Bioresource Technology 107 (2012) 251257

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Ultrasound-assisted compatible in situ hydrolysis of sugarcane bagasse

in cellulase-aqueousN-methylmorpholine-N-oxide system
for improved saccharication
Qiang Li ,1, Geng-Sheng Ji ,1, Yu-Bin Tang, Xu-Ding Gu, Juan-Juan Fei, Hui-Qing Jiang
Jiangsu University of Science and Technology, Zhenjiang 212003, PR China

a r t i c l e

i n f o

Article history:
Received 29 August 2011
Received in revised form 13 December 2011
Accepted 13 December 2011
Available online 22 December 2011
Keywords:
Sugarcane bagasse
Enzymatic hydrolysis
Compatible saccharication system
NMMO
Ultrasound intensication

a b s t r a c t
To fully exploit the benets of N-methylmorpholine-N-oxide (NMMO) in lignocelluloses bioconversion, a
compatible system was established for efcient in situ saccharication of cellulose in NMMO-aqueous
media in which the NMMO is able to activate and solubilize the cellulose, and the cellulases possess high
stability and activity. Cellulase retained its original activity after being pre-incubated in 15% and 20% (w/
v) NMMO solutions. After optimization of reaction parameters, high saccharication rate (96.5%) was
obtained in aqueous-NMMO media by ultrasound assisted treatment of cellulose. The viscosity and FTIR
analysis revealed that NMMO-treated cellulose under ultrasonic condition was porous and amorphous,
which led to improved saccharication. The addition of trie lignin in lower concentration improved
the saccharication efciency of sugarcane bagasse, while higher concentration interferes with hydrolysis. In conclusion, these ndings provided great implications to develop a continuous process NMMO-cellulases system for transformation of native biomass.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Sugarcane is main sugar crop with a production of more than
77.4 million metric tons per sugar production season in the south
of China (Rong and Yong, 2006). As a result, the sugarcane residue
is abundant, inexpensive and readily available source of lignocellulosic biomass in China. However, lignocellulosic biomass is recalcitrant to effective enzymatic hydrolysis due to its highly lignied
and crystalline structure (Galbe and Zacchi, 2002; Kuo and Lee,
2009). To make cellulosic materials more susceptible for hydrolysis, an effective pretreatment is required to soften its tough assembled structure of cellulose crystallinity (Heinze and Liebert, 2001)
and increase the cellulose porosity (Chandra et al., 2010; Zhang
and Lynd, 2003). Pretreatment processes that increase the surface
area accessible to cellulases and water are expected to generate
improvements in efciency of hydrolysis and conversion of cellulosic biomass to sugars (Sun and Cheng, 2002; Zhang and Lynd,
2003). Different pretreatment methods like dilute acid, steam
explosion, ammonia ber explosion, lime and organosolvent pretreatments were employed to improve enzymatic saccharication
(Sindhu et al., 2011. Mosier et al., 2005; Wyman et al., 2005).
Corresponding authors. Tel.: +86 511 85639697 (Q. Li), +86 511 85632660
(G.-S. Ji).
E-mail addresses: comeonareup@yahoo.com.cn (Q. Li), jigsheng@126.com
(G.-S. Ji).
1
The two authors contributed equally to this work.
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.12.068

However, these pretreatment methods produce undesirable

byproducts which inhibit downstream fermentation (Hendriks
and Zeeman, 2009; Mosier et al., 2001, 2005).
To help meet the challenge of sugarcane residue conversion,
NMMO have attracted substantial research interest for N-methylmorpholine-N-oxide (NMMO) is a crystalline compound that is
melting at 170 C, implying low volatility, ammability (Fink
et al., 2001). Recently, several studies demonstrated that NMMO
can effectively solubilize lignocellulosic biomass such as Avicel
and sugarcane bagasse (Biganska and Navard, 2005; Kuo and Lee,
2009; Petrovan et al., 2001; Ramakrishnan et al., 2010). NMMO is
able to dissolve cellulose due to the high polarity of its NO bond,
which breaks the hydrogen bond network of the cellulose and
forms new hydrogen bonds with the solute. The operation conditions for these pretreatments are much milder (<100 C and atmosphere pressure) as compared to the conventional pretreatment
methods. NMMO retains all the advantages of the ionic liquids
ability to dissolve a variety of lignocellulosic substrates (up to
20% by weight) (Petrovan et al., 2001) without the need to chemically modify them and more than 99% of the solvent can be recovered due to its low vapor pressure (Kuo and Lee, 2009). It is also
non-toxic and biodegradable as proven by the work of Lenzig
researchers (Ramakrishnan et al., 2010). Cellulose withdrawn from
NMMO solutions has also generated increased rates of hydrolysis
by cellulases thus implying its potential use in pretreating lignocellulosic biomass for biofuels (Biganska and Navard, 2009; Kuo and

252

Q. Li et al. / Bioresource Technology 107 (2012) 251257

Lee, 2009). Cellulase activity may be inhibited in presence of

NMMO due to high polarity of NAO bond in cellulose (Kuo and
Lee, 2009). But there is no report on systematic investigation of
NMMO compatibility on cellulase and fermentation. Moreover,
no literature was published on the case of natural lignocellulose
in situ enzymatic hydrolysis in NMMO solution which is signicant
to the production of biofuel from natural lignocellulosic materials.
The objective of the present study was to evaluate and optimize
ultrasound intensied cellulase-NMMO system for efcient in situ
saccharication of natural lignocellulosic materials in NMMOaqueous media in which the NMMO is able to activate and solubilize the cellulose, and the cellulases possess high stability and
activity. Different treatment like, conventional heating-NMMOtreatment (mode 1), ultrasound heating-NMMO-treatment (mode
2), ultrasound heating-NMMO-in situ enzymatic hydrolysis-treatment (mode 3) were performed to nd out the fermentable sugar
production from sugarcane bagasse. Moreover, physical characterizations of native sample, and different mode treated samples were
carried out by viscosity and FTIR analysis to elucidate the structural modications.
2. Methods
2.1. Feed stock
The sugarcane bagasse (SB) was collected from a local sugarcane juice shop in Zhenjiang (China). The collected bagasse was
thoroughly washed with distilled water to remove residual soluble
sugars. The raw material was air dried, milled to a size less than
0.5 cm and were stored at room temperature until further use. Cellulase from T. reesei strain was supplied by Solarbio Inc (Japan Yakult Honsha Co., Ltd., E.C. 3.2.1.4, slightly brown powder, 30 U/mg
solid, lyophilized powder). The following chemicals were purchased from SigmaAldrich: b-glucosidase (from Aspergillus niger
strain, 60 U/mg). Avicel PH-101 cellulose (microcrystalline cellulose/MCC, particle size 50 lm, DP 225), N-methylmorpholine-Noxide (NMMO) (Aldrich). Lignin (low sulfonate content, Aldrich).
The lipid accumulating bacterial strain is Rhodococcus opacus
ACCC41043 from Agricultural Culture Collection of China. All other
reagents and chemicals used were of analytical grade.
2.2. Stability of cellulases in NMMO
Stability of cellulase in NMMO was evaluated. The cellulase
mixture, a combination of cellulase and b-glucosidase, was used
in our case with the concentration of 35 FPU cellulase and 60 Cellobiose Unit (CBU) b-glucosidase per gram of cellulose. The cellulase mixture was loaded into NMMO media. Stability tests were
carried out in a total volume of 3 mL containing the cellulase complex and various concentrations of the NMMO (5%, 10%, 15%, 20%,
and 30% w/v) in citrate buffer (50 mM, pH 4.8). The mixture was
pre-incubated at 0 C and 50 C, respectively. Samples were collected at different time points (1, 4 and 24 h) and reducing sugar
analysis was carried out by 2, 5-dinitrosalicylic acid method
(Miller, 1959). The cellulase mixture in the citrate buffer without
addition of NMMO was used as a control. All experiments were
run in triplicate. Relative activity of cellulase% = [cellulase activity
in NMMO solution]  100%/[cellulase activity in citrate buffer].
Reducing sugar analysis in presence of NMMO has not been previously reported, hence reducing sugar analysis in presence of
NMMO were carried out to nd out whether there is any interference in reducing sugar analysis in presence of NMMO. Standard
D-glucose solutions (0.21.4 mg/mL) were mixed with varied concentrations of NMMO (530%, and NMMO free sample as a control)
and subsequently analyzed using the DNS assay. No obvious

interference was observed in the DNS assay for the range of NMMO
concentrations used in this work. Based on these results, DNS assay
was conducted for the measurement of cellulase mixture activity
as follows: 5 mg/ml MCC in 50 mM citrate buffer (pH 4.8) with
the cellulase mixture was incubated at 50 C, followed by mixing
with two volumes of DNS reagent. The reaction mixture was
further incubated at 100 C for 5 min and cooled in an icewater
bath before the absorbance was measured at 540 nm. Control reactions with substrate MCC without addition of NMMO and enzymes,
and enzyme controls in NMMO solution but without loading of
MCC were subtracted from each measurement.
2.3. Hydrolysis of Avicel with the addition of NMMO
2.3.1. Ultrasound intensied Avicel treatment With NMMO
A 3% (w/w) Avicel solution was prepared by combining 0.9 g of
Avicel or sugarcane bagasse with 30 ml NMMO in a 250 ml roundbottom ask and stirred at the speed of 500 rpm. Then the Avicel
solutions were treated with the help of an ultrasonic generator
(TEA-1004, Shanghai TIME Sonication Co., China) at a frequency
of 45 kHz and the sonication power was 100 W. The temperature
was maintained at 90 C during the ultrasonic heating experiments. Sample treated at 90 C by conventional heating served as
the control group. All experiments were run in triplicate.
2.3.2. Enzymatic saccharication of treated Avicel
In the following incubation, the Avicel/ NMMO solution was diluted with 50 mM citrate buffer (pH 4.8) and the nal concentration of Avicel and NMMO was 0.6% and 20% (w/v), respectively.
Three modes of NMMO involved lignocellulosic bres treatment
have been established (mode 1, mode 2 and mode 3). The cellulase
mixture was loaded into Avicel/ NMMO solution and stirred at the
speed of 500 rpm. The enzymatic hydrolysis was carried out at
50 C with the help of an ultrasonic generator at a frequency of
45 kHz. Enzymatic hydrolysis of the cellulosic samples was carried
out at 50 C using conventional heating served as the control
group. The untreated Avicel was hydrolyzed using the same concentration of cellulase mixture and run in parallel with the Avicel/
NMMO system in ultrasound heating bath or conventional heating
bath. The enzymatic reaction was monitored by withdrawing samples from the supernatant periodically and measuring release of
soluble reducing sugars by the DNS assay. Yield of reducing sugars
from sugarcane bagasse was calculated as follows:
Yield of released sugars% = [Reducing Sugars released]  100%/
([sugarcane bagasse weight]  [cellulose ratio in sugarcane
bagasse])
2.3.3. Effect of lignin on the hydrolysis of Avicel in NMMO solution
Effect of lignin on enzymatic saccharication of cellulose were
evaluated by conducting hydrolysis with different concentrations
of lignin. Lignin was mixed with 0.2 g Avicel in 10 ml NMMO,
resulting in 0.06%, 0.60%, 0.86% or 1.20% of lignin and 2.0% (w/v)
cellulose in NMMO, respectively. Then the mixtures were incubated for the treatment, followed by enzymatic hydrolysis as described in section of Avicel Treatment with NMMO and
Enzymatic Saccharication of Treated Avicel with the modication
of time and temperature (72 h, 55 C) in the treatment step.
2.4. Saccharication of sugarcane bagasse in aqueous-NMMO solution
2.4.1. 1Sugarcane bagasse treatment with NMMO
A 3% (w/v) sugarcane bagasse (SB) solution was prepared by
combining 0.9 g of SB (as prepared above) with 30 ml NMMO in
a sterile ask. The SB / NMMO mixture was incubated and stirred
at 80 C for 72 h.

Q. Li et al. / Bioresource Technology 107 (2012) 251257

2.4.2. 2Ultrasound intensied saccharication of sugarcane bagasse

In the following incubation, the SB/NMMO mixture was diluted
with 50 mM citrate buffer (pH 4.8) and the nal concentrations of
SB and NMMO were 0.6% (w/v) and 20% (w/v), respectively. The
cellulase mixture was loaded into SB/NMMO and the enzymatic
hydrolysis was carried out at 50 C with ultrasound intensication.
The enzymatic reaction was monitored by withdrawing samples
from the supernatant periodically and measuring release of soluble
reducing sugars by the DNS assay. All assays were performed in
triplicate. The conversion rate of cellulose was calculated as the total reducing sugar produced in the extracted SB in the initial reaction mixtures.
2.5. Fermentability of the hydrolyzates from saccharication process
After UHT-NMMO-cellulase process, the mixture of NMMO and
hydrolyzates from enzymatic in situ hydrolysis were ltered and
was then passed through a column lled with neutral Al2O3 and concentrated according to our reported method (Xian et al., 2009) and
The hydrolyzates (containing 5.2 g/L glucose) were separated from
the NMMO. Then the hydrolyzates (recovered sugars) were used
for biodiesel preparation according to the our reported method (Li
et al., 2010) using R. opacus ACCC41043. The NMMO was recovered
by heating under reduced pressure condition (Kuo and Lee, 2009).
2.6. Analysis
The constituents of sugarcane bagasse were determined by the
standard analysis procedure of biomass composition (Gouveia
et al., 2009). It was composed of cellulose 34.8%, hemicellulose
28.3%, lignin 19.4% and others 17.5%. Cellulase activity was determined by the standard lter paper assay and expressed as lter paper units per gram of glucan (FPU) (Ghose, 1987). One FPU is
dened as the enzyme that releases 1 lmol of glucose equivalents
per minute from lter paper. The released reducing sugars were
measured by the DNS method using D-glucose as a standard (Miller,
1959). Glucose released by cellulosic biomass hydrolysis was
quantied using an SBA-40D 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 viscosity of cellulose or lignin/cellulose solutions in NMMO
was measured following the method of Evlampieva with some
modications (Evlampieva et al., 2009). Microcrystalline cellulose
or lignin/cellulose mixture was added to NMMO and dissolved by
conventional heating or ultrasound heating pretreatment mentioned above, respectively. After cooling to room temperature, pyridine was added dropwise into the solution, resulting to the nal
stable mixture. An ubbelohde viscometer was used to perform
the viscosity measurement. The viscosity of these concentrationgradient solutions was measured at 30 C. The treated and
untreated cellulosic biomass was measured by Bruker IR spectrometer Tensor 27. The spectra (4000400 cm 1) were recorded with a
resolution of 4 cm 1 and 64 scans per sample. About 2.0 mg samples were prepared by mixing with 120.0 mg of spectroscopic
grade KBr then pressed in a standard device using a pressure of
6000 psi to produce 13 mm diameter pellets. The background spectrum of pure KBr was subtracted from that of the sample spectrum.
3. Results and discussion
3.1. Stability of cellulases in the presence of NMMO
The cellulase was active and stable in the presence of the NMMO
when the temperature was maintained at 4 C (Fig. 1A1B1). Following

253

the rst hour of incubation in 5%, 10%, 15%, 20%, 25%, and 30% of
the NMMO, the cellulase retained 96.2%, 95.0%, 92.2%, 85.0%, and
72.0% of its initial activity, respectively. The enzyme activity decreased with an increase of incubation time; however, the residue
activity still retained approximately 70% of original activity after
24 h of incubation in 30% NMMO (Fig. 1A1B1) at 4 C. Particularly,
b-glucosidase maintained 100%, 99.2%, 99.0%, 97.8% and 91.5% initial activity in rst hour of incubation (as shown in Fig. 1B1) and retained high activity in the incubation process. There are three
constituents of cellulolytic enzymes that are required for the complete breakdown of cellulose to simple sugars. Endoglucanase (EG)
(1, 4-b-D-glucan-4-glucano-hydrolases; EC 3.2.1.74) cleaves glycosidic bonds randomly within the interior of cellulose polymer
chain. Exoglucanases (EC 3.2.1.91 and EC 3.2.1.74) act progressively
on the reducing or non-reducing ends of cellulose chains, releasing
either cellobiose or glucose as major products. The b-glucosidases
(BGL) (EC 3.2.1.21) hydrolyze soluble cellodextrins and cellobiose
to glucose (Saha et al., 1994). In this work, cellobiose was loaded
as substrate to investigate the b-glucosidase activity in presence
of NMMO. Probably, the cellobiose is easily to be degraded as a
disaccharide (Xiao, 2005), while cellulose recalcitrant to enzymatic
hydrolysis due to its highly crystalline structure (Galbe and Zacchi,
2002). As a result, b-glucosidase showed high relative activity in
NMMO system when compared to cellulase (Fig. 1B).
A similar pattern was observed in the 50 C pre-incubation treatment (Fig. 1A2B2). Following 24 h of incubation at 50 C, the cellulases retained 84.7% and 85.6% of the activity in the presence of
5% and 15% NMMO, respectively. The cellulases displayed 84.6% of
the original activity after 24 h of incubation in 20% NMMO. However, the activity signicantly decreased to 40.0% when the cellulases was exposed to 30% NMMO solution for 24 h (Fig. 1A2).
b-glucosidase retained 100%, 98.6%, 98.5%, 98.1% and 85.7% initial
activity in rst hour of incubation, which was lower than that in
4 C pre-incubation treatment. The results indicated that cellulase
was more stable in NMMO at 4 C when compared to cellulase
activity at 50 C. Cellulase buffer solution containing 1520% of
NMMO was suitable for in situ hydrolysis of lignocellulosic biomass.
3.2. Enzymatic saccharication of Avicel
For the enzymatic saccharication, 20% Avicel-NMMO was prepared in 50 mM citrate buffer and the complex was incubated at
80 C for 72 h. Saccharication was carried out using commercial
cellulase in an ultrasound eld and the reducing sugar yield was
monitored at different time points (Fig. 2). Approximately 77.2%
of cellulose was hydrolyzed in the presence of 20% NMMO during
the rst 4 h. After 24 h of digestion, 95.9% of treated Avicel was
converted to reducing sugars compared to 45.3% and 51.6% of the
untreated cellulose and ultrasound heating treated (UHT) Avicel.
The enhancement of conversion in ultrasound heating-NMMOin situ enzymatic hydrolysis (UH-NMMO-ISEH) treated cellulose
indicated that the NMMO-treatment disrupted crystalline structure in cellulose and improved accessibility of the enzymes to cellulose. Moreover, near complete conversion (96.5%) of cellulose in
the aqueous-NMMO system indicated that the cellulase mixture
retained high activity in NMMO. The data clearly elucidated that
the aqueous NMMO cellulase system in ultrasound eld worked
effectively for the hydrolysis of pure cellulose.
To elucidate the effect of ultrasound heating and the concentration of cellulose on in situ enzymatic hydrolysis of cellulose, the
viscosities of cellulose/NMMO treated by conventional and ultrasound heating were analyzed according to the methods mentioned
in the experimental section. It was illustrated in Fig. 4 that the gsp/
cc ow curves of cellulose/NMMO solutions at different concentrations. The intrinsic viscosity [g] was deduced from the classical
way of double extrapolation to zero concentration. It is obvious [g]

100
80
1h

60

4h
24h

40
20

Relative activity of cellulase(%)

Q. Li et al. / Bioresource Technology 107 (2012) 251257

100
80
1h

60

4h
40

24h

20

30
%

M
M

M
M

O
%

20

25

M
M

M
M

M
M
O

NM

5%
N

15

O
M

O
30
%

NM

20
%

25
%

NM
15
%

5%

O
M

O
M

M
NM

NM

80
1h
4h
24h

60
40
20
0

Relative activity of -glucosidase (%)

(A.2)

100

100
80
1h

60

4h
24h

40
20

(B.1)

M
M
O
N
30
%

N
25
%

20
%

M
M
O

M
M
O

M
M
O
N
15
%

5%

M
M
O

M
M
O
N
30
%

25
%

20
%

M
M
O

M
M
O
N
%
15

M
M
O

M
M
O

Relative activity of -glucosidase (%)

(A.1)

5%
N

0
O

Relative activity of cellulase (%)

254

(B.2)

100
80
60
40
untreated Avicel
UHT-treated Avicel
NMMO-treated Avicel
UHT-NMMO-treated Avicel

20
0
0

12

24

36

48

Time(hours)

Conversion rate of cellulose in SB(%)

Conversion rate of Avicel(%)

Fig. 1. The cellulases mixture (A) or b-glucosidase (B) activity after pre-incubation with various concentrations of NMMO. The pre-incubation temperature was 0 C (A.1, B.1)
and 50 C (A.2, B.2) with samples taken at 1, 4, and 24 h of incubation. Enzymatic hydrolysis conditions were 50 C, pH 4.8, 1% Avicel (substrate of cellulases mixture) or 5%
cellubiose (substrate of b-glucosidase). The enzymatic activity was calculated as mg of reducing sugars produced per hour in various NMMO solutions (cellulase
concentration 35 FPU and 60 CBU per gram of substrate, the enzymes in citrate buffer was normalized as 100% activity). Error bars represent standard deviation of the means
(n = 3).

100
80
untreated SB
UHT-treated SB
NMMO-treated SB
UHT-NMMO-treated SB

60
40
20
0
0

12

24

36

48

Time(hours)

Fig. 2. Cellulase-catalyzed saccharication of Avicel in 20% NMMO solution.

Enzymatic hydrolysis of untreated Avicel and SB, NMMO-treated Avicel and SB in
50 mM citrate buffer and in situ enzymatic hydrolysis of Avicel and SB in ultrasound
intensied 20% (v/v) aqueous NMMO system. Reaction conditions were pH 4.8,
50 C; enzyme loadings were 50 FPU cellulase and 50 CBU b-glucosidase per gram of
cellulose. UHT means ultrasound heating treatment; CHT means conventional
heating treatment. Error bars represent standard deviation of the means (n = 3).

Fig. 3. In situ saccharication of sugarcane bagasse in 20% NMMO solution.

Enzymatic hydrolysis of untreated Avicel and SB, NMMO-treated Avicel and SB in
50 mM citrate buffer and in situ enzymatic hydrolysis of Avicel and SB in ultrasound
intensied 20% (v/v) aqueous NMMO system. Reaction conditions were pH 4.8,
50 C; enzyme loadings were 50 FPU cellulase and 50 CBU b-glucosidase per gram of
cellulose. UHT means ultrasound heating treatment; CHT means conventional
heating treatment. Error bars represent standard deviation of the means (n = 3).

of solution prepared with ultrasound treatment (60 cm3/g) was

much lower than that of solution prepared under conventional
heating condition (70 cm3/g) (Fig. 4). An identical observation
was earlier reported by Yang et al., 2010 for ultrasound assisted

ionic liquid pretreatment of cellulose (Yang et al., 2010). Cellulose

in situ treatment by ultrasound was found to be better than conventional heating. This was evident from the viscosity data presented in Fig. 4. The ultrasound pretreated samples showed a
lower viscosity when compared to conventionally heated samples.

255

Q. Li et al. / Bioresource Technology 107 (2012) 251257

500

nsp /C(cm /g)

400
300
200

Avicel in NMMO-CHT
Avicel in NMMO-UHT

100

Avicel and lignin mixture NMMO-CHT

Avicel and lignin mixture in NMMO-UHT

0
0

0.2

0.4
3

0.6

0.8

C10 (g/cm )
Fig. 4. Viscosity of Avicel solution, Avicel and lignin mixture (4.3:10) in NMMOpyridine mixture (NMMO:pyridine = 1:9, v/v) with conventional and ultrasonic
heating treatment respectively. (C represent the concentration of cellulose , gsp is
the specic viscosity).

3.3. Enzymatic saccharication of sugarcane bagasse

The saccharication rate of NMMO-treated sugarcane bagasse
was signicantly increased compared to the untreated sample
(Fig. 3). Approximately 54.6% of cellulose in the treated SB was
converted to reducing sugar in the rst hour of ultrasound intensied in situ enzymatic hydrolysis, while only 5.7% and 6.6% conversion was observed for the untreated SB and ultrasound treated SB.
After 24 h of digestion, the conversion efciency for ultrasound
intensied in situ enzymatic hydrolysis, untreated SB and UHTtreated SB were 90.4%, 31.5% and 34.6%, respectively (Fig. 3). The
ultrasound treatment without NMMO lead to no obvious enhancement of enzymatic hydrolysis (less than 2%). Approximately 95.9%
of cellulose in SB was obtained after of enzymatic hydrolysis for
12 h, indicating the complete digestion time altered from 24 to
12 h with the intensication of ultrasound.

3.4. Effect of lignin on the hydrolysis of Avicel

The lignin effect on cellulose enzymatic hydrolysis in natural
biomass in presence of NMMO was simulated by mixing different
amount of lignin with cellulose. Enzymatic saccharication rate
of UHT-NMMO treated Avicel was higher (96.5%) when compared
to SB (90.5%). Batch addition of enzyme showed an improvement
in the hydrolysis rate of SB. However, no obvious enhancement
of SB hydrolysis (less than 1%) was detected after additional enzymatic hydrolysis for 48 h. While, the results from the experiment
showed that the conversion efciency of sugarcane bagasse was
lower than Avicel, amendment with additional cellulase did not
improve the conversion rate of cellulose in SB, which indicated that
the incomplete conversion was not caused by the deactivation of
cellulases in aqueous-NMMO mixtures.
However, previous work has found that cellulase activity was
reduced during the hydrolysis of lignocellulosic biomass due to
the interaction with lignin or lignin-carbohydrate complexes
(Berlin et al., 2006; Kumar and Wyman, 2009; Wang et al., 2011).
Extensive efforts have been placed on lignin effect on cellulose
hydrolysis in natural biomass (Lee et al., 2008; Binod et al.,
2011). To reveal how the lignin or lignin-carbohydrate complexes
interfere the saccharication of SB, further experiments were carried out in the presence of various amount of lignin to investigate
the effect of other biomass constituents on hydrolysis of cellulose
in aqueous NMMO system. Because there was 19.4% lignin and
34.8% cellulose in SB as measured by the method described in analysis section, the ratios of lignin to cellulose in this work were
determined to imitate the natural constituents (lignin:cellulose = 5.6:10) of SB biomass. Various amounts of lignin (0.06%,

0.60%, 0.86%, or 1.20%) were mixed in NMMO, implying lignin to

cellulose was in the ratio of 0.3:10, 3:10, 4.3:10 or 6:10. After
24 h of hydrolysis, 96.5% of Avicel (control, without lignin) was
converted to reducing sugar while Avicel containing 0.06%, 0.60%,
0.86%, and 1.20% lignin showed conversion rate of 97.9%, 91.9%,
91.8% and 89.3% respectively after enzymatic hydrolysis for 72 h,
indicating the lignin in low concentration favor the enzymatic
hydrolysis compared to interfering the reaction in high concentration. NMMO is a strong oxidant, antioxidants is added with NMMO
in Lyocell process to stabilize the NMMO/cellulose mixture (Rosenau
et al., 2002). Lignin has been demonstrated to be a potential radical
scavenger and antioxidant (Pan et al., 2006). Lignin in low concentration act as antioxidant to protect the cellulase from inactivation,
resulting in relatively high conversion rate of cellulose. To our
knowledge, the 97.9% conversion rate is the highest cellulase
enzymatic hydrolysis conversion rate in aqueous-NMMO compared with experiments reported such as about 93% conversion
rate in Ramakrishnans work (Ramakrishnan et al., 2010).
[g] of solution prepared with lignin in conventional heating
condition (76 cm3/g) was higher than that of solution prepared
without lignin (70 cm3/g) and with lignin under ultrasound heating condition treatment (68 cm3/g) (Fig. 4). Probably, the decrease
of conversion rate was partly caused by viscosity enhancement of
solution with lignin loading. The lignin enhanced the viscosity of
solution, resulting in increased mass transfer resistance of the cellulase enzymatic hydrolysis system.
3.5. FTIR analysis of sugarcane bagasse in various mode of treatment
FTIR spectra of native and treated SB was carried out to elucidate the structural modications. The band near 1160 cm 1 is representative of the antisymmetric bridge stretching of CAOAC
groups in cellulose and hemicellulose, and the band near
1318 cm 1 can be ascribed to CH2-wagging vibrations in the cellulose and hemicellulose (Liu and Chen, 2006). The 895 cm 1 band
which is characteristic of b-linkages, especially in hemicellulose,
was reduced after NMMO treatments. The band at 1635
1640 cm 1, which is attributed to the absorbed water bending
vibrations, decreased after NMMO treatments.
From these spectra, two infrared ratios were calculated: (1)
a1430 cm 1/a900 cm 1, which is referred as the crystallinity index
(OConnor et al., 1958) or lateral order index (LOI) (Hurtubise
and Krsig, 1960), (2) a1372 cm 1/a2900 cm 1, which is known as
the total crystallinity index (TCI) (Nelson and OConnor, 1964).
The higher index value represents the material has a higher crystallinity and ordered structure. As shown in Table 1, after various
mode of treatment, the LOI of SB decreased from 1.441 to 1.122
(CH-NMMO-treated), 0.982 (UH-NMMO-treated) and 0.906 (UHNMMO-IEH-treated), while the TCI of SB decreased from 1.393 to
1.112 (CH-NMMO-treated), 0.978 (UH-NMMO-treated) and 0.878

Table 1
The infrared ratios of FTIR spectroscopy measured for untreated bagasse, enzymatic
hydrolyzed untreated bagasse and bagasse treated by three mode.

Untreated
Mode1treated
Mode2treated
Mode3treated

Lateral order index (LOI)

A1430 /A900

Total crystalline index (TCI)

A1372/A2900

1.441
1.122

1.393
1.112

0.982

0.978

0.906

0.878

Mode 1: Conventional heating-NMMO-treatment; mode 2: ultrasound heatingNMMO-treatment; mode 3: ultrasound heating-NMMO-in situ enzymatic hydrolysis-treatment.

256

Q. Li et al. / Bioresource Technology 107 (2012) 251257

(UH-NMMO-IEH-treated). As a result, the cellulosic biomass, after

UH-NMMO-IEH-treatment, is less crystalline compared to the untreated one and the sample treated in other modes. Similar results
could be observed in the FTIR data of Avicel (data not shown). The
results illustrated that the two indexes of UH-NMMO-IEH-treated
SB were lower than those of untreated SB, UH-NMMO-treated SB
and CH-NMMO-treated SB, indicating some of cellulose in SB was
degraded and the UH-NMMO-IEH-treatment can efciently degrade the tight structure of SB better than conventional treatment.
The similar decreases of these two indexes have also been reported
for sugarcane bagasse withdrawn from conventional NMMO treatment (Kuo and Lee, 2009). Probably, ultrasound-assisted NMMO
treatment can efciently prevent the dissolved SB from restructuring into its original crystalline structure. Moreover, cellulosic biomass dissolved by ultrasound heating displayed a lower
molecular weight than that dissolved by conventional heating.
More disruption must occur during ultrasound treatment, resulting
in the lessening in crystalline and degree of polymerization (Yang
et al., 2010). Consequently, the fragmental and porous treated SB
with amorphous structure could provide more surfaces for enzymes to attack on. It is obvious that cellulase was not able to penetrate into the intact structure of untreated bagasse for hydrolysis.
Thus, a low conversion rate and yield was obtained (as shown in
Fig. 3). Conversely, the intact structure was disrupted by UHNMMO-IEH treatment and resulted in a porous and amorphous bagasse that signicantly enhanced conversion rate of cellulosic
biomass.

3.6. Fermentability of hydrolyzates

The fermentability of the hydrolyzates after in situ saccharication was evaluated using R. opacus. Our data suggested that the R.
opacus could accumulate 4042% lipid of cell dry matter (CDM)
using simple mineral salt medium with hydrolyzates as carbon
source. In contrast to the control test, there was no obvious negative effect observed in reducing sugars consumption and lipid production in the fermentation (Table 2).
Furthermore, the effect of NMMO on fermentation was investigated. As shown in Table 2, about 1.9 g/l CDM and 3941% CDM lipid content were obtained after 30 h of cultivation using
hydrolyzates (containing 5.2 g/l glucose) with the addition of
1020 g/l NMMO. Further increases of the NMMO to the concentration of 50 g/l caused negative effect on CDM and lipid accumulation, but the withdrawn microbial oil had no inuence on
esterication (Table 2). Probably, due to the high polarity of its
NAO bond (Kuo and Lee, 2009), R. opacus was inhibited in the presence of high concentration of NMMO. In the contrast to 10 g/l ionic
liquids [Bmim]Cl could completely inhabit the growth of R. opacus
ACCC41043 (Li et al., 2010), NMMO mediated the obvious advantage as biocompatible solvent for in situ enzymatic hydrolysis.

Table 2
Biodiesel production from hydrolyzates.
Carbon source

Lipid
content
(%CDM)

Biomass
(g/l)

Esterication of
microbial oil
(wt%)

Control (5.2 g/l Glucose)

Recovered sugars
Recovered sugars with 10 g/l NMMO
Recovered sugars with 20 g/l NMMO
Recovered sugars with 50 g/L NMMO

40.05
41.16
41.02
39.28
30.02

1.92
1.91
1.93
1.90
0.92

91.1
91.5
91.6
91.3
91.6

R. opacus was cultivated for 30 h in low-nitrogen medium using hydrolyzates

(containing 5.2 g/l glucose) from in situ saccharication process as carbon source.
Yield of biodiesel was measured by estercation of microbial oil using IL N-methyl2-pyrrolidonium methyl sulfate ([NMP][CH3SO3]) as catalyst.

Our data revealed that NMMO residual in low concentration will

not pose negative effect on fermentation and biofuel production.
4. Conclusions
Ultrasound intensied aqueous-NMMO in situ enzymatic
hydrolysis system mediated the obvious advantage as compatible
system for lignocellulose saccharication. The cellulose conversion
rate increased by 51.2% and 57.8% compared to that of the untreated. Trie lignin protect the cellulase as antioxidant and enhanced the conversion rate (97.9%). Viscosity and FTIR analysis
revealed that ultrasound treatment in NMMO system degraded lignocellulosic structure and decreased the degree of polymerization
of cellulose, which contribute to signicantly increased saccharication rate. NMMO residual in low concentration will not pose negative effect on biofuel production. In brief, our work provided an
opportunity for compatible in situ hydrolysis of natural lignocellulosic biomass using aqueous-NMMO system.
Acknowledgements
We wish to express our thanks for the support from the Natural
Science Foundation of Jiangsu Province (No. BK2011527) and the
Startup Project of Doctoral scientic research of Jiangsu University
of Science and Technology (No. 35211003).
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