southern pine based biorefinery centerbiorefinery … pine based biorefinery centerbiorefinery...

Southern Pine Based Biorefinery CenterBiorefinery Center

Art J. RagauskasInstitute of Paper Science and Technology

School of Chemistry and BiochemistryGeorgia Institute of Technology, Atlanta, GA

1

Project Overview• Technical hurdle to be

overcome?– Optimal material science for

pyrolysis and pretreatment reactors

– Viable generation ofViable generation of green/biodiesel

– Optimized bioethanol generation integrated with pulp/paper/modified cellulosics pathways

– Integration into a modern SW kraft pulp mill operationkraft pulp mill operation

2

Quad Chart Overview

• Project start date • Research studies have been initiatedTimeline Project Development

j– Oct. 1, 2010

• Project end date

Research studies have been initiated and on track

• Project Scope is following program deliverables

– Sept. 30, 2012

• Percent complete: 12%• Completion of deliverables projected

for Sept/30/2012.

Project ParticipantsCo-PI: Dr. P. Singh, MSE/GTIndustrial kick-off meeting

Project managed by deliverables

Initial testing facilities been ordered

3

g

Exploratory research studies on pine conversion technologies initiated

Approach• Develop on the GA Tech campus an integrated southern pine wood to

biofuels/biomaterials processing facility that will test advanced integrated wood processing exploratory technologies at the benchintegrated wood processing exploratory technologies at the bench scale.

Exploratory research program is structured to evaluate key chemical/enzymatic conversion processes needed to convert southern pine to biofuels and novel biomaterials in a pulp millpine to biofuels and novel biomaterials in a pulp mill.

Research program is task/milestone orientated Focus on Process Chemistry of Conversion & Material

4 4

Focus on Process Chemistry of Conversion & Material Science of Conversion Reactors

Technical Accomplishments/ pProgress/Results

Tasks Completedp

• Task A: Collect GA pine woodchips, residues and bark – Feedstock for laboratory studies for next duration of two year projectFeedstock for laboratory studies for next duration of two year project

• Task A.1: Determine biomass constituents

% ComponentPine wood

chipsPine wood

barkPine wood residues% Component chips bark residues

Holocellulose 68.5 50.9 65.9α-cellulose 45.4 29.3 42.1hemicellulose 12.2 13.7 12.5

Lignin 28.0 46.3 30.4acid insoluble lignin 27.3 45.5 29.4acid soluble lignin 0.69 0.81 1.02

-Basics constituents info needed for all studies

5

Ash 0.27 0.91 0.81

Extractives in DCM 2.5 3.6 1.5Tannin − 11.6 −

stud es


• Task A.2: Structural analysis of pine - ongoing

Cellulose

C1

C6C4

Crystallinity & DP – key parameter forenzymatic deconstructionC2,3,5enzymatic deconstructionof cellulose for bioethanol

Data needed for upgradingLignin

Data needed for upgradingLignin studies for Green Diesel/biodiesel

6102030405060708090100110120130140150160170180190200

f1 (ppm)

1313413513613713813914014114214314414514614714814950f1 (ppm)


• Task A.2: Structural analysis of pine - ongoing

St t l tTannins

Structural components mycontribute to corrosion &inhibit biofuel productionby biological routeby biological route

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• Task C.2: Microbial upgrading lignin and pyrolysis oil from pine pg g g py y presidue - ongoing

Single cell oils produced by heterotrophic microorganisms

TAG

Oleaginous species under growth restricting conditions (high C:N ratio) are capable to produce large quantities of TAG.Using agricultural wastes to grow two high yielding strains Rhodococcus opacus

8 8

g g g g y g pPD630 and Gordonia sp DG, the latter can produce TAGs up to 14.45 mg L-1 d-1 on orange waste while earlier converted carob waste with 59.27 mg L-1 d-1 productivity

Technical Accomplishments/ Progress/ResultsProgress/Results

• Task C.2: Microbial upgrading lignin and pyrolysis oil from pine residue - ongoing

DSM 1069 - OD [600]

3.0

residue ongoingMicrooragnisms Under Study1.Rhodococcus opacus DSM 1069 (DSM 1069)2.Rhodococcus opacus PD 630 (PD 630)

2.0

2.5

1.0

1.5

OD

[600

]

0.0

0.5

0 50 100 150 200 250 3000 50 100 150 200 250 300

hours

Glucose PHA VanA UE Gluc-0.5 VanA-0.5 UE-0.5

9 9p-hydroxy benzoate Na+ salt (PHA); 4-hydroxy-3-methoxy benzoate (vanillic acid) (VanA)Ultrasonicated lignin (UE)

R. opacus DSM 1069 growth on different substrates as measured by optical density (OD) at 600 nm.


• Task C.2: Microbial upgrading lignin and pyrolysis oil from pine residue - ongoing

PD 630 - OD [600]

residue ongoingMicrooragnisms Under Study1.Rhodococcus opacus DSM 1069 (DSM 1069)2.Rhodococcus opacus PD 630 (PD 630)

2.5

3.0

3.5

1.5

2.0

OD

[600

]

0.0

0.5

1.0

0 20 40 60 80 100 120 140 160 180

hours

Glucose PHA VanA UE Gluc-0.5 VanA-0.5 UE-0.5

10

Both strains are growing on lignin like structuresNeed determine TAG production, my require reduction in lignin DP


• Task C.GN.2 Pine residue and bark pyrolysis - ongoing

Pine residue pyrolyzed at 550 oC with 0.5 L/min nitrogen flow into the tubeg

Pyrolysis oil 45%Char 40%Non-condensable: 15%

Structure characterization ongoing

Examining role of lignin in pyrolysisExamining role of lignin in pyrolysis

Lignin yields a light/heavy oilP f d t 550 600 C

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Preferred temp 550-600 oC



Light oil is primarily waterChange in pyrolysis temp increases catechols & reduces aliphatic hydroxyls

12 12



350400

Heavy Oils

200250300350

Mn(g/mol)

50100150200

Mw(g/mol)

050

400 500 600 700 Ongoing Studies:Improved reactor designCatalysis pyrolysis reactions

13 13

Technical Accomplishments pProgress/Results

• Task D 2: Cellulosic derivatives ongoing• Task D.2: Cellulosic derivatives – ongoingObjective: Develop new high-valued cellulosic co-products based on cellulosic

whiskers

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Technical Accomplishments pProgress/Results

• Task D 2: Cellulosic derivatives ongoing• Task D.2: Cellulosic derivatives – ongoingObjective: Develop new high-valued cellulosic co-products based on cellulosic

whiskers

a b

AFM image of 0.5 wt% suspensions (a)

0 2μm 0 2μm

dc

whisker (b) DAC (c) DAC-MA (d) DAC-BA

- Testing physical properties- Testing physical properties - Composite applications

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0 2μm 0 2μm

RelevanceRelevance

Fossil Energy Ratio (FER) = Energy Delivered to Customer/Energy Used

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Th Bi E S i C tThe BioEnergy Science CenterBrief Overview

BESC A lti i tit ti l DOE f d d tBioEnergy Science CenterBESC: A multi-institutional DOE-funded center dedicated to understanding and modifying plant biomass recalcitrance

Oak Ridge Samuel Roberts Noble Foundation Oak Ridge National Laboratory

University of GeorgiaUniversity of Tennessee

Samuel Roberts Noble FoundationNational RenewableEnergy LaboratoryBrookhaven National University of Tennessee

Dartmouth CollegeGeorgia Institute of Technology

Brookhaven NationalLaboratoryUniversity of California–Riverside Cornell University West Virginia University

ArborGen, LLCCeres, Incorporated

Cornell University Washington State UniversityUniversity of Minnesota

322 people in 20 institutions

Mascoma CorporationVerenium Corporation

North Carolina State UniversityVirginia Polytechnic InstituteU i it f C lif i L A l

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University of California–Los Angeles

Access to the sugars in lignocellulosic biomassin lignocellulosic biomass is the current critical barrier

• Overcoming this barrier

Butanol

Alcohols

Consolidatedbioprocessing

Hydrocarbons

will cut processing costs significantly and be used

Conventionalenzyme fermentation

Ethanol Fermentation Chemicalcatalysis

Syntheticbiology

Hydrocarbons

be used in most conversion processes

enzyme fermentation

• This requires an integrated multidisciplinary

Recalcitrance Time frame Planned ActualModified plants to field trials Year 5 Year 4

multidisciplinary approachNew or improved

microbes to development

Year 4–5 Year 3–4

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Analysis and screening technologies

Year 3 on

Year 2 on

A Two-pronged Approach to Increase the Accessibility of Biomass Sugars

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Comparative impacts of R&Dp pon biomass processing cost

A1 I h d l i i ldA1: Increase hydrolysis yield

A2: Halve cellulase loading

A3: Eliminate pretreatment

A: Conversion of biomassinto available sugars

A3: Eliminate pretreatment

A4: Consolidate bioprocessing

B1: Simultaneous C5 and C6 use

B2: Increased fermentation yield B: Conversion ofsugars into biofuels

B3: Increased ethanol titer

0 10 20 30 40 50 60 70% of processing cost reduction

Without overcoming biomass recalcitrance (A), cellulosic biofuels will be more expensive than corn biofuels Improved sugar conversion (B) is not enough

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expensive than corn biofuels. Improved sugar conversion (B) is not enough.Ref: Lynd, L.R., M.S. Laser, D. Bransby, B.E. Dale, B. Davison, R. Hamilton, M. Himmel, M. Keller, J.D. McMillan, J. Sheehan, C.E. Wyman, "How Biotech can transform biofuels," Nature Biotechnology 26:169-172 (2008)

BESC Will Revolutionize How Biomass is Processed

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Targeted cell wall synthesis approach:• Test known putative recalcitrance genes in via

Populus and switchgrass transgenics (TP)• Basic research to identify unknown genes and

decipher how they effect recalcitrance

Discovery-based natural variation approach:• Identify natural variation in recalcitrance• Identify gene responsible • Test via Populus and switchgrass transgenics (TP)Test via Populus and switchgrass transgenics (TP) • Activation tagging

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HTP Characterization Pipeline for the Recalcitrance Phenotypefor the Recalcitrance Phenotype

S i f 1000’ f l• Screening of 1000’s of samplesPre-treatment

th d ithComposition analytical

pyrolysis IR confirmed byEnzyme digestibility

lnew method with dilute acid and steam

pyrolysis, IR, confirmed by wet chemistry

sugar release with enzyme cocktail

24Detailed chemical and structural analyses of specific samples

Mining Variation to Identify Key Genes in Biomass Composition and Sugar Release

HTS PipelineCollected ~1300 samples for HTS Pipeline

Skagit (Sedro Woolle )

Collected 1300 samples for Populus Association and

Activation-tag Study

Sugar Release Assay

Analytical Pyrolysis

Skagit (Sedro-Woolley)

Skykomish (Monroe)

Puyallup (Orting)Columbia (Longview)

Cell Wall Biosynthesis

Database

Create Genetic Marker Map to identify allelic variation

Identify Marker Trait Association100 mi

Existing collections (N = 500; 1-12 trees/site)

New collections (N = 580; 140-160 trees/site)

Existing collections (N = 500; 1-12 trees/site)

New collections (N = 580; 140-160 trees/site)

Establish common d f i ti

Trait Association200 km

gardens for association and activation tag

populations with 1000s of plants

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Association Study Association Study ––Composition DataComposition DataComposition DataComposition Data

•The association samplesThe association samples display extreme variation in lignin, S/G ratio, and sugar 80g , , gcontent

•Extreme phenotypes have 30

40

50

60

70

Cou

nt

Extreme phenotypes have been characterized for detailed pretreatment and

0

10

20

1.00

1.17

1.35

1.52

1.69

1.87

2.04

2.21

2.39

2.56

2.73

2.90

S/G ratiop

sugar release assays•All sampled genotypes are

S/G ratio

2 5

3.0

3.5

being replicated and will be established in a common garden experiment 1 0

1.5

2.0

2.5

S/G

Rat

io

26

garden experiment0.5

1.0

15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00

Lignin (%)

Populus Association Study0.8

mas

s]

Populus Association Study• Tested for enhanced sugar release

characteristics through pretreatment and

0.6

gar /

g b

iom

characteristics through pretreatment and enzymatic hydrolysis

– Hot water pretreatments at 160 and 180oC

0.4

0.2yiel

d [g

sug

• HTP pretreatment and co-hydrolysis in 96 well-plates

• Preliminary observations:0.0su

gar

30252015

li i t t [%]

0.8

iom

ass]

Preliminary observations:– Sugar yield increases with S/G ratio– Lignin content has minimal effect

S tli l l hibit

lignin content [%]

0.6

0.4suga

r / g

b – Some outlier poplar samples exhibit very high sugar release

• Characterization pipeline works

0.2

gar y

ield

[g

Pretreatment conditions: 180oC, 18Min160oC, 68Min

Standard BESC poplar

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0.0sug

3.02.01.0

lignin S/G ratio [-]

Standard BESC poplarTheoretical sugar yield

Studer, Wyman et al.

Establishment of a transformation protocol for switchgrass

A B CA B C

D E

28 Stewart et al. - UTn

Genetic block in lignin biosynthesis in switchgrass increases biofuel yieldsPhenylalanine PAL

HOOC

cinnamate LigninAgrobacterium-

mediated Ethanol yield

in switchgrass increases biofuel yields

R- OCoAS

HOOC OH

4-coumaric acid

C4H

4CL

Ligninpathwaytransformation

of switchgrass

Ethanol yield

0.25

0.30

0.35

Wei

ght

g/g)

O

HOH

R OOH

4-coumaroylshikimic acidor quinic acid

OOH

O

CoASOH

4-coumaroyl CoA HCT

C3HCCR

0 10

0.15

0.20

ol Y

ield

per

WB

iom

ass

(g

4-coumaraldehyde

HOH2C OH

4-coumaroyl alcohol

O

R- OOH

caffeoylshikimic acidor quinic acid

HCT

CAD

0.00

0.05

0.10

Wild-type

Eth

ano of

Noble

CoASOCH3

H lignin O

CoASOH

OH

caffeoyl CoA

CCoAOMT G ligninOCH3

switchgrass Foundation transgenic

switchgrass

feruloyl CoAO

CoASOH

HOH

OCH3

OCH3

coniferyl alcohol

OHHOH2C

CAD 5-hydroxyconiferyl alcohol

OCH3

OH

OH

HOH2C

OCH

F5HCCRCOMT

COMT

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coniferaldehydeO

OH

5-hydroxyconiferaldehyde

O

HOH

OCH3

OHsinapaldehyde

O

HOH

OCH3

CH3O sinapyl alcohol

OCH3

OH

OCH3

HOH2C S ligninF5H CAD

The Samuel Roberts

NOBLEFoundation

COMT

29See Fu, C.; Mielenz, J.R.; Xiao, X.; Ge, Y.; Hamilton, C.Y.; Rodriguez Jr. M.; Chen, F.; Foston, M.; Ragauskas, A.J.; Bouton, J.; Dixon, R.A.; Yu, Z.; Wang, Z.Y. PNAS, DOI; 10.1073

Understanding the Structure/Function of the Cellulosome - CbhA is a Critical Enzymey

C. thermocellum CbhA

DockerinCBM3b

Bayer et al 2009CBM4

Fn3 Fn3PDB

New structures solved at NREL

Bayer et al. 2009(unpublished)NREL 2009 Fn31- Fn32

NREL 2009Ig-GH9

PDB

New structures solved at NREL

A bl d 7 d i

30 30Assembled 7 domain enzyme• Ready for computer simulations

New tools from “Systems Biology” offer confidence for conversion successconfidence for conversion success

250

kDa Run 1Run 2

250

kDa Run 1Run 2

Shot-gun Proteomic LC-MS/MS IdentificationAffinity Purification

400

500

(mg/

L)

Fermentation

Metabolic Labeling Quantitation

3750

75100150

3750

75100150

0

100

200

300

Pelle

t Pro

tein

(

0 5

1.0

1.5

2.0Log2(Switchgrass/Cellulose)Log2(Cellobiose/Cellulose)

0 5

1.0

1.5

2.0Log2(Switchgrass/Cellulose)Log2(Cellobiose/Cellulose)

10152025

10152025

00 5 10 15 20 25 30

Time (h)

80

120

160

Prot

ein

(mg/

L)

-1.0

-0.5

0.0

0.5

CelSCelACthe0CelKCelQ

CelRXynDXynZ

-1.0

-0.5

0.0

0.5

CelSCelACthe0CelKCelQ

CelRXynDXynZ

0

40

0 5 10 15 20 25 30Time (h)

Supe

rnat

e

• C thermocellum alters its cellulosome

-2.0

-1.5

S (GH48)A (GH8)e0821 (GH5)K (GH9)Q (GH9)R (GH9)D (GH10)Z (GH10)-2.0

-1.5

S (GH48)A (GH8)e0821 (GH5)K (GH9)Q (GH9)R (GH9)D (GH10)Z (GH10)

Time (h)

• Pretreated Switchgrass• Cellobiose• Amorphous Cellulose• Avicel - 14N

• C. thermocellum alters its cellulosome catalytic composition depending upon the growth biomass substrate

• We identified and experimentally verified Avicel N• Avicel - 15N • Avicel-Pectin• Avicel-Xylan• Avicel-Pectin-Xylan

16 “new” cellulosome components

31Citation: “Raman B, et al. (2009) Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum ATCC 27405 Cellulosome Composition: A Quantitative Proteomic Analysis. PLoS ONE 4(4): e5271. doi:10.1371/journal.pone.0005271”

BESC Will Revolutionize How Biomass is Processed and ConvertedBiomass is Processed and Converted

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Industrial partners facilitate strategic commercializationstrategic commercialization

Thank You!33

southern pine based biorefinery centerbiorefinery … pine based biorefinery centerbiorefinery...

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