Targeted Conversion Research
Biochemical Platform Review
August 7-9, 2007
Michael Himmel
National Renewable Energy Laboratory
This presentation does not contain any proprietary, confidential, or otherwise restricted information
Overview
Timeline
•
•
•
Budget
Project start date – 10/1/2000
Project end date – 9/30/2012
Percent complete – 60%
FY04
FY05
FY06
FY07
FY08
$8,000
$7,000
•
•
•
Bt.C Biomass Recalcitrance
Bt.D Pretreatment Chemistry
Bt.H Enzyme Biochemistry
Partners
•
•
CRADAs: Genencor, Syngenta, Poet,
Edenspace
Subcontracts: Colorado State University,
Colorado School of Mines, Cornell University,
Oklahoma State University, South Dakota
School of Mines, UC San Diego Supercomputer
Center, University Georgia (CCRC), Vanderbilt
University, Weizmann Institute of Science
Stage of R&D
•
A,B – Exploratory & Development Research
Thousands of dollars per year
Barriers Addressed
$6,000
$5,000
$4,000
$3,000
Capital
$2,000
$1,000
Subcontracts
$0
F
Targeted
Conversion
Research
Goals and Objectives
Project Background
– This task was initiated in 2000 and has steadily
adopted a leadership role in critical fundamental
research important for DOE’s near and mid term
goals.
– It is clear that industry is reluctant to conduct
fundamental research and thus DOE led work is
important to both the public and private sectors.
Barriers to Lower Cost
• Feedstock Cost
9 Biomass Pretreatment
9 Enzymatic Hydrolysis
• Fermentation
Overall goals and objectives
– The near term correlative development of more active cellulases and
improved pretreatment processes will ensure attainment of DOE’s
2012 goals.
– This work will ensure the availability of new science knowledge needed
by industry to develop 2017 biorefinery processes.
– To enable the dramatically new technologies providing 60 billion
gallons of bioethanol by 2030, considerable improvement in
understanding of biomass recalcitrance must be achieved in
collaboration with the Office of Science.
Approach
• Detailed description of tasks
– Chemical Process Fundamentals. To develop sufficient understanding of key
barrier related processing challenges to enable the reduction of pretreatment severity as
well as maximization of xylose yields (e.g., > 90%).
– Biological Process Fundamentals.
A) To enable industry to achieve a 2x
reduction (~$0.10/gallon ethanol) in enzyme cost and 90% glucose yield. B) To
determine if non cellulase enzymes, such as xylanases, can be used to improve xylose
yields and reduce costs associated pretreatment.
– Plant Cell Wall Characterization.
To acquire knowledge about the structure
and chemistry of the energy plant cell wall that helps us overcome biomass recalcitrance.
The considerable improvements in optimizing pretreatment conditions and enzyme
performance must be achieved in the context of understanding the cell wall.
• Organization of the tasks
Edenspace
CAFI
CPF
UGa
OSU
Vandelbilt Genecor
Cornell
BPF
BNL
CSM
ADM
PCWC
SDSU
Broin
UCSD
Syngenta
Targets
Explain economic and/or technical metrics used to measure progress.
–
–
–
2012 Target: >90% xylan to xylose and <5% xylan degradation in
integrated pilot operation with >30% solids.
•
Define the relationships between pretreatment conditions and the
chemical ultrastructural changes in corn stover resulting in biphasic
xylan release. (2007)
•
Reduce sugar degradation to <8% by understanding the kinetic
mechanisms that lead to the undesirable degradation products and
then systematically blocking these pathways. (2008)
2012 Target: Validate $0.10/gal ethanol cost of enzyme used in
integrated pilot operation.
•
Generate the first computer model of key enzymes. (2007)
•
Determine how cellulase enzymes move along the cellulose chain
and the respective roles of the different enzyme substructures.
(2008)
•
Define cellulase interactions at the plant cell wall (2010)
2012 target: >85% cellulose (and >85% non-glucose sugars) to
ethanol in a total of 3 days in integrated pilot operation with >20%
total solids.
•
Define the relationships between lignin redeposition and enzyme
kinetics (2009)
Chemical Processing Fundamentals
-Approach
•
We must understand the chemistry and structure of plant
materials changed by pretreatment chemistries and processing
conditions (MS 2007-2010 Pretreatment)
– What governs the hydrolysis and solubilization of
hemicellulose in pretreatments?
– What is the fate of xylan after solubilization (xylooligomers,
xylose, degradation and reversion products)?
– How is lignin redistributed by pretreatment and how does
this affect cellulose digestibility?
– How do pretreatments affect cellulose digestibility (cellulose
crystallinity, accessibility, degree of polymerization,
porosity, particle size)?
– How can pretreatment decrease biomass recalcitrance?
Chemical Processing Fundamentals
Which Anatomical Fractions Are Most Recalcitrant?
Rationale
• Reported differences in pretreatability and
digestibility of corn stover anatomical
fractions having impact on process design.
• Corn husks and cobs released xylose more
easily from dilute acid pretreatment.
• Corn sheaths, leaves, nodes, internodes and
rind released xylose less easily.
• Corn rinds, internodes, and cobs required
higher severity factor to get cellulose
digestion compared to the nodes.
70%
60%
50%
Husks
Cobs
Kramer
Sheaths
Leaves
Nodes
Pith
Internode
Rinds
40%
30%
20%
10%
0%
0.0
0.5
1.0
1.5
Combined Severity Factor (Log Ro - Initial pH)
2.0
Digestibility of Pretreated Anatomical Fractions
90
Cellulose Conversion (%)
Outcome & Relevance
80%
Xylan Solubilization (wt%)
• Define the relationships between variations in
the feedstock composition and key
processing parameters (MS 2009 Feedstock
Interface).
• Does corn stover fraction pre-sorting make
sense?
Fraction of Xylan Solubilized (Total Sugar Basis)
90%
80
70
60
50
40
30
20
10
0
Pith
(0.61)
Rinds
(1.06)
Rinds
(1.86)
Internodes
Leaf
(1.79)
Sheaths
(0.61)
Leaves
(0.61)
Husks
(0.61)
Fraction (Combined Severity Factor)
Cobs
(1.06)
Chemical Processing Fundamentals
How is Lignin Distribution Changed by Pretreatment?
Rationale
•
Define the relationships between
lignin redeposition and enzyme
kinetics (MS 2009
Saccharification)
Outcome & Relevance
•
•
•
•
SEM and TEM of Maize Internodes
Examined temperatures from 80
to180oC in dilute acid PT.
Found EM evidence for
coalescence of lignins within cell
wall at ~140oC followed by
expulsion into liquor.
Redeposition as droplets poses
problems from perspective of
mass transfer and adsorptive
enzyme loss.
This problem may be solved by
process alterations or addition of
lignin adsorbents (proteins, etc).
.
Native ; 1M KMnO4 for 10 min
2% H2SO4; 150 oC for 20 min
Abundant in cell types with secondary cell walls
Intracellular spaces – apoplast
Chemical Processing Fundamentals
What is the Cause of Biphasic Xylan Hydrolysis?
3.0
Rationale
•
Define the relationships between
pretreatment conditions and the
chemical/ultrastructural changes
in corn stover stems (MS 2007
Pretreatment).
Need improved pretreatment
processes with increased yields.
2.5
LN (1/(1-Xylan Conversion))
•
Outcome & Relevance
•
•
•
•
160
160
140
140
Xylan redistribution changes
during dilute acid PT was
observed using xynAb probe!
Implications for mass transfer
limitations within the cell wall
were discovered.
Native
Linkage between lignin migration
and xylan hydrolysis probable.
Impact on process design
includes PT soak time,
temperature fine tuning, etc.
C, 1.2% H2SO4
C, 0.8% H2SO4
C, 1.2% H2SO4
C, 0.8% H2SO4
2.0
1.5
1.0
0.5
0
2
4
6
8
10
Pretreatment Time (min)
1st Order Kinetic Plots of Xylan Conversion
Xylan redistribution at 140° C
2m
10 m
12
Chemical Processing Fundamentals
How Do Pretreatments Affect Cellulose Digestibility?
13C Solid
Differences in
Rationale
• Define cellulase interactions at the
plant cell wall (MS 2010 Enzyme
Production).
• Directly impacts an improved
pretreatment process, increased
sugar yields, and digestibility of
pretreated biomass.
Outcome & Relevance
• Studied changes in cellulose and
biomass properties caused by dilute
acid pretreatment
• Determined that pore size distribution
is likely of lesser importance for
digestion.
• Determined that crystallinity, reducing
ends, particle size, xylan/lignin
compositions are important.
State NMR crystalline/amorphous cellulose
Digestibility 73%
97% xylan removed
lignin
C1
C4
C6
OCH3
Digestibility 73%
90% xylan removed
Digestibility 67%
85% xylan removed
Digestibility 43%
68% xylan removed
200
150
100
ppm 50
0
Chemical Processing Fundamentals
How Can We Improve Yields of Solubilized C5?
Rationale
xylose to furfural: quantum MD
•
•
To understand sugar degradation
pathways occurring in parallel to xylose
formation during pretreatment (MS 2009
Pretreatment).
Require understanding of two critical
competing pathways for xylose
(dehydration and reversion reactions)
Outcome & Relevance
•
•
•
Experimentally measured competing
reactions for xylose.
Computationally calculating the kinetic
and thermodynamics constants for
these reactions.
Discovery that furfural product formation
are exponentially temperature and
linearly pH dependent has process
design implications.
OH
OH
OH
OH
CHOH
H2O
O
O
OH
OH
OH
OH
H2O
OH
OH
OH
OH
OH
H2O
OH
O
O
O
H2O
H2O
Biological Processing Fundamentals
-Approach
•
Cellulase cost reduced to
~$0.10/gal EtOH (MS 2011
Enzyme Production)
– Need deeper understanding
of enzyme action to meet
DOE’s goals (FY08 Enzyme
Solicitation).
Protein engineering
for improvements
– Cel7A is most critical
cellulase component enzyme;
however, the mechanism of
this enzyme is not known.
Corrected model
– What is the biochemical
relationship between structure
and function?
– What are the experimental
strategies for improving
Cel7A?
– What model of cellulose best
Protein Tomography and XRD
fits the cell wall microfibril?
Approach
Classical biochemistry
Imaging
Computational science
Bioinformatics
Molecular dynamics
models
Cellulose (cell wall)
modeling
Experimental
design
Experimental
observations
Testing key mutations
suggested from modeling
Biological Processing Fundamentals
•
Although closely related, not all Cel7A
enzymes are alike!
– Activity - P. funiculosum is more
than 2X more active than T. reesei
enzyme.
– Stability - Cel7A from P.
funiculosum is also more thermal
stability
Percent conversion of corn stover
Comparative/bioinformatics approach to understanding action
Rationale
•
20
Native T. reesei Cel7A
recombinant T. reesei Cel7A
1/2 loading P. funiculosum Cel7A
P. funiculosum Cel7A
0
20
40
60
80
100
120
140
Time (hours)
5e-5
T. reesei Cel7A
P. funiculosum Cel7
66.8oC
65.0oC
4e-5
3e-5
Cp (cal/oC)
•
Identified several key Cel7A enzymes
that display divergent specific activity.
Initiated a plan to take advantage of
natural diversity toward the
understanding structure/function of
cellobiohydrolases.
New diversity information benefits
industry’s enzyme engineering efforts.
pH 5.0, 38oC:
CBH 27.8 mg/g cellulose
E1cd 1.13 mg/g cellulose
40
0
Outcome & Relevance
•
60
2e-5
1e-5
0
-1e-5
56
58
60
62
64
66
68
o
Temperature C
70
72
74
Biological Processing Fundamentals
Cellobiohydrolase database: applying bioinformatics tools
Rationale
Cel7A Sequence Dendrogram
•
Evaluation of naturally evolved
homologous sequences to identify
key residues
–
–
–
Extract mutational data to identify
residues important to protein function
Correlate residue conservation to
functional importance
Distinguish between functional
residues and residues that are
important for maintaining structural
integrity
PF
TR
Outcome & Relevance
•
•
•
Identified loop regions that evolved
naturally more rapidly.
Identified highly conserved residues.
Permits basis for engineered
evolution improvements.
PC
Evolutionary trace of Cel7A enzymes
Biological Processing Fundamentals
Molecular Dynamics Modeling of Cellulases
Rationale
•
CBH I with microfibril
Develop understanding of CBH I action
through MD simulation combined with
mutational studies.
Outcome & Relevance
Binding Domain
Catalytic Domain
• Built first computational
model of entire enzyme.
• Permits initiation of
mechanistic model
• Model will encourage or
discourage, but inform,
enzyme engineers.
Biological Processing Fundamentals
Family One Cellulose Binding Modules
Rationale
•
•
CBM’s play a major role
of improving activity of
enzyme against insoluble
substrates.
Pretreatment may impact
cellulase action through
altered cellulose
morphology.
Outcome & Relevance
•
•
•
New functional model of Family
one CBM’s developed.
Currently testing site directed
modifications of CBM’s based on
new computational model.
This work should tell us if CBM
can be improved by enzyme
engineering.
Biological Processing Fundamentals
Understanding the Catalytic Domain
Rationale
•
•
The processive mechanism for the
CD of Cel7A is not known. Can it
be improved?
What role does water play in this
process?
Outcome & Relevance
•
•
•
Models of the active site tunnels of
key CDs have reveled differences
in both surface area and volume of
the active site tunnel.
Contours of water density inside
the active site tunnel of the
catalytic domain of Cel7A may
also impact activity.
Knowledge of processive action of
the CD tunnel will aid researchers
interested in improving enzyme
activity.
The protein surface is rendered in purple, and the white
clouds are water density contours (arbitrary cutoff),
calculated between 500 and 700 ps, with the protein
structure taken as that at 500 ps
Biological Processing Fundamentals
Cellulase/Hemicellulase Synergy
Enhancement of Optimal Cel7A Loading on Hot Water Pretreated Corn
Stover by Addition of Non-Cellulolytic Activities
Rationale
– vary by pretreatment
chemistry/severity
– feedstock dependent
Outcome & Relevance
•
160.00
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
Cel7A
Xylanase is the key accessory
enzyme tested
– synergy w/ esterases is strong on
substrates pretreated at low/mild pH
– Enzymatic xylan removal directly
improves cellobiohydrolase
performance
•
glucan to glucose
glucan to cellobiose
180.00
mg/g biomass
Obtain/identify activities and
synergies key to specific substrates
(MS 2009 Pretreatment)
Correlate hemicellulase and
accessory enzyme impact on
cellulase activity
– linear dependency between xylan
removal and cellulose hydrolysis
+XynA
+Axe1
+FaeA
+Axe1
+XynA
+FaeA
+XynA
+XynA
+Axe1
+FaeA
Correlating Enzymatic Xylan Removal to Glucan Conversion
200.00
Enzymatic Glucan Conversion (mg/g
biomass)
•
200.00
180.00
160.00
140.00
120.00
R2 = 0.9971
100.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Enzymatic Xylan Conversion (mg/g biomass)
10.00
11.00
12.00
Plant Cell Wall Characterization
-Approach
Rationale
•
•
•
•
•
Developing new methods for
biomass characterization
Imaging molecular structure of
plant cell wall
Mapping structural changes
during pretreatments
New labeling techniques for
cellulases acting on cell wall
Generating hypotheses for
testing
What do
microfibrils
really look
like?
Biomass Surface
Characterization Lab (BSCL)
AFM
TIRF
TEM
SEM
CFM
Plant Cell Wall Characterization
Outcome & Relevance
• Molecular Labeling
– Map cell wall by
molecular
recognition
– Limited by
availability of probes
and knowledge of
specificity
– Holds promise for a
new correlative
imaging approach
where chemical
mapping is needed.
Plant Cell Wall Characterization
Outcome &
Relevance
• Single Molecule
Tracking
– Determine linear
rate of motion of
cellulases for
model.
– Determine
association of
critical enzyme
components.
– Uncertainty in
affects of protein
modification
must still be
resolved.
Plant Cell Wall Characterization
Outcome & Relevance
•
•
•
•
•
Imaging plant cell wall
structure at the nanometer
scale by AFM
New concepts for the cell
wall microfibril.
Affects of in-field drying
reflected in aggregation of
elementary fibrils in cell
wall.
Some PT practices also
leave microfibrils in
aggregated state.
Can we actually visualize
residual matrixing
polymers and apply to PT
improvement?
Field dried Corn
stover parenchyma
cell wall
Never dried maize
coleoptile scanned
under water
Only 3 nm!
Plant Cell Wall Characterization
New Models for the structure of cellulose in the cell wall microfibril
Outcome
• Challenges to
existing dogma
regarding cellulose
structure are
emerging!
New ultra-sharp
probes
Critical Success factor
and/or Show-stoppers
•
Explain potential show-stoppers
– Dramatic reduction in the estimated cost of feedstocks or introduction of
superior genetically modified plants would eliminate need for pretreatment.
– Successful demonstration that CBP is effective and economic could replace
current SSCF paradigm.
– Our effort to seek fundamental understanding of pretreatment and enzyme
digestion processes may determine that the known conversion steps are near
theoretical and cannot be improved to the degree predicted.
•
What is the duration of the window of opportunity?
– The advent of the BP Center and the OBER Centers (the “1+3 Centers”)
demonstrates the need enhanced fundamental studies in the next 10 -15
years.
– Note that the DOE BER Biomass Research Centers were directed to operate
as “Startup Biotech Companies” where R&D plans work towards specific
business objectives.
•
What are the critical performance parameters that must be met
for the project to be considered successful?
– The 2012 technology technical targets for cellulose conversion to glucose,
glucose conversion to ethanol, xylan conversion to xylose, xylose conversion
to ethanol, cellulase cost, fermentation time, and solids concentrations for
pretreatment.
– Future targets for 2017 and 2030 technology.
Future Work
•
Next 14 months we intend to:
– Confirmed that in dilute acid pretreatment, hemicellulose/lignin migration and reprecipitation is both necessary and problematic (this may be attenuated by
engineering approaches).
– Confirm that cellulose in microfibrils undergoes undesirable “hardening” reactions
upon dehydration and as consequence of most chemical pretreatments.
– Show that the formation of non fermentable sugars from reversion reactions can
impact process yield and must be deeply understood.
– Show that the key cellulase enzyme, Cel7A, which appears to display limited
natural activity diversity (range as yet unknown) benefits from protein engineering.
– Demonstrate that AFM and other advanced imaging tools hold promise for
enhancing our understanding of the conversion processes.
•
Highlight upcoming key milestones (FY07)
– 30x30 DMS: Define the relationships between pretreatment conditions and the
chemical/ultrastructural changes in CS that result in biphasic xylan hydrolysis.
– 30x30 DMS: Generate first computer model of key enzymes.
•
Address how you will deal with any decision points during that time
– Imaging studies of new feedstocks will require new base line studies and guidance
regarding initiation of this work will be sought from DOE.
– The choice of pretreatments currently conducted at NREL is limited primarily to
dilute acid and this may changes depending on new findings from CAFI.
– New enzymes entering the public sector (and thus available for testing) may affect
the direction of enzyme research.
Summary
Summarize the key points
•
We seek to understand the scientific basis for biomass recalcitrance
at the level of the plant cell wall. We will use existing and develop
new chemical and biophysical mapping tools to generate models for
cell wall deconstruction.
•
We plan to elucidate chemical mechanisms for parasitic reactions of
sugars. Make recommendations to teams conducting pretreatment
scale up for implementation of possible solutions.
•
We intend to understand the mechanism of cellobiohydrolase
enzymes so that industry can improve cellulase cocktail
performance.
•
We will evaluate mixed chemical/biological pretreatments and
provide insights regarding biomass handling, storage, pretreatment,
and saccharification that can reduce conversion cost.
TCR
• END
• Following slides are for Appendix
Outline
•
•
•
•
•
•
•
•
•
Background, Goals and Objectives
Scope of Work
Targets
Feasibility and Risks
Accomplishments
Issues to Overcome
Critical Issues
Planning
Summary
Accomplishments
•
Accomplishments
– Targeted Conversion Research
• Chemical Processing Fundamentals (CPF)
• Biological Processing Fundamentals (BPF)
• Plant Cell Wall Characterization (PCWC)
Feedstock
Process
Interface
Pretreatment &
Enzymatic
Hydrolysis
Biochemical
Processing
Integration
Targeted
Conversion
Research
Enzyme
Solicitation
Validation
Biochemical
Platform
Analysis
Critical Targets (T) and Activities
T1:
Complete evaluation
of alternate pretreatments
T3:
Optimized accessory
enzymes developed
T2:
Pretreatment yields
better than 75% xylose
1
2007
2008
Strain contract(s) start
2
2009
T4:
2-5x reduction in
cellulase cost to
$0.10/gal
3
2010
Feedstock systems identified
to provide $45/ton to reactor
T5:
Robust co-fermenting
strain with 85% C5 & 95%
C6 yields
4 5
2011
G
2012
Pilot runs
commence
Enzyme contract(s) start
Project target
Project activity
Feedstocks target
Goal:
State of Technology case with pilot
data resulting in $1.31/gal ethanol
with model $44/ton feedstock
The Power of the Combined Approach
Targeted Conversion Research
-Publications 2006-2007
Decker, SR, Sheehan, J, Dayton, DC, Bozell, JJ, Adney, WS, Hames, B, Thomas, SR, Bain, RL,
Czernik, S, Zhang, M and Himmel, ME. Biomass Conversion, in Reigel’s Handbook of
Industrial Chemistry,” (J.A. Kent, Ed.), Kluwer Academic/Plenum Publishers, New York,
~200 pages, 2007, In press.
Jeoh, T, Ishizawa, CI, Davis, MF, Himmel, ME, Adney, WS, and Johnson, DK. Cellulase Digestibility
of Pretreated Biomass is Limited by Cellulose Accessibility, Biotechnol. Bioeng, 2007, In
press.
Zhang, Y-HP, Ding, S-Y, Mielenz, JR, Cui, J-B, Elander, RT, Laser, M, Himmel, ME, McMillan, JR,
and Lynd LR. Fractionating recalcitrant lignocellulose at modest reaction conditions.
Biotechnology and Bioengineering 2007, in press.
Porter, SE, Donohoe, BS, Beery, KE, Xu, Q, Ding, S-Y, Vinzant, TB, Abbas, CA, Himmel, ME.
Microscopic analysis of corn fiber using starch- and cellulose-specific molecular probes.
Biotechnology and Bioengineering 2007, in press.
Nimlos, MR, Matthews, JF, Crowley, MF, Walker, RC, Chukkapalli, G, Brady, JW, Adney, WS,
Cleary, JM, Zhong, L, and Himmel, ME. Molecular Modeling Suggests Induced Fit of Family
I Carbohydrate Binding Modules with a Broken Chain Cellulose Surface, Protein
Engineering and Design 2007, In press.
Himmel, ME, Ding, S-Y, Johnson, DK, Adney, WS, Nimlos, MR, Brady, JW and Foust, TD. Biomass
Recalcitrance: Engineering Plants and Enzymes for Biofuels Production. Science 2007, 315,
(5813), 804-807.
Ishizawa, CI, Davis, MF, Schell, DF, and Johnson, DK. Measurement of Porosity in Dilute Sulfuric
Acid Pretreated Corn Stover, J. Agr. and Food Chem. 2007, 55(7), 2575-2581.
Decker, SR, See, CH, Himmel, ME and Williford, CW. Characterization of Lignin Using Multi Angle
Laser Light Scattering and Atomic Force Microscopy, Analytica Chimica Acta 555(2): 250258, 2006.
Gidh, A, Decker, SR, Vinzant, TB, Himmel, ME and Williford, CW. Determination of Lignin by
HPSEC using Multi Angle Laser Light Scattering, J. Chromatogr. A 1114(1), 102-110, 2006.
Ding, S-Y, Xu, Q, Ali, MK, Baker, JO, Bayer, EA, Barak, Y, Lamed, R, Sugiyama, J, Rumbles, G,
and Himmel, ME. Versatile derivatives of carbohydrate-binding modules for imaging of
complex carbohydrates at the molecular level of resolution. BioTechniques 2006, 41(4),
435-443.
Ding, S-Y, and Himmel, ME. The Maize Primary Cell Wall Microfibril: A New Model Derived from
Direct Visualization. J. Agric. Food Chem. 2006, 54, 597-606.
Nimlos, MR, Qian, X, Davis, M., Himmel ME, and Johnson, DK. Energetics of Xylose
Decomposition as Determined Using Quantum Mechanical Modeling, J. Phys. Chem. A,
2006, 110, 11824-11838.
Targeted Conversion Research
- Blackwell Book
Chapter 1 – Introduction – Michael E. Himmel
Chapter 2 - The Biorefinery - Thomas D. Foust, Kelly N. Ibsen, David C. Dayton, J.
Richard Hess, Kevin E. Kenney
Chapter 3 - Anatomy and Ultra Structure of Maize Cell Walls: An Example
Energy Plant - Shi-You Ding and Michael E. Himmel
Chapter 4 - Chemistry and Molecular Organization of Plant Cell Walls - Philip J.
Harris and Bruce A. Stone
Chapter 5 - Cell Wall Synthesis - Debra Mohnen, Maor Bar-Peled, and Chris
Somerville
Chapter 6 - Structures of Plant Cell Wall Celluloses - Rajai H. Atalla, John W.
Brady, James F. Matthews, Shi-You Ding & Michael E. Himmel
Chapter 7 - Lignins: A 21st Century Challenge - Laurence B. Davin, Ann M.
Patten, Michaël Jourdes, and Norman G. Lewis
Chapter 8 - Computational Approaches to Study Cellulose Hydrolysis - Michael F.
Crowley and Ross C. Walker
Chapter 9 - Mechanisms of Xylose and Xylooligomer Degradation During Acid
Pretreatment - Xianghong Qian and Mark R. Nimlos
Chapter 10 - Enzymatic Depolymerization of Plant Cell Wall Hemicelluloses Stephen R. Decker, Matti Siika-aho, and Liisa Viikari
Chapter 11 - Aerobic Microbial Cellulase Systems - David B. Wilson
Chapter 12 - Cellulase Systems Of Anaerobic Micro-Organisms From The
Rumen And Large Intestine - Harry J. Flint
Chapter 13 - The Cellulosome: A Natural Bacterial Strategy to Combat Biomass
Recalcitrance - Edward A. Bayer, Bernard Henrissat, and Raphael Lamed
Chapter 14 - Pretreatments for Enhanced Digestibility of Feedstocks – David K.
Johnson and Richard T. Elander
Chapter 15 - Understanding the Biomass Decay Community - William S. Adney,
Daniel van der Lelie, Alison Berry, and Michael E. Himmel
Chapter 16 - New Generation Biomass Conversion: Consolidated Bioprocessing Y.-H. Percival Zhang and Lee R. Lynd
Biomass
Recalcitrance:
Deconstructing the Plant Cell
Wall For Bioenergy
M.E. Himmel, Ed., Blackwell Publishing,
London, UK, est. 2007
TCR – Key Reviewers Comments 2004
Summary of Recommendations:
“A plan for the project that clearly identifies the work to be performed in each task and how
they integrate to a single project.” Beginning in FY05, wording in the AOP defines the
demarcation between Tasks and Subtasks more clearly. In FY06, joint milestones brought
together critical resources for conducting cross cutting work.
“Although most of the proposed work is unique, much related work has been done or is being
done. The available resources should be leveraged by development of collaborations with
academic, government, and industrial scientists.” Work proposed for FY06-07 is entirely
unique. The reviewer is referring to plant structure studies and we propose to conduct work that
leverages known aspects of plant structure against unknown relationships between enzymes,
pretreatment, and sugar yield. To help us understand more about what is known regarding plant
structure, we are collaborating with Chris Somerville (Stanford) and Alan Darvill (CCRC
UGA); as well as 8 other subcontractors with expertise ranging from biochemistry to botany.
“Much of the work is centered around surface characterization. Well annotated images should
be available, from a relational database, to the public over the internet.” The construction of
such a data base of images in underway at NREL in the BSCL.
“Quarterly updates of progress in the Fundamentals and New Concepts Project should be
generated and shared with the interested public.” We strive to continue an excellent publication
and public presentation record for this purpose.
Targeted Conversion Research
• Backup Accomplishment cases
Biological Processing Fundamentals
Fundamental research has directly impacted industrial progress
Rationale
•
Cellulase cost reduced to ~$0.10/gal
EtOH (MS 2011 Enzyme Production)
Outcome
•
•
•
•
CRADA collaborations with
Genencor International led to the
development of a new, hyper
effective commercial cellulase
preparation, called Advanced
Biomass Conversion (ABC)
Cellulase.
NREL contributed a bacterial,
thermal tolerant endoglucanase EI to
the fungal enzyme mixture.
The gene coding this enzyme was
used to transform a leading
commercial T. reesei strain.
This work was recognized by an
R&D 100 Award in 2004.
EI
GH5
CBDI II
GH12
CBDII
CBDI I
GH6
PS-rich
Linker
CBDII I
GH48
CBDI II
GH74
Chemical Processing Fundamentals
How Can We Improve Yields of Solubilized Sugars?
Rationale
•
•
xylobiose: barriers to reaction
To understand sugar degradation
pathways occurring in parallel to
xylose formation during pretreatment
(MS 2009 Preteatment).
Require understanding of two critical
competing pathways for xylose
(dehydration and reversion reactions)
Ea = 17.8 kcal mol-1
OH
OH
O
Ea = 19.8 kcal mol-1
•
•
Experimentally measured formation of
reversion products from xylose (~10%
= 20 g/L from a high solids
hydrolysate)
Computationally calculating the kinetic
and thermodynamics constants for
these reactions.
Determined that reversion products
formation are strongly temperature
and reactant concentration dependent
OH
O
OH
Ea = 0.0 kcal mol-1
hydrolysis
Outcome
•
Ea = 4.7 kcal m
OH
O
Ea = NA
Metadynamics
F
OH
Biological Processing Fundamentals
Cel7A glycosylation: Steric hindrance by hyperglycosylation?
•
•
Investigate “aging effects”
for T. reesei cellulase
preparations.
Heterologous expression
systems will be used for
enhanced enzyme
production.
Outcome
•
•
•
Showed that glycosylation
and “hyper” can impact
activity and stability
Identified key N-linked
glycan sites and eliminated
deleterious effects through
protein engineering.
Demonstrated that the Olinked linker regions can be
interchanged.
Cellulose Conversion (%)
Rationale
25
20
15
10
native T. reesei Cel7A
recombinant Cel7A
recombinant N384A
5
0
0
20
40
60
80
100
Time (hours)
CHARMM simulation
8 mannose residue on N384
Active site tunnel shown with cellotetraose (yellow)
120
Biological Processing Fundamentals
Cellobiohydrolase database: applying bioinformatics tools
Rationale
•
•
Loop 1
Identify un-conserved loops between
key domains
Correlate loop architecture with
activity
Outcome
•
Identified loop regions that may
contribute to increased activity and
stability
–
–
Engineered loop 1 out of P.
funiculosum and inserted similar loop
into T. reesei Cel7A
Engineered loop 2 out of T. reesei and
inserted into P. funiculosum Cel7A
Loop 2
Plant Cell Wall Characterization
• Mapping cell wall
structure changes
during pretreatment
Native
H2SO4,, 140 ºC
H2SO4,, 200 ºC
• TEM tomography of
lignin migration during
dilute acid
pretreatment
100 nm
ce l
lw
all
su
lignin droplets
mid
d
le l
am
e ll a
rfa
c
e
co
a
les
ce
d
lig
ni
n