INTERNATIONAL BIOREFINERY
CONFERENCE
(IBC 2009)
Clean Conversion to Fermentable Glucose
from????
Wheat Straw
Lu Lin
South China University of Technology, Guangzhou, China
Outline
1. Introduction
2. Experimental
3. Results and discussions
4. Conclusion
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1. Introduction
CO2
Industry
Car
Tree
Wheat straw
Reed
Ethanol
Acid
Cellulose
Cellulase
Fermentation
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1. Introduction
Fermentable sugars
Tree
Wheat straw
Reed
Key Step
Ethanol
Acid
Cellulose
Cellulase
Fermentation
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2. Experimental
2.1 The experimental route:
Structural analysis of cellulose
Eliminate inhibitor
Wheat straw
Formic Acid
hydrolysis
Sugar solution
Fermentable Sugar
Acid recycle
Formic acid system
9 Easy recycle
9 Mild reaction temperature
9 High yield of glucose
The analysis of reduced sugar and glucose
The analysis of hydrolysis kinetics
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2. Experimental
2.2 The hydrolysis system of cellulose
Material: wheat straw ;
Formic acid system: 78.22 % formic acid, 4 % hydrochloric
acid and 17.78 % water;
Reaction temperature: 60°C , 65°C , 70°C , 75°C ;
Reaction time: 1-9 h;
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2. Experimental
2.3 13C Solid State NMR Analysis
Frequency:100.61 MHz
Probe: 4 mm MAS BB-1H
25 ºC. The contact time: 1.5 ms
Angle: 90°
Pulse:5.6 μs.
MAS speed was 5 kHz.
delay time : 3s.
Bruker Avance 400 spectrometer
Cross polarization/magic angle spinning
(CP/MAS) 13C solid-state NMR
The crystallinity (Cr.I.) and amorphicity (Am.I.) indexes, given as a percentage
of the integrals of the C4 peaks at 86–92 (a) and 80–86 ppm (b), were calculated
by the following relations.
Cr .I .(%) = 100 × a (a + b )
Am.I .(%) = 100 − Cr .I .
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2. Experimental
2.4 FTIR Analysis
FTIR spectra are recorded between 4000
and 400 cm-1, using a NEXUS spectrometer.
Discs have been prepared by first mixing 2 mg
of dried sample with 200 mg of KBr (for
spectroscopy) in an agate mortar.
The resulting mixture is successively pressed
at 10 MPa for 3 min.
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2. Experimental
2.5 X-Ray Diffraction Analysis
Wide-angle X-ray diffraction was conducted with
a D/MAX-III, an instrument with 12° / min scan
speed. Cellulose powder samples were laid on the
glass sample holders (35×50×5 mm) and analyzed
under plateau conditions. Cu radiation was generated
at a voltage of 40 kV and a current of 30 mA. The
scan scope was between 2° ~ 50°.
The degree of crystallinity (χc) was calculated by
χ c = FC (Fa + Fc ) × 100%
The crystallites sizes were determined from
D(hkl ) =
Kλ
β 0 cosθ
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2. Experimental
2.6 HPLC Analysis
The HPLC system consists of Waters 600E
system controller, Waters 717 automatic sampler,
Waters 410 differential refractometer, Waters
Sugar pak I column. The mobile phase is distilled
water and is run at a flow rate of 1.1 ml/min. The
LC system is operated at 90°C. The sample
volumes injected are 10μl. Standard samples and
hydrolyzate samples have been filtrated by 0.45μm
filter before analysis.
Glucose
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3. Results and Discussions
3.1 CP/MAS 13C-NMR Analysis
Sample
Control
C1
200
180
160
140
160
140
C6
C4
120
100
80
105.93
Treatme
nt for 3 105.78
hours
C2，3，5
T reatm en t fo r 3 h o u rs
C1
60
40
20
0
120
100
80
60
C h em ica l sh ift ( δ p p m )
40
20
0
C4
C2, 3, 5
C6
89.63
75.73
65.50
84.40
73.51
63.53
89.54
76.07
65.65
84.69
73.20
63.44
C o n tro l
200
180
Fig. 1. CP/MAS 13C NMR spectra of wheat straw
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3. Results and Discussions
3.1 CP/MAS 13C-NMR Analysis
Treatment for 3 h
110
109
108
107
106
105
C2，3，5
200
180
160
140
120
100
60
103
102
Iβ
C6
80
102
Iβ
Iα
C4
103
Less-ordered
C1
T reatm en t fo r 3 h o u rs
104
Control
40
20
0
110
109
108
107
106
105
104
chemical shift / δ ppm
C o n tro l
200
180
160
140
120
100
80
60
C h em ica l sh ift ( δ p p m )
40
20
0
Fig. 1. CP/MAS 13C NMR spectra of wheat straw
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3. Results and Discussions
3.1 CP/MAS 13C-NMR Analysis
Control
Treatment for 3 hours
Assignment
Chemical
shift (ppm)
Intensity
/%
Chemical
shift (ppm)
Intensity
/%
Ⅰβ
106.87
4.86
106.92
2.87
Ⅰα
106.33
12.42
106.34
18.92
Less-ordered
105.77
21.60
105.79
9.56
Ⅰβ
105.05
61.12
105.33
68.66
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3. Results and Discussions
3.1 CP/MAS 13C-NMR Analysis
Treatment for 3 h
C2，3，5
94
92
90
88
C1
84
82
80
78
Fibril surface
Amorphous
Para-crystalline
C6
C4
T reatm en t fo r 3 h o u rs
86
Iß
I(a +ß )
Ia
Control
200
180
160
140
120
100
80
60
40
20
0
94
92
90
88
86
84
82
80
78
chemical shift /δ ppm
C o n tro l
200
180
160
140
120
100
80
60
C h em ica l sh ift ( δ p p m )
40
20
0
Fig. 1. CP/MAS 13C NMR spectra of wheat straw
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3. Results and Discussions
3.1 CP/MAS 13C-NMR Analysis
Control
Assignment
Chemical
Intensity
shift
/%
(ppm)
Treatment for 3 hours
Cr.I.
/%
Chemical
Intensity
shift
/%
(ppm)
Іα
90.35
2.06
90.17
2.66
І (α+β)
89.77
5.68
89.76
8.31
Para-crystalline
88.99
6.93
89.13
11.21
Іβ
87.97
7.49
88.16
8.61
Amorphous
85.47
21.18
85.62
21.65
Fibril surface
84.51
28.77
84.72
26.56
Fibril surface
82.77
27.89
83.51
21.00
22.74
Cr.I.
/%
29.15
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3.2 FTIR Analysis
4
3
2
-1
-1
1718cm 1630cm
-1
1510cm
-1
1460cm -1
1431cm
1
3340-3412
cm-1
O-H
stretching
2900, 2968
cm-1
C–H
stretching
1431, 1316
cm-1
CH2
wagging
1373, 1281
cm-1
C–H
bending
1201cm-1
C6–O–H, in
plane
bending
1237cm-1
O-H
bending
1158, 901
cm-1
C–O–C
stretching
1061, 1033
cm-1
C3–O, C–C,
C6–O
stretching
672, 711 cm-
C-O-H, outof-plane
bending
-1
-1
1373cm
672cm
-1
1316cm
-1
1237cm
-1
-1
1114cm 1061cm
-1
2900cm
-1
3400cm
4000
3200
2400
1600
800
-1
Wave number / cm
Fig. 4. FTIR spectra of wheat straw. 1, 2, 3,
4- the control and treatment for 1, 3, 5 hours
of wheat straw respectively
1
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3.2 FTIR Analysis
4
0.25
a
0.30
0.25
0.20
1
2
0.10
3
1
0.20
0.15
Absobance /%
b
0.15
3
2
0.10
0.05
3
2
0.05
0.00
0.00
3800
3600
3400
3200
3000
3800
3600
3400
3200
3000
-1
0.20
c
0.30
1
d
0.25
0.15
1
0.20
0.10
-1
2
0.10
0.05
2900cm
3
-1
3400cm
0.05
0.00
-1
1373cm
672cm
-1
1316cm
-1
1237cm
-1
-1
1114cm 1061cm
-1
0.15
2 3
-1
1718cm 1630cm
-1
1510cm
-1
1460cm -1
1431cm
1
4000
3200
2400
1600
800
0.00
-1
3800
3600
3400
3200
3000
3800
3600
3400
3200
3000
Wave number / cm
-1
Wave number /cm
Fig. 5. Resolution of hydrogen-bonded OH
stretching for wheat straw. a: control, b:
treatment for 1 hours, c: treatment for 3 hours,
d: treatment for 5 hours. 1: Valence vibration of
H bonded OH groups and O(2) H-O(6) (intra),
2: O(3)H-O(5) (intra), 3: O(6)H-O(3′) (inter).
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3.2 FTIR Analysis
0.25
a
0.30
0.20
1
2
Absobance /%
0.10
0.15
3
0.05
0.00
0.00
3600
3400
2
0.10
0.05
3800
Bond 2
1
0.20
0.15
0.20
b
0.25
3200
3000
c
3800
0.30
1
3600
3
3200
3000
d
0.25
0.15
3400
1
0.20
0.10
0.15
2 3
2
0.10
0.05
3
Bond 3
0.05
0.00
3800
Bond 1
0.00
3600
3400
3200
3000
3800
3600
3400
3200
3000
-1
Wave number /cm
Fig. 5. Resolution of hydrogen-bonded OH stretching
for wheat straw. a: control, b: treatment for 1 hours, c:
treatment for 3 hours, d: treatment for 5 hours. 1:
Valence vibration of H bonded OH groups and O(2) HO(6) (intra), 2: O(3)H-O(5) (intra), 3: O(6)H-O(3′)
(inter).
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3.2 FTIR Analysis
Treatment for
Control
1 hours
3 hours
5 hours
Wave number /cm-1
Intensity /%
1
3469.3
49.39
2
3350.7
24.38
3
3225.6
26.23
1
3464.4
55.29
2
3344.7
21.53
3
3232.0
23.18
1
3468.7
53.08
2
3351.0
22.12
3
3237.1
24.80
1
3464.2
54.07
2
3344.1
21.77
3
3231.4
24.16
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3.3 X-Ray Diffraction Analysis
Treatment
for
lattice
plane
Crystalline
size /nm
101
1.53
020
2.40
004
0.95
101
1.48
020
2.53
004
2.26
101
2.12
020
2.11
004
2.33
101
1.79
020
2.20
004
1.12
Crystallinity
degree / %
002
101
040
0
5
10
15
20
25
30
35
Treatment for 5 h
40
45
50
Control
Treatment for 3 h
0
5
10
15
20
25
30
35
40
45
50
Treatment for 1 h
0
5
10
15
20
25
30
35
40
45
1 hours
56.92
53.54
50
Control
0
5
10
15
20
25
30
35
Bragg angle (2θ / ° )
40
45
50
3 hours
Fig. 7. The X-diffraction profiles of
wheat straw
5 hours
68.73
61.46
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3.4 The hydrolysis kinetics of wheat straw
Hydrolysis model:
k1
k2
Cellulose ⎯⎯→
Glu cos e ⎯⎯→
Degradation Pr oducts
r1 = k1[C ]
(1)
r2 = k2 [G ]
(2)
[C]: the concentration of cellulose
[G]: the concentration of glucose
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3.4 The hydrolysis kinetics of wheat straw
Mass balances on cellulose and glucose lead to:
d [C ]
−
= r1 = k1[C ]
dt
(3)
d [G ]
= r1 − r2 = k1[C ] − k 2 [G ]
dt
(4)
Integrating equations (3) and (4) lead to
k1[C 0 ] −k1t
[G ] =
((e − e −k2t )
k 2 − k1
XG
k1
[G ]
=
=
(e −k1t − e −k2t )
[C 0 ] k 2 − k1
(5)
(6)
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3.4 The hydrolysis kinetics of wheat straw
32
60 ℃
65 ℃
70 ℃
75 ℃
The yield of glucose / %
28
24
20
16
N
1
σ =∑
[X G − f (T , k1 , k 2 )]2
n =1 N − 1
12
8
4
0
1
2
3
4
5
6
7
8
9
Time / h
Fig. 9 Experimental and predicted dependence of the sugars released with the time during
the hydrolysis in formic acid solution (formic acid to water weight ratio of 0.782: 0.178) with
4% hydrochloric acid
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3.4 The hydrolysis kinetics of wheat straw
N
1
[X G − f (T , k1 , k 2 )]2
n =1 N − 1
σ =∑
k = A0 e
−E
a
Kinetics parameters on the hydrolysis of
wheat straw in formic solution (formic acid
to water weight ratio of 0.782: 0.178) with
4% hydrochloric acid
/ RT
The Arrhenius parameter on the hydrolysis
of wheat straw in formic solution (formic
acid to water weight ratio of 0.782: 0.178)
with 4% hydrochloric acid.
Param
eters
k1, h-1
55°C
65°C
70°C
Value
Parameters
75°C
A0
A1, h-1
9.12×1014
A2, h-1
106.35
σ
0.01
Ea1, kJ/mol 7.08×1015
Ea
Ea2, kJ/mol
111.00
σ
0.01
0.0190 0.0325 0.0683 0.0931
k2 , h-1 0.0285 0.0448 0.1098 0.1436
σ
0.0046 0.0112 0.4390 0.8378
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The influence of Residual formic acid and formyl anions
to the fermentation
Concentration
g/l
Glucose
Concentration
g/l
Concentration
of Ethanol
g/l
1.0
0.13
1.64
2.0
45.60
0.81
13
1.0
0.10
4.26
14
2.0
0.41
1.37
15
3.0
0.41
2.20
16
4.0
0.23
2.85
17
5.0
0.01
2.10
6.0
0.01
3.11
19
7.0
0.01
4.29
20
8.0
0.16
7.58
21
9.0
0.01
8.94
22
9.5
40
7.23
23
10.0
40
1.86
No.
1
2
18
HCOOH
HCOO-
The study on the elimination of residual formic acid by
electrodialysis
Concentration
of residual
formic
acid/l
No.
The equipment of
electrodialysis
Electric
voltage
/V
Treatme
nt
time
/min
Removal
rate
of residual
formic acid
/%
1
10
150
30
4.94
2
10
150
60
16.62
3
30
150
30
5.15
4
30
150
60
18.22
5
50
150
30
5.28
6
50
150
60
21.98
7
100
150
15
15.54
8
100
150
30
39.55
9
100
150
45
43.24
10
100
150
60
44.02
11
100
150
75
44.79
4. Conclusion
1. The hydrolysis of wheat straw take place, at first, on the surface of
cellulose.
2. When the formic penetrate into the inner of cellulose,the new
hydrolysis of formic between cellulose are formed, the structure of
cellulose was broken, the crystalline degree decreases, and eventually
the rigid framework of crystalline lattice of cellulose is crushed.
3. The hydrolysis took place on the amorphous zone and crystalline
zone at the same time.
4. After treatment, the absorbency of hydrogen bonds stretching shifted
to higher wave number and decreased to some extent, indicated the
breaking of hydrogen bonds structure.
5. The intermolecular hydrogen bond of 6-OH…O-3′ decreased first,
and then increased gradually, which indicated that the cellulose
bundled together during hydrolysis.
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4. Conclusion
5. The intermolecular hydrogen bond of 6-OH…O-3′ decreased first,
and then increased gradually, which indicated that the cellulose
bundled together during hydrolysis.
6. The hydrolysis velocity of wheat straw in formic acid with 4%
hydrochloric acid are 0.0190 h-1, 0.0325 h-1, 0.0683h-1, 0.0931h-1
respectively at 55°C, 65°C , 70°C and 75°C. The activation energy
is 106 .35kJ/mol.
7. During the hydrolysis of wheat straw, the degradation velocity of
glucose is 0.0285h-1, 0.0448h-1, 0.1098h-1 and 0.1436 h-1
respectively at 55°C, 65°C and 75°C, the activation energy is 111.0
kJ/mol.
South China University of Technology
INTERNATIONAL BIOREFINERY
CONFERENCE
(IBC 2009)
Thank You for Your Attention!
Contact Information:
Lu Lin
Professor
Department of Resources Science and Engineering
College of Light Industry and Food Engineering
State Key Laboratory of Pulp and Paper Engineering
South China University of Technology
Guangzhou, Guangdong Province 510641, China
TEL:86-20-22236719 (Mobile Phone:13054460958)
FAX:86-20-22236719
EMAIL:lclulin@scut.edu.cn