Download - 2011 Highlight Slides
Center for Direct Catalytic Conversion
of Biomass to Biofuels (C3Bio)
RESEARCH PLAN AND DIRECTIONS
We will maximize the energy and carbon efficiencies of advanced biofuels production by the
design of both thermal and chemical conversion processes and the biomass itself. Impacts
are to more than double the carbon captured into fuel molecules and expand the product
range to alkanes and other energy-rich fuels.
C3Bio develops transformational knowledge
and technologies for the direct conversion
of plant lignocellulosic biomass to
advanced (drop-in) biofuels and other
biobased products, currently derived from
oil, by the use of new chemical catalysts
and thermal treatments.
Mahdi Abu-Omar and Hilkka Kenttämaa / Department of
Chemistry, Purdue University
Catalytic conversion of lignin
Lignin is a major component of lignocellulosic
biomass. It is an aromatic rich polymer that is
essential for plant’s life. Lignin poses the problem of
recalcitrance as well as an opportunity for making
aromatic-rich liquid fuels and valuable chemicals. A
desirable catalyst is one that can depolymerize lignin,
remove oxygens, and retain the aromaticity. Another
challenge in this area is the analysis of complex
mixtures. We have developed a catalyst Zn/Pd/C that
cleaves aromatic ether linkages while leaving the
aromatic group unscathed. We have also implemented
mass spectrometry methods that enable the
quantitative analysis of lignin products. We are now
poised to apply these methods of catalysis and
analysis to engineered lignin biomass.
OH
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We are involved in the synthesis and application
of organic-inorganic hybrid materials that
ultimately will become single site catalysts.
Using a well developed synthetic methodology,
we have created high surface area catalysts
functionalized with aryl sulfonic acids. These
catalysts are being tested for their ability to
hydrolyze cellobiose into glucose. This is a
model study that has implications for the
eventual conversion of cellulose from biomass
into viable fuels and other high value chemicals.
In a parallel line of research, we are
investigating the selective oxidation of lignin
models to produce quinones which may be
easily transformed into value added chemicals.
We are exploring a number of titanium-on-silica
catalysts created through targeted synthetic
methods that will allow for the determination of
which active site is optimal for oxidation. Early
results have been promising for conversion of
the lignin models to benzoquinones in high yield
and with good selectivity.
C Barnes, J Abbott, D Taylor, S Chen - Univ. of Tennessee, Knoxville
Catalytic hydrolysis of cellulosic materials
& selective oxidation of lignin models
A Olek1, S Ding
2, B Donohoe
2, L Makowski
3, L Paul
1, and N Carpita
1
Biochemical mechanism of cellulose synthesis
• Synthesis of cellobiose units eliminates the steric problem of iterative
synthesis of a single unit. because the O-4 would always be in the
same location in the non-reducing end of the growing chain.
• A channel of 8 x 2 = 16 membrane spanning domains would be
equivalent to callose synthase and most sugar transport proteins.
• The dimer produces two Zn-finger domains to recruit into larger
complexes.
1Purdue University,
2NREL,
3Northeastern University/ANL
The 55 kDa catalytic domains of CesA
spontaneously dimerize when a thiol-reducing
agent is depleted from the reaction mixture.
The dimerization is reversible and can be
shown by high-performance size-exclusion
chromatography, analytical ultracentrifugation,
atomic-force microscopy, and X-ray scattering
experiments.
The 55 kDa monomer is predicted by WAXS to be 30.0Å,
where the 110 kDa dimer is a more spherical 34.0Å. The
ratio of the monomer : dimer estimates a distance
between centers of mass to be 41.3Å
J I Kim and C Chapple, Purdue University
Understanding cell wall assembly using
Arabidopsis lignin mutants
Lignin is a major component of the plant
cell wall and understanding how, when and
where it is deposited is critical to being
able to catalyze its conversion to useful
products such as biofuels.
We have capitalized on our suite of lignin-
deficient mutants of Arabidopsis to
generate plant lines in which lignin
biosynthesis, which is normally blocked in
these mutants, can be turned on by
application of a chemical inducer. Normally,
lignin deficiency leads to dwarfing, but
when lignification is induced in these lines,
they again grow normally. We are now
using this system to study the early stages
of lignification, where lignin is first
deposited and how cell wall assembly is
altered when lignification is uncoupled
from cell wall polysaccharide synthesis.
-DEX +DEX
C4H-deficient Control
ControlC3’H-deficient
C4H-deficient
C3’H-deficient
“Research Goes to School”
An outlet for EFRC science
K Clase, K Goodpaster, O Adedokun, L Kirkham, P Ertmer, G Weaver, M Abu-
Omar, N Carpita, H Kenttämaa, M McCann and N Mosier, Purdue University
In June 2011, 21 in-service and pre-service teachers participated in an intensive 2-week
workshop From Field to Fuel - The Science of Sustainable Energy to help educators develop
biofuels curricula specifically to increase the relevance of STEM subjects for rural students.
“Research Goes to School” is an NSF Innovations through Institutional Integration grant to
Purdue in collaboration with the Woodrow Wilson STEM Goes Rural Initiative, National Rural
Education Association, I-STEM Resources Network, and Purdue Rural Schools Network.
C3Bio investigators Abu-Omar, Carpita, Kenttämaa,
McCann and Mosier assisted Dr. Clase through
presentations on their state-of-the-art research in
advanced biofuels. McCann is a co-PI on the NSF grant.
The teachers developed problem-based learning units
for classroom curricula, mapped to educational
standards using C3Bio content.
The educators completed pre- and post- science teaching self-efficacy and
content knowledge measures, and participated in a post-workshop focus
group. Preliminary results indicate that the workshop enhanced participants’
knowledge of biofuels concepts and their beliefs that student learning can be
influenced by effective teaching. Furthermore, participants expressed that
the workshop enhanced their understanding of the applications of biofuels
concepts to STEM content areas and enhanced their sense of purpose for
teaching.
P Ciesielski, J Matthews, M Crowley, M Himmel, B Donohoe (NREL)
• Transmission electron tomography is used to obtain
3D data sets (tomograms) of thermochemically
deconstructed plant cell walls. A single slice from a
tomogram (top left) shows 2 intertwined cellulose
microfibrils.
• The geometry of the microfibrils is determined by
fitting parametric equations to the 3D dataset (top
right).
• Atomistic, macromolecular models (bottom) are
constructed by building the molecular structure of
cellulose around the determined geometry of the
microfibrils.
• These structures will allow for molecular dynamics
simulations that more accurately reflect the structure
of biomass and are highly relevant to real processing
conditions.
Macromolecular modeling of cellulose
microfibrils from electron tomography
H Kenttämaa, M Abu-Omar / Purdue Univ.
HPLC/MS analysis of degradation
products of lignin model compounds
HPLC
chromato-
graph
(detection
by MS)
Retention Time:
6.35 min
Retention Time:
7.75 min
Retention Time:
10.82 min
The development of chemical methods for the direct
catalytic conversion of biomass to high value organic
molecules is an area of increasing interest. The plant
matter component known as lignin is a polymer
containing many aromatic rings. Hence, it could
provide a means of obtaining aromatic chemicals
currently derived solely from petroleum. We have
developed a catalytic system that selectively breaks
down dimeric lignin components. A high-pressure
liquid chromatography tandem mass spectrometric
(HPLC/MSn) method was devised for the determination
of the products of these catalytic reactions. This
method first separates the degradation products and
subsequently ionizes all components for detection by
mass spectrometry, yielding molecular weight
information. In MSn experiments, the ions are
subjected to several consecutive collision-activated
dissociation (CAD) steps to determine their structures.
0.1 eq 5% Pd/C
0.1 ZnCl2300psi H2
MeOH,150°C, 8h
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-CH3
-CH2 CH2OH
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MSn spectra: consecutive CAD
of ions of m/z 181, 166 and 121
Mass/Charge80 100 120 140 160 180 200
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MW 182 Da
Mass spectra
MW 124 Da
MW 166 Da
L Makowski, H Inouye (Northeastern); R Harder, J Lal (Argonne);
L Yang (Brookhaven)
in situ analysis of cellulose crystallite structures
Data collection at 34IDC (APS)
Bragg peak from
single crystallite
Image of crystallite
from maize
SAXS data from X9 (NSLS)
CDI - Coherent diffraction imaging of cellulose crystals in situ has
been used to demonstrate that acid pretreatments lead to changes in
cellulose morphology.
SAXS - small-angle x-ray scattering - provides quantitative estimates
of the average length and diameter of the crystallites.
Comparative studies of the effect of treatments on a variety of
cellulosic materials are underway.
N Mosier, M Abu-Omar, Purdue Univ.
Ultraselective catalysts for
hydrolysis of cellulose
The major goal of this project is to develop catalytic
processes that enable the extraction, fractionation, and
depolymerization of carbohydrates from biomass into
aqueous solutions. The secondary goal is to couple this
catalytic fractionation/depolymerization with catalytic
transformation of carbohydrates into hydrocarbons and
valuable chemicals.
A new reaction method (microwave heating) was validated
against results obtained from previously used reaction
method (sand bath heating). The data indicate that the
results between the two methods were not different to any
statistical significance. The new method offers advantages
of more rapid and accurate temperature
Detailed kinetics of xylose and furfural degradation in the
presence of maleic acid (250mM) allow for optimization of
conditions to achieve high yields of furfural directly from
biomass.
Reaction of xylose with furfural was minimal when maleic
acid is the catalyt, in contrast to significant coupling
reactions when sulfuric acid is used.
Switchgrass
Xylose
Xylose
Recovery
(%)
Selectivity
(%)
Xylose
Conversion
(%)
Furfural
Yield (%)
HPLC
1st Run
2nd
Run
3rd
Run
> 90
> 85
>85
67
65
60
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80
57
51
48
Recycling
Maleic Acid
to Convert
Switchgrass
Xylose to
Furfural
Kinetic
Modeling
M Easton, J Nash, Purdue University
Levoglucosan May Not Be the Product of
Fast Pyrolysis of Cellulose
For many years, levoglucosan has been thought to be the
product of fast pyrolysis of cellulose. High level calculations
show that a number of isomers of levoglucosan are considerably
more stable than levoglucosan itself. The correct assignment of
the cellulose pyrolysis product is essential for designing practical
and efficient methods for its conversion to biofuel.
Note: G4MP2 relative free energies (kcal/mol) are shown in black and red.
H Yang, G Ma, X Liu, A Murphy, W Peer, Purdue University
Expression of metal-binding proteins in cell
walls as Trojan horse catalysts
Engineering peptide transporters & metal binding peptides to deliver
iron catalysts to the cell wall for biomass conversion
• Transition metal accumulation in living biomass: Generate
transgenic plant materials that express genes encoding
plasma membrane-localized metal transporters.
• Significance: Rice lines that lack a metal transporter have been
generated. The lines will be tested for Fe accumulation in the cell wall.
This is enhance biomass to biofuel catalysis.
• Engineer catalysis-enhancing proteins: Construct transgenes
that encode chimeric proteins with specific metal binding
peptide motifs and have affinity for particular wall
components, in order to target metal ions more specifically
within the structure of the cell wall.
• Significance: Iron binding peptides (IBP) that can bind Fe at cell wall
pH (pH 5.5) have been identified.
• Significance: Carbohydrate binding motifs (CBMs) targeted to the cell
wall have been identified. These motifs will be combined with the IBP
and the minimal secreted Fe-binding peptide.
• Identify cellular delivery routes to enable the secretion/self-
assembly of catalyst-ready tailored biomass.
• Significance: A secreted metal-binding protein is being modified to
bind Fe instead of Zn. The minimal protein required for secretion is
being identified.
Carbohydrate Binding Module (CBM) Fluorescently tagged, targeted to cell wall
Iron Binding Peptides (IBP)
DLGEQYFKG & LAEEKREGYER
pH
5.5
p
H 7
.0
F Ribeiro, H Kenttämaa, Purdue University
Fast pyrolysis for direct production
of molecules in the fuel range
Pyrolysis, heating to temperatures where biomass forms a
gas and then condenses to form a bio-oil, is a relatively
simple process for fuel production. However, the bio-oil
contains too much oxygen and is corrosive. We need to
reduce the oxygen content of bio-oil as it forms, and also
the huge range of undesirable products, in order to make an
energy-rich fuel. We have developed a method to measure
the reaction products in the gas phase by mass
spectrometry. Using a pyroprobe instrument with which the
rate of heating and final temperature can be precisely
controlled, we can evaluate the reaction products from
cellulose and other model compounds in the presence of
hydrogen or other gas. Instead of the thousands of
products observed during conventional pyrolysis of
cellulose, we observe a few discrete masses using fast-
hydropyrolysis (a heating rate of 1000K per second in the
presence of hydrogen). We can control the types of
products by varying gas temperature and flow rate.
Removing the unwanted oxygen from biomass may now be
feasible to produce diesel-like products.
J Madden, G Simpson, Purdue University
Compact second harmonic generation (SHG)
microscope for combined optical and x-ray
analysis
•Synchrotron sources provide high X-
ray intensities, enabling nanoscale
characterization of biomass cellulose
structure over a limited field of view.
Complementary methods for rapid initial
characterization over large fields of view
are under development to guide
positioning for X-ray analysis and
improve the overall throughput.
•Second harmonic generation (SHG), or
the frequency doubling of light,
provides highly selective contrast for
crystalline cellulose domains.
X-ra
y C
CD
Dete
cto
r
Synchro
tron X
-
ray ra
dia
tion
Cryogenic
sample
handling robot
Goniometer
for sample
positioning
Cryo-stream
SONICC
microscope
Observed areas of
fiber diffraction100 µm
•An initial low-footprint prototype SHG microscope employing an
ultrafast (<100 fs) fiber laser source has been designed, assembled,
integrated into a synchrotron source, and used for combined X-ray
and SHG analysis for localization of crystalline -cellulose. Regions
of bright SHG correlated with regions of cellulose X-ray diffraction.
C Staiger & J Henty, Purdue University
Fast filament dynamics remodel
the cortical actin cytoskeleton
The cytoskeleton provides a filamentous framework that
serves as “tracks” for a variety of intracellular organelle
movements, including trafficking of membrane bound,
polysaccharide precursor delivery to the cell wall. In the
cortical array of epidermal cells, these tracks are of at least
two types: massive filament bundle superhighways, and fine
individual filaments. The latter population of tracks is under
constant rearrangement by a process of rapid growth
balanced by stochastic severing events and disassembly. To
study the interplay between these populations and to dissect
the molecular mechanism, we examined cytoskeletal
dynamics in a homozygous mutant for an adf4 knockout
using state-of-the-art live cell imaging. The adf4 knockout
has a 3-fold reduction in severing frequency, longer filament
lengths and lifetimes, as well as increased number of
filament bundles. This provides compelling evidence for the
contribution of a key actin-binding to filament dynamics and
reveals a mechanism for the interplay between single
filaments and bundled actin arrays. Future work will examine
whether both populations support movement of secretory
vesicle cargo to the cell wall.
Figure. Actin filament dynamics in the cortical array
of Arabidopsis epidermal arrays imaged with
variable angle epifluorescence microscopy. (A)
Wild-type cell. (B) adf4 knockout mutant cell.
Henty et al., (2011) The Plant Cell, submitted
Delivering metal co-catalysts to plant cell walls
for the deconstruction of engineered biomass
Incorporating iron ions into dilute acid pretreatment of
biomass is a promising technology for increasing sugar
yields. We are developing approaches to express metal-
binding or storing proteins into plant cell walls to enhance
biomass deconstruction. One technique being developed is
to down regulate an oligopeptide transporter gene (OPT3),
leading to the accumulation of iron and other metals in stem
and likely apoplast of model plants (Stacey et al., 2008). In
addition, we have tested 6 cellulose binding modules
(CBMs), among which CBM11 was the most efficient in
attaching to plant cell walls, making it a good candidate for
combining with iron binding peptides for precise delivery of
metal ion co-catalysts into cell walls during plant growth.
One concern with this approach is that in some metal-
storing proteins such as ferritin, iron exists as ferric oxide
nanoparticles which are stable up to 300-400 oC, raising a
question about their bio-availability. We have confirmed that
ferritin-Fe3+ can be released, and at a concentration of 2
mM, it’s incorporation into corn stover significantly
enhances both glucose and xylose monomer releases by
14% and 29%, respectively, in dilute acid pretreatment.
H. Wei1, H. Yang2, J. Cox2, P.N. Ciesielski1, B.S. Donohoe1, A.S. Murphy2,
W. Peer2, M.E. Himmel1, M. McCann2, M.P. Tucker1 1NREL, 2Purdue Univ.
CBM11-IBPs fused genes are being
engineered into plants to deliver
iron catalysts to cell walls.
Iron co-catalyst pretreatment using an iron storage
protein (ferritin) increases biomass digestibility
ferritin protein
(~4500 iron ions
/molecule) (modified from Masuda et
al. 2010)
impregnated
with ferritin
corn
stover
pretreated in
dilute acid
160 oC, 20 min
*
*
CBM11 was shown to be the most
effective at attaching to cell walls of
live plants.
Iron binding
peptide (IBP)
CBM11
Binding curve
Corn
Red
fluorophore Arabidopsis
Cellulose binding
module (CBM)
CBM3-mCherry
bright field
Metal catalysts delivery to plant cell walls
Fabio Ribeiro, rocket scientists, Purdue Univ.
Fast hydropyrolysis – closing the
mass balance
In February, we highlighted a breakthrough in measuring the primary reaction products of pyrolysis of
cellulose using mass spectrometry. Instead of the thousands of products observed during conventional
pyrolysis, we observed only a few discrete masses using fast-hydropyrolysis (a heating rate of 1000K per
second in the presence of hydrogen). However, we were limited to micrograms of material by our
commercial pyroprobe, making it impossible to conduct a mass balance. In collaboration with rocket
scientists at Purdue, our chemical engineers have built and tested a high-pressure, low-residence time,
hydropyrolysis reactor at a safe distance from main campus buildings. Good news – at this much larger
scale of tens of grams, we can close the mass balance to within 20%.
Product distribution from cellulose
pyrolyzed in the rocket reactor