Lignin Composition’s impact on
Enzymatic Hydrolysis
Crystal Oldfield1 and Ganti Murthy, PhD2
Bioresource Research1, Biological and Ecological Engineering2
Oregon State University, Corvallis, OR 97330
Utilizing Plant Biomass: A Global Concern
Agricultural importance
We can utilize residues from crops
for chemicals, energy, & materials
Bioenergy & Biochemicals
U.S. 20% fuel energy and
25% chemicals from biomass by 2030
Efficiency & Cost
Replacement of nonrenewable
petroleum products
US DOE. 2009. Bioenergy Research Centers: An Overview of the Science, DOE/SC0116, US Department of Energy.
Zakzeski et al., 2010
Biomass Process and the Biorefinery
Forestry and
Agricultural
Residues
Pretreatment
Hydrolysis and
Conversion
Chemicals and
Fuels
U.S. DOE. 2006. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda, DOE/SC/EE-0095, U.S. Department of Energy Office of Science and
Office of Energy Efficiency and Renewable Energy. (p. 120)
Structural
Components
Building Blocks
Cellulose
• Crystalline homopolymer
• 6-carbon monomeric
Hemicellulose
• Highly branched heteropolymer
• Both 5- and 6-carbon
monomeric sugars
• Xylan backbone
Lignin
Rubin, E. Genomics of cellulosic biofuels. Nature 454, 841–845 (2008).
Lignin
Monolignols
• P-Coumaryl alcohol (H)
• Coniferyl alcohol (G)
• Sinapyl alcohol (S)
Polymerized
• P-Hydroxyphenyl (H)
• Guaiacyl (G)
• Syringal (S)
U.S. DOE. 2006. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda,
DOE/SC/EE-0095, U.S. Department of Energy Office of Science and Office of Energy Efficiency and
Renewable Energy. (p. 94)
What Pretreatment Does
Breaks apart cell wall
Makes cellulose available
Cellulose can further
break down with enzymes
during hydrolysis
US DOE. May 2007. Biofuels Primer Placemat: From Biomass to Cellulosic Ethanol and
Understanding Biomass: Plant Cell Walls, US Department of Energy Office of Science.
Hydrolysis and Enzymes
Crystalline
Non-reducing End
Reducing End
Amorphous
Glucose
Cellobiose
Oligosaccharides
Beta-Glucosidase
Exoglucanase (Cellobiohydrolase)
Endoglucanase
Lignin
Coalescence
Known to solubilize,
precipitate and
redistribute as droplets
Acts as a physical barrier
Goal of the Study
To analyze lignin droplets on pretreated biomass in order to understand
how the different chemical structures of lignin might impact hydrolysis.
Do certain ratios of lignin composition slow hydrolysis?
Do lignin droplet amount and size differ
between different biomass and temperatures?
A higher syringyl (S) lignin content and larger percent of lignin, will
potentially form more droplets on the cellulose.
Methods
Phase I:
Organosolv
Pretreatment of pure cellulose
Scanning Electron Microscope (SEM)
Biomass
Biomass
Wheat
Straw
Scientific
Name
Triticum
aestivum,
var.
Thunderbird
Sugarcane
Saccharum
Bagasse
spp.
Eastern
Populus
Cottonwood deltoides
(Poplar)
% Glucan
% Xylan
% Total
Lignin
S/G Ratio
Range
33.61
19.3
18.1
0.50-0.69
40.5
22.04
24.4
0.83-1.4
44.6
13.39
27.2
1.30-2.0
Composition of wheat straw, bagasse, and poplar;
Biomass obtained from NIST(National Institute of Standards and Technology)
Organosolv
Hydrophobic ethanol organosolv lignin:
Isolated Lignin
Lignin isolated from Bagasse, Poplar, & Wheat
Straw
1.2% w/w H2SO4 catalyst and 65% Ethanol
190 oC for 60 minutes in fluidized sand bath
Lignin is filtered, precipitated,
centrifuged, and then dried
Quarts Tube Reactors
Pretreatment
Lignin and Whatman #42 ashless
cellulose filter paper
Temp: 120oC,150oC, & 180oC
0.75% w/w H2SO4
10:1 liquid to solid ratio
0.25 mL total in 0.50 mL tube
Pretreated Samples
Cellulose Filter Paper
5 replicates; 45 samples
Samples coated with electrically
conductive film of gold/palladium alloy
using a Sputter Coater
Wheat Straw 120 C at 20,000x magnification
FEI QUANTA
600F
environmental
SEM
Methods
Phase II:
Pretreatment of Whole Biomass
HPLC
Scanning Electron Microscope (SEM)
Slurry
Liquid
Steel Tube Reactors
Pretreated Biomass
HPLC
High Performance
Liquid
Chromatography
Results - Wheat Straw
The larger droplets shown have diameters up to 5.0 µm while
the smallest can go well below 0.10 µm.
Results - Bagasse
Many small droplets at and below 0.10 µm diameter.
Results - Poplar
Many droplets sized from 5 µm to less than 0.10 µm in diameter.
Biomass
Biomass
Wheat
Straw
Scientific
Name
Triticum
aestivum,
var.
Thunderbird
Sugarcane
Saccharum
Bagasse
spp.
Eastern
Populus
Cottonwood deltoides
(Poplar)
% Glucan
% Xylan
% Total
Lignin
S/G Ratio
Range
33.61
19.3
18.1
0.50-0.69
40.5
22.04
24.4
0.83-1.4
44.6
13.39
27.2
1.30-2.0
Composition of wheat straw, bagasse, and poplar.
Results
Showing 3
different lignin
compositions on
pure cellulose
Lignin
Type
Wheat
Straw
Bagasse
Shown at
10,000x
magnification
10µm scale bar
Poplar
120 C
150 C
180 C
Results
Showing 3
different
pretreated
biomass at 3
temperatures
Shown at
10,000x
magnification
Biomass
Type
Wheat
Straw
Bagasse
Poplar
120 C
150 C
180 C
Lignin Morphology
SEM of whole wheat straw pretreated at 150 oC for 30 min; Image 1 showing thicker cell wall
area with droplets; Image 2 showing concentrated small lignin droplets.
Lignin Morphology
SEM of whole wheat straw pretreated at 180 oC for 10 min; Image 3 and Image 4 show the
variation of droplets.
Lignin Morphology
SEM of whole poplar biomass pretreated at 180 oC for 10 min.
Summary
There is a visual difference between
each biomass and at each
Temperature
Larger droplet size did not
correlate to a greater amount of
lignin, S/G ratio, or temperature
Variability in droplet morphology
and size has been observed for
each biomass and temperature
Limitations
Numerous lignin droplets that were much smaller than
originally expected made quantification difficult
There was not enough time for enzymatic hydrolysis
Future Study
Investigating other biomass with similar S/G ratios
Quantitative analysis and image processing of the droplets could
be completed with programming or future automation
Enzymatic hydrolysis should be performed for further analysis
Acknowledgements
Thanks to the Oregon State University
Electron Microscope Facility
John Simonsen, PhD.
Kate Field, PhD.
Wanda Crannell
Glen Li, PhD.
Allex McDaniels
Deepak Kumar, PhD.
Haider Jawad Kadhum
S.M. Hossein Tabatabaie
William Hohehschuh
Ankita Juneja
Lily Xu
Scott Oldfield
USDA funded
OSU Bioenergy
Education Program