Enhancement of enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media by ultrasonic intensification

Article abstract of DOI:10.1016/j.carbpol.2010.02.031

A new approach for in situ enzymatic saccharification of cellulose in ionic liquids (ILs)-aqueous media is presented in which ultrasonic pretreatment was used to enhance the conversion of cellulose. For this purpose, the solubility of cellulose and the activity of cellulase were investigated in six alkylphosphate ILs. 1-Methyl-3-methylimidazolium dimethylphosphate ([Mmim][DMP]) giving favorable solubility and biocompatibility was selected to establish aqueous-ILs system for enzymatic in situ saccharification of cellulose. After further optimization of reaction parameters concerning cellulase concentration, temperature and IL concentration, higher conversion (95.48%) of cellulose was obtained in the media of aqueous-[Mmim][DMP] by conducting the pretreatment of cellulose with ultrasonic heating, whereas the conversion of cellulose untreated was 42.77%. Scanning electronic microscopy (SEM) and viscosity analysis indicated that IL-treated cellulose under ultrasonic condition was subjected to depolymerization, which led to more efficient saccharification. The findings of this study would have great implications for developing a continuous process for transformation of biomass such as straw cellulose to ethanol or other hydrocarbons.

Full text of DOI:10.1016/j.carbpol.2010.02.031

Carbohydrate Polymers 81 (2010) 311–316
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Enhancement of enzymatic in situ saccharification of cellulose in aqueous-ionic
liquid media by ultrasonic intensification
Fang Yang1, Liangzhi Li1, Qiang Li, Weiqiang Tan, Wei Liu, Mo Xian∗
Key Laboratory of Biofuel, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new approach for in situ enzymatic saccharification of cellulose in ionic liquids (ILs)-aqueous media
is presented in which ultrasonic pretreatment was used to enhance the conversion of cellulose. For this
purpose, the solubility of cellulose and the activity of cellulase were investigated in six alkylphosphate
ILs. 1-Methyl-3-methylimidazolium dimethylphosphate ([Mmim][DMP]) giving favorable solubility and
biocompatibility was selected to establish aqueous-ILs system for enzymatic in situ saccharification of
cellulose. After further optimization of reaction parameters concerning cellulase concentration, tem-
perature and IL concentration, higher conversion (95.48%) of cellulose was obtained in the media of
aqueous-[Mmim][DMP] by conducting the pretreatment of cellulose with ultrasonic heating, whereas
the conversion of cellulose untreated was 42.77%. Scanning electronic microscopy (SEM) and viscosity
analysis indicated that IL-treated cellulose under ultrasonic condition was subjected to depolymeriza-
tion, which led to more efficient saccharification. The findings of this study would have great implications
for developing a continuous process for transformation of biomass such as straw cellulose to ethanol or
other hydrocarbons.
Received 27 January 2010
Received in revised form 5 February 2010
Accepted 11 February 2010
Available online 4 March 2010
Keywords:
Cellulose
Enzymatic hydrolysis
In situ saccharification
Ionic liquid
Pretreatment
Ultrasonic
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
order to overcome these economic and environmental disadvan-
tages mentioned above, scientists have embarked on the research
Cellulose is the most abundant carbohydrate component of
biomass. Along with the diminishing of fossil fuel resources
and global heating warnings caused by greenhouse gas emis-
sions, the efficient utilization of cellulosic biomass is gaining
increasing attention (Kilpeläinen et al., 2007). However, the high
crystallinity of cellulose makes it difficult to hydrolysis into indi-
vidual glucose subunits (Dadi, Varanasi, & Schall, 2006; Finkenstadt
& Millane, 1998). Therefore, the cellulose pretreatment process
has been extensively promoted as one of the solutions to the
energy crisis. Hydrolysis of cellulose is generally catalyzed by
mineral acids. Unfortunately, the chemical saccharification of cel-
lulose using inorganic acids produces abundant by-products that
may increase the environmental load (Schäfer et al., 2007). Dur-
ing the past decades, considerable attention has been paid to
developing novel solvents for dissolution of cellulose, such as N-
methyl-morpholine-N-oxide, LiCl/N,N-dimethylacetamide, ammo-
nium fluorides/dimethylsulfoxide (Gericke, Schlufter, Liebert,
Heinze, & Budtova, 2009; Kuo & Lee, 2009). Nevertheless, these
solvents possess several undesired properties (e.g., high toxicity,
volatility and high costs), limiting their commercial application. In
to find efficient solvents for cellulose (Hermanutz, Gähr, Uerdingen,
Meister, & Kosan, 2008).
Ionic liquids (ILs) are environmentally friendly molten salts,
most of which have the virtues of excellent solvency, low melting
point, nonvolatility and designability (Ohno & Fukaya, 2009). The
physical and chemical properties of ILs can be adjusted to adapt to
different reactions by the choice of cations, anions and substituents
(Yang & Pan, 2005). Due to these unique qualities, IL is taken as a
promising alternative to traditional solvents, especially as a new
reaction medium for biocatalysis (Rantwijk & Sheldon, 2007). Enzy-
matic hydrolysis of cellulose in ILs has become a research hotspot in
recent years (Watanabe, 2010), but being hampered by the incom-
patibility of ILs with cellulase. It has been demonstrated that many
ILs would induceinactivation andunfoldingofthecellulase (Turner,
Spear, Huddleston, Holbrey, & Rogers, 2003). One strategy to solve
this problem is to pretreat the cellulose with ILs and then subject
to enzymatic hydrolysis. Previous work in this lab has shown that
wheat straw recovered from the IL 1-ethyl-3-methylimidazolium
diethylphosphate ([Emim][DEP]) was enzymatic-hydrolyzed more
easily than the untreated substrates (Li et al., 2009). However, it
is a painstaking and time consuming task to regenerate cellulose
hydrolytes from ILs. A potential approach to overcome the above
drawback is to in situ hydrolyze dissolved cellulose in ILs, which
requires to develop ILs compatible with both cellulose solubility
and cellulase activity. Recently, Kamiya et al. (2008) reported that
∗
Corresponding author. Tel.: +86 532 8066 2766; fax: +86 532 8066 2765.
E-mail address: xianmo@qibebt.ac.cn (M. Xian).
1
Both authors contributed equally to the paper.
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.02.031

312
F. Yang et al. / Carbohydrate Polymers 81 (2010) 311–316
Fig. 1. Chemical structure of six ILs: [Mmim][DMP] (1-methyl-3-methylimidazolium dimethylphosphate); [Meim][DMP] (1-methyl-3-ethylimizolium dimethylphosphate);
[Maim][DMP] (1-methyl-3-allylimidazolium dimethylphosphate); [Emim][DEP] (1-ethyl-3-methylimidazolium diethylphosphate); [Eeim][DEP] (1-ethyl-3-ethylimizolium
diethylphosphate); [Eaim][DEP] (1-ethyl-3-allylimizolium diethylphosphate).
byadjustingtheratioof [Emim][DEP]to water, the enzymaticin situ
saccharification of cellulose in aqueous-ILs media was made possi-
ble. Over 50% of the cellulose could be converted to glucose in 24 h,
indicating that [Emim][DEP] shows good compatibility with cellu-
lase. But in aqueous-[Emim][DEP] system, the efficiency of cellulose
hydrolysis into glucose is still not high enough. In-depth studies
concerning biocompatibility of ILs and process intensification of in
situ enzymatic saccharification would be necessary to make the
hydrolysis more applicable.
Here, we screened ILs with good performance in both dissolu-
tion and in situ enzymatic hydrolysis of cellulose. After systematic
optimization of the reaction parameters, an in situ saccharifica-
tion process in aqueous-ILs media was established. Especially, the
significant effects of ultrasound pretreatment on the enzymatic
hydrolysis were investigated in detail. Scanning electron micro-
scope (SEM) and viscosity analysis were applied to elucidate the
possible reasons for the enhancement of enzymatic hydrolysis.
a magnetic stirrer and placed into a heating oil bath. Small precise
amounts of microcrystalline cellulose (about 10 mg) were added
discretely into the vials. The mixture was stirred for dissolution
until the solution was just within the boundary of transparency.
Experiments were conducted at different heating temperatures
(from 40 ◦C to 90 ◦C increased by 10 ◦C every time) and the solubility
of cellulose was calculated by the following formula:
Weight of dissolved cellulose
Solubility =
100%
Weight of ILs
2.4. Ultrasonic pretreatment experiments
Five milligram of microcrystalline celluloses was dissolved in
0.5 ml of six different ILs to equal concentration. Then the cellulose
solutions were pretreated with the help of an ultrasonic genera-
tor (KQ-300VED, Kunshan Sonication Co., China) at a frequency of
45 kHz. The sonication power was 100 W and the sonication time
was 30 min. The temperature was maintained at 60 ◦C during the
ultrasonic heating experiments. Cellulose solutions treated at 60 ◦C
by conventional heating for 30 min served as the control group.
2. Material and methods
2.1. Reagents
Microcrystalline cellulose was purchased from Sigma-Fluka
Chemical Co. Cellulase from Trichoderma reesei was obtained from
Beijing Solarbio Science & Technology Co., Ltd. (China). All other
reagents were of analytical grade and dried or distilled before use.
2.5. Enzymatic in situ saccharification of cellulose in aqueous-ILs
media
Sodium acetate buffer (pH 4.8) was added to the cellulose
solutions prepared by ultrasonic pretreatment, resulting in a het-
erogeneous suspension. The final reaction system was a total 0.5 ml
solution containing various concentrations of ILs, namely 20%, 50%
and 100% (v/v). Enzymatic reaction was initiated by addition of
cellulase to the aqueous-ILs mixture. The enzymatic solution was
vortex mixed and then the enzymatic hydrolysis was carried out
in a constant temperature incubator shaker at 160 rpm. The effi-
ciency of enzymatic saccharifcation was evaluated by quantifying
the glucose released after 24 h. Then optimization experiments
were designed to investigate the effect of cellulase concentration,
temperature and ILs concentration on the enzymatic hydrolysis.
2.2. Synthesis of ILs
Six imidazolium-based ILs (Fig. 1) were synthesized as follows:
equal molar amounts of alkylimidazole and trialkylphosphate were
added to a round-bottom flask and stirred vigorously at 150 ◦C for
15 h (Nie, Li, Sun, Meng, & Wang, 2006). The reactions were car-
ried out under nitrogen atmosphere to prevent the uptake of water
from the air. After cooling to room temperature, the mixture was
washed with ether for three times to remove the unreacted start-
ing material. Then the volatile residues were evaporated from the
crude product at 80 ◦C for 6 h with a rotary evaporator. All of the
ILs were dried under vacuum at 80 ◦C for 24 h before use.
2.6. Analysis methods
2.3. Dissolution of cellulose in ILs
Glucose released by cellulose hydrolysis was quantified using an
SBA-40C Biological Sensing Analyzer (Biology Institute of Shandong
Academy of Sciences, China). One milligram per milliliter glucose
solution was used as a standard for the measurement.
The solubility of cellulose in each IL was measured by a stan-
dardized procedure (Mazza, Catana, Vaca-Garcia, & Cecutti, 2009).
A weighed amount of IL was added to clear glass vials equipped with

F. Yang et al. / Carbohydrate Polymers 81 (2010) 311–316
313
Table 1
Zhao et al., 2009). As for the alkylphosphate type ILs, initial research
which received beneficial results of in situ enzymatic saccharifi-
cation was proceeded using [Emim][DEP] (Kamiya et al., 2008).
The results were consistent with the rule that the effect of ions on
enzyme activity and stability usually follows the Hofmeister series,
that is to say, anions exert their adverse effect on enzyme as follows:
Solubility (wt%) of microcrystalline cellulose in six ILs at different temperature.
ILs
40 ◦
C
50 ◦
C
60 ◦
C
70 ◦
C
80 ◦
C
90 ◦
C
[Mmim][DMP]
[Meim][DMP]
[Maim][DMP]
[Emim][DEP]
[Eeim][DEP]
[Eaim][DEP]
4.65
5.09
0.56
5.30
6.43
1.80
5.11
–
7.36
7.15
3.00
6.20
1.17
2.04
8.75
9.42
6.24
8.96
2.08
4.46
9.84
12.96
7.86
9.60
5.86
5.25
10.64
16.18
9.06
11.48
9.13
F
− < PO43− < SO42− < CH3COO− < Cl− < Br− < I− < SCN− (Zhao, 2005).
Based on previous results, we performed enzymatic saccha-
3.84
a
–
–
–
6.25
rification using alkylphosphate type of ILs-aqueous system to
investigate the impact that ILs brought to cellulase. The biocompat-
ibility of six ILs with cellulase was evaluated by glucose released
during the reaction. Table 2 revealed the amounts of glucose
released at the three given concentrations of ILs. For all of the
six ILs, 20% was the best concentration for enzymatic saccharifi-
cation. Especially, [Mmim][DMP] showed the best performance at
this concentration, the conversion could reach 39.68% after 24 h,
almost three-fold higher than that of the control (11.05%). At two
other concentrations (i.e., 50%, 100%), no activity of cellulase was
detected.
The cellulase activity in 20% ILs descended in the fol-
lowing sequence: [Mmim][DMP] > [Meim][DMP] > [Maim][DMP] >
[Eeim][DEP] > [Eaim][DEP] > [Emim][DEP], indicating that ILs with
the dimethylphosphate anion were more compatible with cellulase
than diethylphosphate ones. According to the theory of Hofmeis-
ter series, the effects of [DMP]− on the solubility of proteins is
greater than [DEP]− due to its smaller side chain. Cellulase in the
[DMP]− solutions persists much more stable secondary and tertiary
structure, and thus exhibits a more efficient catalytic activity.
a
Means not detected.
The viscosity of cellulose solutions in ILs was measured
following the method of Evlampieva with some modifications
(Evlampieva, Vitz, Schubert, & Ryumtsev, 2009). Microcrystalline
cellulose was added to ILs and dissolved by conventional heating
or ultrasonic heating pretreatment mentioned above, respectively.
After cooling to room temperature, pyridine was added dropwise
into the solution, resulting to the final stable mixture. An ubbelohde
viscometer was used to perform the viscosity measurement. The
viscosity of these concentration-gradient solutions was measured
at 30 ◦C.
A microscopic evaluation of morphological changes occurring
during ultrasound pretreatment of microcrystalline cellulose was
studied using a Hitachi S-4800 FE Scanning Electron Microscopy,
where samples were coated with a thin layer of gold in an auto-
matic sputter coater before observation. The accelerating voltage
was 5 kV and images of the samples were acquired at 5000 magni-
fication.
3. Results and discussion
3.3. Effect of ultrasonic heating treatment on enzymatic
hydrolysis of cellulose in ILs
3.1. Solubility of cellulose in ILs
Former research has demonstrated that ultrasound pretreat-
ment could bring about enhancement of cellulose dissolution in
ILs (Mikkola et al., 2007). Cellulose dissolved in ILs was pretreated
by ultrasound to investigate its effect on enzymatic saccharification
of cellulose. As the results showed in Table 2, ultrasonic treatment
exhibited a significant improvement on conversion of cellulose to
glucose. In contrast to cellulose with conventional heating pretreat-
ment, the conversion of cellulose pretreated by ultrasonic heating
increased notably, especially when ILs concentration was 20%.
Activities were detected in all of the three dimethylphosphate type
ILs at the concentration of 50%, whereas activity was only detected
in [Eeim][DEP] among diethylphosphate ILs at this concentration.
None but [Mmim][DMP] showed tiny compatibility with cellulase
when ILs concentration reached 100%. The conversion of cellulose
treated with ultrasound reached 53.15% in [Mmim][DMP] after
24 h, while that were 16.09% for control and 39.68% for cellulose
treated with conventional heating, suggesting that ultrasonic pre-
treatment contributed to saccharification assuredly. In addition,
ultrasonic heating pretreatment also enhanced cellulose hydrol-
The mechanism of cellulose dissolution in ILs has been stud-
ied in detail by Remsing, Swatloski, Rogers, and Moyna (2006). The
dissolution of cellulose in 1-butyl-3-methylimidazolium chloride
([Bmim]Cl) involved hydrogen-bonding between the carbohydrate
hydroxyl protons and the chloride ions of IL in a 1:1 stoichiometry.
ILs dissolve cellulose by weakening the inter- and intra-molecular
hydrogen bonds of the cellulose chains with its hydrogen bond
acceptability, especially from anion. ILs that can dissolve cellulose
always consist of anions such as Cl−, HCOO−, CH3COO−. Car-
boxylate salts such as 1-allyl-3-methylimidazolium formate can
dissolve 10 wt% of cellulose at 60 ◦C and 20 wt% at 85 ◦C, but they
were defective in poor thermal stability and tedious synthesis pro-
cedure (Fukaya, Sugimoto, & Ohno, 2006).
In contrast, alkylphosphate type ILs can be easily synthesized
through one-step method and persist perfect thermal stability.
All the six ILs synthesized above were liquids at room tempera-
ture with lower viscosity than the corresponding halogen type ILs.
Owing to the strong hydrogen bond acceptability of the phosphate
anion, alkylphosphate type ILs showed considerable solubility of
cellulose at different temperature compared to chloride-based ILs
which could only dissolve cellulose above 70 ◦C (Fukaya et al., 2006;
Zhao et al., 2008). As listed in Table 1, all the six ILs dissolved cel-
lulose over 70 ◦C, while only [Eeim][DEP] and [Eaim][DEP] showed
no significant solubility at temperatures below 60 ◦C. As a whole,
[Mmim][DMP] and [Meim][DMP] performed better than other ILs,
having solubility of 4.5–7.5% within the range of 40–60 ◦C which
was just around the operative temperature for cellulase.
Table 2
The conversion of cellulose to glucose in ILs after 24 h with conventional heating
pretreatment (CHP) and ultrasonic heating pretreatment (UHP).
ILs concentration
20%
CHP
50%
100%
CHP
UHP
CHP
UHP
UHP
a
[Mmim][DMP]
[Meim][DMP]
[Maim][DMP]
[Emim][DEP]
[Eeim][DEP]
[Eaim][DEP]
39.68%
35.86%
26.32%
1.64%
12.55%
9.55%
53.18%
48.14%
34.36%
2.18%
18.55%
11.00%
–
1.00%
0.70%
0.30%
–
0.10%
–
–
–
–
–
–
–
0.30%
–
–
–
–
–
–
–
–
–
–
3.2. Evaluation of biocompatibility of different ILs
High melting point, strong polarity and cumbersome synthesis
process have hindered the application of in situ cellulose enzymatic
hydrolysis in chloride- and carboxylate-type ILs (Turner et al., 2003;
a
Means not detected. The conversion of cellulose to glucose during enzymatic
hydrolysis in aqueous media with CHP and UHP were 11.05% and 16.09%, respec-
tively.

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F. Yang et al. / Carbohydrate Polymers 81 (2010) 311–316
SEM analyses were also employed to investigate the changes in
the structure of cellulose regenerated from [Mmim][DMP] under
conventional and ultrasonic heating pretreatment conditions,
respectively. SEM graphs suggested that in original microcrys-
talline cellulose, the major component present was ordered and
condensed fibrils (Fig. 3a). After being regenerated from ILs (Fig. 3b),
the surface became rough and swollen. When the cellulose was
subjected to ultrasonic pretreatment, the structure of cellulose
regenerated further got even loosen and less compact (Fig. 3c), illus-
trating the disruption of linkages in cellulose to a certain extent.
More accessible external and internal surface area of cellulose was
attainable as binding site for cellulase, leading to the enhancement
of enzymatic saccharification (Singh, Simmons, & Vogel, 2009).
3.4. Optimization of reaction conditions for enzymatic hydrolysis
of cellulose
Based on the preliminary findings obtained above,
[Mmim][DMP] which showed good performance in both sol-
ubility and biocompatibility was selected for the next optimization
experiments under ultrasonic conditions.
Fig. 2. Viscosity of cellulose solution in [Mmim][DMP]-pyridine mixture
([Mmim][DMP]: pyridine = 1: 8, v/v) with conventional and ultrasonic heating pre-
treatment respectively.
3.4.1. Effect of cellulase concentration on enzymatic
saccharification of cellulose
ysis in aqueous media. The conversion of cellulose to glucose was
raised from 11.05% (cellulose pretreated with conventional heat-
ing in aqueous system) to 16.09%. Zhang, Fu, and Liang (2008)
have reported similar findings that ultrasonic wave-assisted alkali
pretreatment improved the enzymatic hydrolysis rate of lignocel-
lulosic substrates.
To explain why ultrasonic heating enhanced enzymatic hydrol-
ysis of cellulose, the viscosities of cellulose/ILs pretreated by
conventional and ultrasonic heating were analyzed according to
the methods mentioned in the experimental part. Fig. 2 illustrated
the ꢀsp/c–c flow curves of cellulose/[Mmim][DMP] solutions at dif-
ferent concentrations (c is the concentration of cellulose and ꢀsp
The cellulase concentration was an important factor for enzy-
matic saccharification of cellulose. In order to enhance the
conversion of cellulose to glucose, the effect of cellulase concentra-
tion was investigated in detail. As shown in Fig. 4a, the conversion of
cellulose after 24 h was enhanced markedly along with the increase
of cellulase concentration from 1 mg/ml to 8 mg/ml, and the corre-
sponding maximal conversion reached 82.77%. Further increase of
cellulase concentration from 8 mg/ml to 12 mg/ml did not bring
obvious changes to the hydrolysis efficiency. Previous literature
illustrated that despite the enzyme concentration increased, no
more glucose released during the enzymatic hydrolysis of cellulose
in [Bmim]Cl (Turner et al., 2003). According to the study of Zhao
et al. (2009), 5.0 mg/ml cellulase loading did not bring out better
results, but even slower reaction rate compared to 3.0 mg/ml cellu-
lase loading. Considering the cost of the enzyme, we chose 8 mg/ml
as the optimal cellulase concentration.
is the specific viscosity). The intrinsic viscosity [ꢀ] ([ꢀ] = limꢀsp/c)
c→0
was deduced from the classical way of double extrapolation to zero
concentration. It is obvious [ꢀ] of solution prepared with ultrasound
treatment (60 cm3/g) was much lower than that of solution pre-
pared under conventional heating condition (75 cm3/g). According
to the Mark–Houwink equation:
3.4.2. Effect of temperature on enzymatic saccharification of
cellulose
[ꢀ] = KM˛
The catalytic activities of cellulase were severely diminished
at high temperature (Andreaus, Azevedob, & Cavaco-Paulo, 1999),
whereas low temperature is not conducive to the solubility of cel-
lulose in aqueous-ILs system. Thus, we chose a range from 40 ◦C to
60 ◦C to investigate the effect of temperature on hydrolysis of cel-
lulose. As suggested in Fig. 4b, the reaction temperature appeared
to obviously affect the conversion of cellulose to glucose. Like
most enzyme-catalyzed reactions, the rate of cellulose hydrolysis
increased as the temperature was raised. After 24 h, approximately
K and ˛ are the parameters depending on the particular polymer-
solvent system; M is the mean molecular weight of a polymer.
The molecular weight of cellulose was positively correlated
with [ꢀ]. It indicated that cellulose dissolved by ultrasonic heating
displayed a lower molecular weight than that dissolved by con-
ventional heating. More disruption must occur during ultrasonic
treatment, resulting in the lessening in crystalline and degree of
polymerization (DP).
Fig. 3. SEM micrographs of cellulose: (a) untreated cellulose; (b) regenerated cellulose with conventional heating pretreatment; and (c) regenerated cellulose with ultrasound
heating pretreatment.

F. Yang et al. / Carbohydrate Polymers 81 (2010) 311–316
315
Fig. 5. Time course of enzymatic saccharification of cellulose. Reaction conditions:
10 mg/ml cellulose, 8 mg/ml cellulase, 20% [Mmim][DMP], 50 ◦C.
mum at the optimal temperature, whereas high temperature would
inactivate enzymes.
3.4.3. Effect of ILs concentration on enzymatic saccharification of
cellulose
According to the preliminary experiment results, the enzymatic
saccharification of cellulose was the most efficient when ILs were
20% of the total volume. Intensive study was carried out to find the
optimal concentration of[Mmim][DMP] around20%. Fig. 4cshowed
the conversion of cellulose at different ILs concentrations after a
24-h reaction. When the proportion of ILs was within 20%, glu-
cose formation was enhanced along with the ILs concentration. The
conversion of cellulose raised from 70.82% to 95.55% by increasing
[Mmim][DMP] concentration from 10% to 20%. One possible reason
was that ILs pretreatment of cellulose generated more adsorption
sites for the cellulase. On the other hand, any increase of ILs con-
centration above 20% caused a sharp reduction in the conversion
of cellulose. When ILs concentration was 40%, the conversion was
only 42.09%. This might be explained by the adverse effect of high
ILs concentration on cellulase. The above results were similar to
the aqueous-[Emim][DEP] system in a former study (Kamiya et al.,
2008), that is, the conversion of cellulose to glucose was prefer-
able at an IL to water ratio of 1:4 (v/v). Any further increase in
the concentration of ILs would lead to decreasing of conversion
dramatically.
3.5. Time course of in situ enzymatic saccharification of cellulose
Finally, to demonstrate the enhancement of cellulose conver-
sion based on the optimal conditions obtained above, the time
course of enzyme reactions proceeded in aqueous-[Mmim][DMP]
mixture (pretreated with ultrasonic heating or conventional heat-
ing) and aqueous media, respectively. All of the reactions were
heterogeneous solid-liquid system which is the classic character-
istic of enzymatic hydrolysis of cellulose (Gan, Allen, & Taylor,
2003). The glucose released during the enzymatic hydrolysis was
monitored continuously (Fig. 5). Cellulose pretreated with ultra-
sonic heating and hydrolyzed in aqueous-ILs media exhibited the
best conversion at each predetermined time. After 24 h, the reac-
tion solution became clear and the conversion of cellulose reached
95.48%, compared to 75.57% of cellulose pretreated with conven-
tional heating and 42.77% of the untreated cellulose. Much higher
conversion efficiency was achieved under ultrasonic conditions in
Fig. 4. Effect of reaction parameters on the saccharification of cellulose: (a) cellu-
lase concentration (10 mg/ml cellulose, 60 ◦C, 20% [Mmim][DMP]); (b) temperature
(10 mg/ml cellulose, 8 mg/ml cellulase, 20% [Mmim][DMP]); and (c) ILs concentra-
tion (10 mg/ml cellulose, 8 mg/ml cellulase, 50 ◦C).
93.09% of the cellulose was converted to glucose at 50 ◦C compared
to 83.36% at 40 ◦C. However, when the temperature was further
raised to 60 ◦C, only 80.09% of the cellulose was converted. Enzymes
were sensitive to temperature and the thermal stability of cellu-
lase significantly decreased at temperature above 50 ◦C (Eriksson,
Börjesson, & Tjerneld, 2002). Thus, the reaction rate reached a maxi-

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F. Yang et al. / Carbohydrate Polymers 81 (2010) 311–316
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hydrolysis of cellulose: An overview, an experimental study and mathematical
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Moreover, it is worth mentioning that the recovery of glucose
and ILs from the in situ reaction system using neutral alumina
column chromatography is being carried out in our laboratory.
Encouraging results have been obtained and will be reported later.
All these findings will provide a good basis for reducing the cost
and promote the integrated utilization of cellulose.
Gericke, M., Schlufter, K., Liebert, T., Heinze, T., & Budtova, T. (2009). Rheological
properties of cellulose/ionic liquid solutions: From dilute to concentrated states.
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Six imidazolium-based alkylphosphate type ILs were inves-
tigated in this study, and all of them could dissolve cellulose.
Especially, [Mmim][DMP] showed favorable solubility and biocom-
patibility simultaneously. Thus, a novel aqueous-[Mmim][DMP]
reaction system was established for in situ enzymatic sacchar-
ification of cellulose. After optimizing the reaction parameters,
the conversion of cellulose with ultrasonic heating pretreatment
increased by 52.71% compared to that of cellulose untreated. The
results of viscosity and SEM analysis showed that pretreatment
with ultrasonic heating in ILs decreased the crystallization and
degree of polymerization of cellulose, which might contribute to
increased rate of enzymatic hydrolysis of cellulose. Overall, our
work offered a new reaction system aqueous-[Mmim][DMP] which
was qualified to be perfect candidate as alternative media for
in situ enzymatic hydrolysis of cellulose. Additionally, this study
highlighted the role of ultrasound pretreatment in enhancing the
conversion of cellulose during the enzymatic reaction. Direct in situ
enzymatic hydrolysis of cellulose using aqueous-ILs system would
finally lead to efficient utilization of the cellulosic biomass.
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105, 59–131.
Acknowledgement
Singh, S., Simmons, B. A., & Vogel, K. P. (2009). Visualization of biomass solubiliza-
tion and cellulose regeneration during ionic liquid pretreatment of switchgrass.
Biotechnology and Bioengineering, 104, 68–75.
We thank the CAS 100 Talents Program (KGCX2-YW-801) for the
financial support of this investigation.
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liquid salt-induced inactivation and unfolding of cellulase from Trichoderma
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