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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
252
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.
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
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
(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
(A.1)
5%
N
0
O
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)
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)
255
500
400
300
200
Avicel in NMMO-CHT
Avicel in NMMO-UHT
100
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).
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
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
Table 2
Biodiesel production from hydrolyzates.
Carbon source
Lipid
content
(%CDM)
Biomass
(g/l)
Esterication of
microbial oil
(wt%)
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
257
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