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

O

OH

O

O

HO

OH

O

OH

O

T: ITMS - p ESI Full ms [50.00-750.00]

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lative

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un

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nce

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[M-H]-

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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10.85

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6.35

8.14 11.24

164.91

-CH3

-CH2 CH2OH

-CH2OH

-CO

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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tive A

bundance

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166

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93

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

85

80

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