulose (20100 mm dia.) from Daejung

Chemical (Korea) was pretreated with three different chemicals, acid (H2SO4), alkali (NaOH) and ionic liquid
([C4mim]Cl). Five grams of cellulose flour was hydrolysed
with 50 mL (1:10 w/v) of various concentrations of H 2SO4
and NaOH for 30, 60, 90 and 120 min at 80 C under

5163

i n t e r n a t i o n a l j o u r n al of hy d r o g en e n e r g y 33
(2008) 51615168

that the boiling temperature of IL is much higher than those

of anti-solvents. In IL recovery process by evaporation, the
anti- solvent molecules are easily stripped off the IL
molecules when the mixture of them is heated at
temperature above boiling point of anti-solvent. Cellulose
pretreated by the recovered [C4mim]Cl was also tested for

H2 content (%)

a 30

H2 production by
T. neapolitana.

2.3.

30 min
60 min
90 min
25
120 min
20

15

Analytical procedure
10

H2 content (%, v/v of headspace)

The amount of H2 was quantitatively measured by gas chro5

matography (GC, HewlettPackard 5890 Series II, USA) with
a thermal conductivity detector (TCD) with N 2 as the carrier
gas. The column used for gas determination was a stainless
0
column (1/8 in 15 ft) packed with 50/80 mesh Carboxen
30
60
90
120
150
180
1000 (Supelco).
Cultivation time (h)
The glucose produced in the liquid medium was
enzymat- ically measured using a Glucose E-Kit (BMI
b 40
Company, Korea). The amounts of cellulose and total soluble
carbohydrates were determined by the anthrone method at
Pretreated 35
cellulose with washing step Pretreated cellulose with neutralization step
630 nm [16].
30
The protein concentration was used as a parameter to estimate the bacterial growth and was measured by the Bradford
25
method with bovine serum albumin as a standard [4].

20

3.

Results and discussion

3.1.

H2 production using cellulose

pretreated by NaOH method

15
10
5

The dissolution and swelling of cellulose are two important

phenomena occurring in cellulose pretreatment. Their common features from the physicochemical point of view are
that the intermolecular forces between the macromolecules
are overcome by stronger interactions leading to the
loosening or eliminating the cellulose supramolecular
structure. The processes of both swelling and dissolution
serve the purpose of enhancing the accessibility of the
hydroxyl groups for sub- sequent reactions. The swelling, not
the dissolution, is the im- portant phenomenon in the
pretreatment of cellulose with NaOH. This process easily
occurs in various regions of the cel- lulose and affects the
crystalline regions by breaking down the intermolecular
bonds between the polymer chains [14,15]. First, the effect of
the pretreatment time on H 2 production was investigated
with 10% NaOH at 80 C for 30, 60, 90 and 120 min.
Hydrolysate mixtures whose color changed from yel- low to
brown depending on the pretreatment time were neutralized with HCl for fermentation. Fig. 1(a) shows an
increase in H2 production when the pretreatment time is
increased. When the pretreatment time was increased
beyond 60 min, the H2 production by T. neapolitana was
insignificantly in- creased. Therefore, 60 min was chosen as
the pretreatment time for further studies with NaOH
pretreatment.

0
Untreated

10

15

20

Various concentrations of NaOH were used to treat cellulose at 80

C before using it for the production of H2 by
T. neapolitana. In case of the pretreated cellulose after the
water-washing step, the H2 production increased as the
NaOH
concentration was increased from 1 to 20%. The highest H2 content was
obtained from the cellulose pretreated with 20% NaOH and amounted
to 20.1% (v/v) of the headspace

5164

internatio nal j ournal of hydr ogen e ne r g y 3 3 ( 2008)

51615168

NaOH concentration (%,

w/v)
Fig. 1 Effect of NaOH pretreatment on
the H2 production by Thermotoga
neapolitana: (a) with different
pretreatment times; (b) with two different
recovering methods of pretreated
cellulose.

not contain glucose (Table 1). The H2 fermentation results for

the pre- treated cellulose subjected to the neutralization step
are pre- sented in Fig. 1(b). The best H2 production (22.7%)
was observed in the case of the cellulose pretreated with 15%
NaOH which also produced the highest amount of soluble
carbohydrates.

3.2.

H2 production using cellulose

pretreated by H2SO4 method

volume (Fig. 1(b)). The amount of soluble

carbohydrates in the hydrolysate solutions from
the NaOH pretreatment in- creased with the
increasing NaOH concentration and showed the
highest content with 15% NaOH, but they did

Acids can hydrolyse cellulose by the cleavage of glycosidic

bonds between the two anhydroglucose units in the cellulose
polymer chains. This cleavage of the b-l,4-glycosidic bond is
catalyzed by the H ions of acids [14,15]. We first used 10%
H2SO4 to hydrolyse cellulose for different pretreatment times

Table 1 Formation of soluble carbohydrates in hydrolysed solution by chemical pretreatment

Concentration
of pretreatment
agent (%)

NaOH

H2SO4

Glucose
(g/L)

Other soluble carbohydrates

(g cellulose equivalents/L)

Glucose
(g/L)

Other soluble carbohydrates

(g cellulose equivalents/L)

0
0
0
0
0

0.4 T 0.024
0.6 T 0.029
4.9 T 0.242
5.9 T 0.304
5.3 T 0.256

0.0 T 0.000
0.1 T 0.003
0.4 T 0.002
0.6 T 0.003
1.0 T 0.004

0.2 T 0.009
0.8 T 0.046
2.5 T 0.130
3.7 T 0.189
5.0 T 0.244

1
5
10
15
20

(30, 60, 90 and 120 min) to determine the optimum residence

time. The hydrolysate mixtures were neutralized with NaOH
before utilizing them for fermentation. Hydrogen production
by T. neapolitana showed the best value in the case of the
cel- lulose pretreated for 120 min (Fig. 2(a)). H2SO4 solution at
con- centrations ranging from 1 to 20% was used to treat
cellulose at 120 min. As in the case of the NaOH
pretreatment, the H2 production from the cellulose
H2 content (%, v/v of headspace)

pretreated with H2SO4 followed

a 40
35
30
25

by a subsequent water-washing step increased in proportion

to the acid concentration (Fig. 2(b)). The best H2 production
of 23.2% was obtained using pretreatment with 20% H2SO4.
Glucose was present in small amounts in the dissoluble
carbo- hydrates of the hydrolysate solutions. The content of
dissolu- ble carbohydrates was also found to increase with
increasing H2SO4 concentration (Table 1). In the case of the
hydrolysate obtained from 10% H2SO4 pretreatment subjected
to a neutral- ization step, T. neapolitana gave the highest H2
production of 24.9% (v/v) of headspace volume. The
cellulose degradability was 77.2% which is higher than that
obtained from the un- treated cellulose (12.2%) and the
cellulose pretreated with 15% NaOH (54.6%) (Table 2). The
H2 production with the cellu- lose pretreated with 20% H 2SO4
was decreased, although the concentration of dissoluble
carbohydrates in the hydrolysate solution was the highest.
This appears to have an inhibitory effect on the fermentation,
resulting from the too high salt concentrations produced by
the neutralization.

20

3.3.

15
30
60
90
120

10
5

min
min
min
min

H2 production using cellulose

pretreated by ionic liquid
method

Cellulose pretreated by an ionic liquid was tested in the

enzy- matic hydrolysis of pure cellulases from T. reesei [7].
30
60
90
120
150
The pre- treatment step with this chemical was found to
180
enhance the cellulose saccharification activity of the
Cultivation time (h)
cellulases. In our study, the regenerated cellulose from the
ionic liquid pretreat- ment was hydrolysed by a live system,
b 40
the hyperthermophilic eubacterium T. neapolitana. The
of the pretreatment was estimate through H2
Pretreated35
cellulose with washing step Pretreated cellulose with neutralizationefficiency
step
H2 content (%, v/v of headspace)

fermentation with the complex met- abolic pathway in this

bacterial strain.

30

25

3.3.1. Effect of various cellulose-dissolving ratios in

ionic liquid

20

With various cellulose-dissolving ratios in IL which is called

cellulose dissolubility in IL and calculated by the mass of cellulose divided by the mass of ionic liquid, amorphous cellulose shows different changes in molecular structure after
precipitation from the IL solution by the addition of water, an
anti-solvent [9,21]. The cellulose conversion into glucose
catalyzed by bacterial cellulase system is dependent on the
surface area defined by changes in the structures of cellulose

15
10
5
0

Untreated

5
20

10

15

H2 SO4 concentration (%, w/v)

Fig. 2 Effect of H2SO4 pretreatment on the H2
production by Thermotoga neapolitana: (a) with
different pretreatment times; (b) with two different
recovering methods of pretreated cellulose.

fibrils accessible to cellulase. Therefore, H2 production of the

bacterium from cellulose might be affected by different dissolubilities of cellulose in IL. Cellulose dissolubility is a very
im- portant parameter in pretreatment because it has a
significant part of the cost of the whole pretreatment process.
The higher dissolubility
will
lead
the
more
economical process.

Table 2 Comparison of hydrogen production with different kinds of chemical pretreatment methods
H2 content
(%, v/v
headspace)

Methods

Untreated cellulose
NaOH pretreatment
(15%, 80 C, 60 min,
neutralization)
H2SO4 pretreatment
(10%, 80 C, 120 min,
neutralization)
[C4mim]Cl pretreatment
(10%, 80 C, 6 h, EtOH)
without N2 sparging
[C4mim]Cl pretreatment
(10%, 80 C, 6 h, EtOH)
with N2 sparging

Cellulose
degradability
(%, w/w)

H2 yield
mL H2/L
culture

mol H2/mol
glucose equivalent

6.1 T 0.31
22.7 T 1.06

122.0 T 6.2
454.0 T 21.2

1.59 T 0.087
1.22 T 0.061

12.2 T 0.74
54.6 T 2.68

24.9 T 1.45

498.0 T 30.0

0.95 T 0.053

77.2 T 3.78

36.1 T 1.81

720.2 T 34.6

1.22 T 0.067

87.6 T 4.51

36.1 T 1.81

1280 T 58.0

2.2 T 0.10

88.9 T 4.75

100

H2 content
Cellulose utilization

80

30

60

Cellulos
utilization
(%)

1500

120
Cellulose utilization H2 production
H2 prodution rate
90

1000
60

Cellulose utilization (%)

40

H2 production (ml H2/L culture)

H2 content (%, v/v of headspace)

[C4mim]Cl is the most efficient solvent with 18% of cellulose

H2 yield also increased from 0.77 to 0.95 mol H 2/mol hexose
dissolution [2,8]. At 80 C, cellulose was dissolved in
under N2 sparging condition [13]. Therefore, N2 sparging was
[C4mim]Cl with three different concentrations of 5, 10 and
applied in this study for maintaining the continuous H 2 pro15% (w/w; g cellulose/100 g ionic liquid). Complete
duction, thus increasing the H2 yield of T. neapolitana.
dissolution occurred within 6 h. The dissolved cellulose in
the pretreatment solu- tions was regenerated by precipitation
using water via a pref- erential solute-replacement 3.3.2. Effect of anti-solvents on cellulose precipitation
mechanism. The results of the fermentation process with T.
In cellulose hydrolysis, cellulases first absorb on the cellulose
neapolitana on the pretreated cel- lulose are shown in Fig.
surface, partially stripping the individual polymer chains
3. The cellulose derived from the 10% (w/w) solution in the
from the crystalline structure and then cleaving the glycoside
liquid gave the maximum H2 production (34.3%).
bonds [26]. The increase in the surface area accessible to
In our previous research on H2 production by T.
water and cellulases after the pretreatment process would be
neapolitana, the maximum yield of H2 production was 34
expected to improve the cellulose fermentation to H 2. The
36% (v/v of gas headspace). After reaching this H2 content,
structure of regenerated cellulose was found to differ
the bacterium pro- duced H2 at a very low rate or stopped the
depend- ing on the anti-solvents used for cellulose
H2 production be- cause of inhibitory effect by high H 2 partial
precipitation [7]. This difference also had various effects on
pressure. Gas sparging that reduces hydrogen partial
the direct H2 pro- duction from the pretreated cellulose. Fig. 4
pressure in the liquid phase has been a useful method to
shows that EtOH was the most efficient anti-solvent to obtain
remove produced hydro- gen in the gas phase [20]. Mizuno
regenerated cel- lulose from the cellulose pretreated with
et al. demonstrated that low- ering dissolved H2 by sparging
[C4mim]Cl for the purpose of H 2 production under N2
N2 resulted in a 68% increase in H2 yield from a reactor
sparging conditions. The rate of reaching to its maximum H 2
1
operating on an enriched mixed micro- flora with 10 g L
content (36.4%) using the cellulose regenerated with EtOH
glucosemineral salts [17]. On a continuous fermentative H2
was higher than that with MeOH or water. This result is in
production by seed sludge, it was shown that
conformity with the
10

H2
production
rate (mmol
H2

20
40

500
30

20

10

0
10

0
Untreated

15

Concentration of cellulose in [C4mim]Cl (%, w/w)

0
EtOH

MeOH

Anti-solvents

Water

Fig. 3 Effect of [C4mim]Cl pretreatment on the

H2 production by Thermotoga neapolitana.

Fig. 4 Comparison of the effect of various antisolvents in the cellulose recovering step after
[C4mim]Cl pretreatment on the H2 production
under N2 sparging conditions.

saccharification and fermentation processes.

In our data, T. neapolitana showed a cellulose
degradability of greater than 85% when using the
regenerated cellulose from [C4mim]Cl pretreatment, which
was approximately 7-fold higher than that from the
untreated cellulose (12.2%). This value was also much higher
than the conversion yield of the same regenerated cellulose
to glucose by enzyme cellulase (5972%) [7]. The recovered
[C4mim]Cl was tested by reutilizing it in three subsequent pretreatments. It showed the
same results in terms of the growth, cellulose degradability
and H2 production on this bacterium (data not shown).

Glucose concentration (g/L)

research of Dadi et al. [7]. The X-ray powder diffraction

pattern of the cellulose regenerated using EtOH exhibited a
lower de- gree of crystallinity in comparison with that
regenerated with MeOH and water. Therefore, it can be
inferred that the cellu- lose regenerated using EtOH was the
easiest for the bacterium to convert into H2 through

160

140
120

Cel
growth
(mg
protein/L
culture)

100
2

80

60
40

0
Glucose Cell protein
0
5
10

20
15

20

25

30

35

Cellulose concentration (g/L)

Fig. 6 Effect of concentration of [C 4mim]Clpretreated cellulose on the growth and cellulose
hydrolysis of Thermotoga neapolitana.

3.3.3. Effect of pretreated substrate concentrations

H2 production (ml H2/L culture)

H2 production by T. neapolitana mainly depends on its ability

to hydrolyse cellulose. The rate of any enzyme catalyzed
reac- tion is determined by the concentrations of the enzyme
and substrate. Therefore, fermentations at different substrate
concentrations, ranging from 5.0 to 30.0 g/L of pretreated
cel- lulose, were carried out to determine the effect of the
sub- strate concentration on the H2 production. The cellulose
conversion into H2 in T. neapolitana is a two-stage process
in- cluding enzymatic cellulose degradation into glucose by
cellu- lase system and glucose fermentation into H 2. From
Fig. 5, we recognize that the increase in the concentration of
IL-pre- treated cellulose from 15 to 30 g/L does not cease H 2
produc- tion, and it does not also significantly improve H 2
yield. Moreover, there is a significant increase in
concentration of glucose in medium and the growth of the
bacterium shows
a decrease when pretreated cellulose
concentration is in- creased (Fig. 6). The increase of glucose
which is even in the level supporting H 2 production in the
bacterium cannot im- prove the H2 production. Therefore, it
might be that the ceiling of H2 production when increasing
cellulose concentration could be due to the limitation in
activities of hydrogenase sys- tem of the bacterium.

3.4.

Comparison of different chemical

methods of cellulose pretreatment

The conversion of cellulose into H2 via anaerobic

fermentation by T. neapolitana directly depends on the
cellulose utilization of this bacterium [20]. The pretreatment
method influences the H2 production by enhancing the
cellulose hydrolysis activity of the cellulase system available
in the bacterial cells. Table 2 shows the different effects of the
chemical pretreatment methods on the cellulosic H2
production by T. neapolitana. The hydrogen production
from cellulose pretreated with [C4mim]Cl was 88.9% which
was higher than that from the cellulose pre- treated with
NaOH (54.6%) and H2SO4 (77.2%). This means that the
regenerated cellulose from the [C 4mim]Cl pretreat- ment is
more easily enzymatically hydrolysed than the regen- erated
cellulose from the NaOH and H2SO4 pretreatments.
The H2 content obtained when using the regenerated
cellu- lose from the [C4mim]Cl pretreatment was 36.1% (v/v)
of the headspace volume, which was higher than that of the
cellu- lose derived from the NaOH or H2SO4 pretreatment.
This value was also much higher than that of the same
bacterial strain when using glucose, an easily utilizable
carbon source, as the substrate (27%) [24]. The regenerated
cellulose also gave

Cellulos

2000

the highest H2 yield in units of mL H2 produced by 1 L

100 utilization
(%)

1800
80

1600
1400

60

1200
40

1000
800
5

600
0

10
35

15

20

25

Cellulose concentration (g/L)

H2 production Cellulose utilization

30

20

culture (1280 mL H2/L) compared with those of the

untreated and acid- and alkali-pretreated celluloses.
Although the H2 yield in units of mol H2/mol glucose
equivalent in the case of the fer- mentation using untreated
cellulose was shown to be the highest, the production of H 2
bacterium in this case was not efficient because there was an
insufficient amount of ferment- able carbon source for H 2
conversion. The H2 yield in units of mol H2/mol glucose
equivalent of the fermentation using the cellulose pretreated
with H2SO4 was the lowest (0.95 mol H2/ mol glucose)
although its cellulose digestibility was relatively high. This
may be because some byproducts from the pretreat- ment
process or the Na2SO4 concentration formed in the neu-

t
Fig. 5 Effect of concentration of [C4mim]Clpretreated cellulose on the H2 production by
Thermotoga neapolitana under N2 sparging
conditions.

ralization step result in the formation of product other than

H2. In any case, the H2 yield in units of mol H2/mol glucose of
T. neapolitana obtained in this study is still low compared
with that of other Thermotoga strains. The H2 yield from

glucose as a substrate in T. maritima and Thermotoga elfii

was reported to be 4.0 and 3.3 mol H2/mol glucose [22,23],
respec- tively, which are very high. The H 2 yield in units of
mol H2/ mol glucose equivalent of T. neapolitana obtained
from pre- treated cellulose in this study was 2.2, relatively
low compared with that of Thermotoga strains but this was
a potential result for H2 production from recalcitrant
feedstock source.
Utilization of cellulosic material in H 2 production is not
a new trend in biofuel production. However, cellulosic material has not been an economically efficient feedstock source
for biological conversion because a main problem comes
from its accessible structure that leads to a too low
availability of fermentable sugars. Pretreatment is a process
which can al- ter the structure of cellulosic material to make
it more acces- sible to biological hydrolysis. Therefore,
pretreatment is
an
important means to overcome the problem in biological conversion of cellulosic material.
An important contribution of our study is the
suggestion of a new method for pretreatment process of
cellulosic biomass. Because IL can efficiently dissolve
cellulose, a direct and quick cellulose separation from
cellulosic biomass to use as feed- stock for biological
processes might be developed. Pretreat- ment of cellulosic
biomass using IL shows more preeminent features than
ordinary chemical pretreatment methods using acids or
bases. It is a simple, quick and green process with fewer
pretreating steps and shorter pretreating time. Compared with acid- and base-pretreatment methods, IL
pretreat- ment does not contain the system corrosion,
byproduct inhibition from sugar degradation and
environmental toxica- tion. The pretreated cellulose from IL
pretreatment is easily obtained for using in biological
conversion without neutrali- zation. Another important
advantage of this pretreatment process is that IL can be
recovered for subsequent utilizations. At this time, the
commercial price of ILs is not so cheap be- cause ILs are
very new chemicals. However, with many such practical
properties above, the price of ILs will not be a barrier for
application in practical cellulose conversion processes in
the near future. As illustrated in the manuscript, the
proposed cellulose pretreatment process possesses many
technical out- standing characteristics. Thus, the
pretreatment method is strongly believed to be very costeffective and environmental
benign.

4.

Conclusion

In our attempts to improve the cellulose utilization for H2

pro- duction via fermentation using different pretreatment
methods, we found that the ionic liquid to be the most efficient chemical compared with the acid and alkali processes.
The highest H2 production (36.1%) was obtained from the fermentation using the regenerated cellulose resulting from the
[C4min]Cl pretreatment at 80 C in 6 h with ethanol as the
regenerating agent. The process using the ionic liquid has
fewer steps and dissolves large amounts of cellulose under

not affected by inhibitory products resulted from glucose

deg- radation, but H2 partial pressure. Therefore, the
application of this pretreatment in the utilization of cellulosic
biomass source for hydrogen production holds considerable
promise in the near future.

Acknowledgement
This Research was supported by the Hydrogen Energy R&D
Center, one of the 21st Century Frontier R&D Programs,
funded by the Ministry of Science and Technology of Korea.

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resistant construction materials as compared with the acid
method. The H2 yield of 2.2 mol H2/mol glucose equivalent
was obtained from cellulose pretreated by [C4min]Cl. The H2
fermentation using the cellulose pretreated with [C4min] is

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