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Characterizing Lignocellulosics from Pine to Bioethanol
Poulomi Sannigrahi and Arthur J. Ragauskas
School of Chemistry and Biochemistry
Georgia Institute of Technology
Technology, Atlanta
Atlanta, GA.
GA
RESULTS: Enzyme hydrolysis and fermentation of Pine
350
Carbohydrate Profile
% dry weight of untreated
d wood
Inorganic elements
100.0
Lobllolly Pine
Sweetgum
300
Content (mg/K
Kg)
250
Extractives content:
Loblolly pine: 2.9 %
Sweetgum: 0.4 %
200
150
80.0
60.0
40.0
100
20.0
50
70.0
* Medium enzyme loading
60.0
Untreated
Acid
Acid-enzyme
Organosolv
OS-enzyme
50.0
40.0
30.0
20.0
10.0
0.0
Arabinose Galactose GlucoseXylose Mannose
Ba
Cu
Fe
Na
P
1200.0
O
HO
OH
OH
OH
50
40
O
OH
OH
OH
CH2OH
OH
O
O
OH
400.0
 Organosolv
Ca
K
Mg
S
Na
Arabinose Galactose Glucose Xylose Mannose
Loblolly pine
Ash
0.2%
Klason lignin
29.3%
Acid-soluble lignin
0.4%
Cellulose Iβ
Paracrystalline
Inaccessible fibril
surface
 Fermentation with Saccharomyces cerevisiae
pH 5.5; 30 °C; 48 h; 10 g/l yeast
10
10.0
0.0
0
20
Accessible fibril
surface
6
 CH2OH
CH2OH
CH2OH
 CH
CH
CH
 CH
1
CH
1
3
5
C5 (C-C)
C3, C4
C1
C5 (C-H)
3
5
4
OH
OH
C in -O-4
Guaiacyl
C in -O-4
SW
x
HW
x
6
2
OCH3 CH O
3
4
C in -O-4,C in -5 and -
6
2
Syringyl
OCH3
CH
1
2
3
5
4
OH
p-Coumaryl
x
x
60
Time (hours)
80
100
Crystalline cellulose
Cellulose Iα
45
Cellulose Iα+β
40
para-crystalline
35
Cellulose Iβ
30
25
20
15
5
Sweetgum
0.8%
26.0%
2.4%
Comparison to Sweetgum
 Higher degree of crystallinity in Pine
62.5 % vs. 53.3 %
 Smaller proportion of cellulose Iα
 More para-crystalline cellulose
 Greater proportion of cellulose Iα+β
Milled wood lignin: Quantitative 13C NMR
40
10
5
Spectral fitting results of the C4
region of cellulose NMR spectra
ENZYME HYDROLYSIS AND FERMENTATION
Celluclast 1.5L--20 FPU/g cellulose
Novozym 188
188--40
40 IU/g cellulose
50 mM acetate buffer, pH 4.8; 45 °C; 96 h.
15
Mn
C ll l
Cellulose
Iα+ β
65% Ethanol, 1.1% H2SO4, 170 °C, 60 min
 Cellulase and Cellobiase enzymes
Accessible fibril surface
20
0.0
 Aqueous Ammonia
5.6 M NH4OH; 50 °C; 48 h.
25
OH
OH
0
OH
OH
OH
PRETREATMENT STRATEGIES
Step 1: 0.5% H2SO4; 180 °C; 10 min
Step 2: 1% H2SO4; 200 °C; 2 min
20.0
50
CH2OH
OH
OH
600.0
200.0
OH
OH
10
800.0
O
Cellulose Iα
 Dilute sulfuric acid
30.0
Inaccessible fibril surface
Cellulose: Solid-state CP/MAS 13C NMR
Distribution map of Loblolly
Pine
40.0
Accessible fibril ssurface
rface
1000.0
x
OCH3
Comparison to Sweetgum
 Fewer β-O-4 and β-β structures
 More phenolic structures
 More β-5
 Greater no. of syringyl residues
in sweetgum
0
Glucos
se-to-ethanol conversion (%)
HO
HO
20
Acid
Base
OS
50.0
Amorphous cellulose
30
Relative proportion (%)
OH
60
% dry weight of wood
Content (mg/Kg)
C b h d t
Carbohydrates
30
60.0
Si
35
 Widely prevalent softwood
species in Eastern US
 Used for lumber, furniture,
pulpwood and plywood
 Mature tree from Baldwin Co., GA
sectioned, debarked and chipped
 Chips stored at < -5 °C
 Composite sample used for all
analyses
Cellulose glucose conversion
70.0
0.0
0
BIOMASS FEEDSTOCK
80.0
Relative proportion (%)
Content (µg/g) dry wood
Extractives
Cellulose-to-glucose co
onversion (%)
LOBLOLLY PINE vs. SWEETGUM
0
Untreated
Untreated Acid Acid-enz Base Base-enz Organosolv OS-enz
Fermentation results
100
Acid-enzyme
OS-enzyme
80
60
40
20
0
0
10
20
30
40
Time (hours)
50
60
Acid Acid-enz
Base Base-enz Organosolv
OS-enz
Cellulose Crystallinity Index
120
Crystallinity Index (%)
C
INTRODUCTION
The efficient processing of lignocellulosics into bioethanol is
contingent on the characterization of the chemical constituents of
plant carbohydrates and lignin and their changes in structure
throughout the process. This program will evaluate the impact of
process chemistry for three different biomass resources. The first
year of this research program focused on a softwood biomass
resource Loblolly Pine, while the second year is focused on the
hardwood feedstock Sweetgum. Highlights of results from FY
2006-2007 are presented here along with comparison of the two
different biomass feedstocks.
100
90
80
70
60
50
40
30
20
10
0
Untreated
Acid
Acid-enz
Base
Base-enz Organosolv OS-enz
CONCLUSIONS: Loblolly Pine
Organo-solv pretreatment is most effective
Supported by SEM and chemical analysis
Effects of pretreatments
Decrease in ash
•Pretreatments effectively remove metals
Increased condensation of lignin
Lignin molecular weight decreases
Generate new ‘extractives’
Change in cellulose ultrastructure
Effects of enzyme hydrolysis
Increased cellulose crystallinity index
Preferential removal of amorphous cellulose
•Preferential
Data suggests need to develop enzyme capable of
degrading more stable cellulose forms (eg. Iβ)