Refining Biorefining Art J. Ragauskas BioEnergy Science Center Sch oo Ch

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1 Refining Biorefining Art J. Ragauskas BioEnergy Science Center Sh School lchemistry & Biochemistry i Institute of Paper Science & Technology Georgia Institute of Technology Atlanta, GA

2 Motivation: World oil demand/consumption 2

3 Cellulosic Conversion Improvement Strategies State of art versus theory Biological has lower theoretical yield (~100 gal/ton) but higher achieved yields (70-85 gal/ton) and potential co-products Thermochemicalh has highest h theoretical ti yield (~120+ gal/ton) but much lower achieved yield (50-65 gal/ton) and less desirable coproducts (i.e., methanol) Simplified Thermochemical Cellulosic Process Biomass Gasification Syngas Catalysis (modified d FT) Mixed alcohols Clean- up 3

4 Comparative Impacts of R&D on Biomass Processing cost via Biological Platform A1: Increase hydrolysis yield A2: Halve cellulase loading A: Conversion of biomass into available sugars A3: Eliminate pretreatment A4: Consolidate bioprocessing B1: Simultaneous C5 and C6 use B2: Increased fermentation yield B3: Increased ethanol titer B: Conversion of sugars into biofuels % 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. 4 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: (2008)

BioProcessing of Biomass/Recalcitrance Efficient biological conversion

physical and chemical properties of the plant cell wall via plant

Factors Affecting Enzymatic Digestibility: Macroscale: Epidermal

Hemicelluloses Lignin composition Lignin carbohydrate complexes NREL,

5 BioProcessing of Biomass/Recalcitrance Efficient biological conversion of cellulose into fermentable sugars is dependent on modifying the physical and chemical properties of the plant cell wall via plant science, pretreatment and improved organisms. Factors Affecting Enzymatic Digestibility: Macroscale: Epidermal tissue, arrangement and densityof the vascular bundles Cell wall porosity, biomass particle size. Microscale: Cellulose crystallinity & degree of polymerization Hemicelluloses Lignin composition Lignin carbohydrate complexes NREL, Ding et al, unpublished results 5 Himmel, M. E. et al. Science, 2007, 315(5813), Chundawat, S.P. S.et al. Current pinion in Biotechnology, 2009, 20(3),

6 Access to the Sugars in Lignocellulosic Biomass is the Current Critical Barrier Alcohols Butanol Consolidated Bioprocessing Hydrocarbons Ethanol Conventional Enzyme Fermentation Fermentation Chemical Catalysis Synthetic Biology Hydrocarbons 6 Recalcitrance Solving this will cut processing costs significantly and be used in most conversion processes This requires an integrated multidisciplinary approach

The BioEnergy Science Center BESC: A multi-institutional DE-funded center dedicated to

institutions 7 http://www.bioenergycenter.

Renewable Energy Laboratory Georgia Institute of Technology Samuel Roberts Noble

Ceres, Incorporated University of California-Riverside Cornell University Washington

7 The BioEnergy Science Center BESC: A multi-institutional DE-funded center dedicated to understanding and modifying plant biomass recalcitrance BESC is >318 people in 20 institutions 7 ak Ridge National Laboratory University of Georgia University of Tennessee National Renewable Energy Laboratory Georgia Institute of Technology Samuel Roberts Noble Foundation Dartmouth College ArborGen, LLC Verenium Corporation Mascoma Corporation Ceres, Incorporated University of California-Riverside Cornell University Washington State University University of Minnesota North Carolina State University Brookhaven National Laboratory Virginia Polytechnic Institute West Virginia University UCLA

8 The BioEnergy Science Center BESC: A multi-institutional DE-funded center dedicated to understanding and modifying plant biomass recalcitrance 8

BESC: Characterization Studies Characterization

H H H H H H H H H H H H H H H H H Ding S.Y. et al. J.

9 BESC: Characterization Studies Characterization Focus Bulk /Surface Molecular Contribution to Recalcitrance Molecular Contribution to Recalcitrance H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Ding S.Y. et al. J. Agric. Food Chem. 2006, 54, H Ac Ac H H H Ac Xylan 9 H 2 C H Me H

10 Mining Variation to Identify Key Genes in Biomass Composition and Sugar Release Collected ~1300 samples for Populus Association and Activation-tag Study HTS Pipeline Skagit (Sedro-Woolley) Skykomish (Monroe) Columbia (Longview) Puyallup (rting) Sugar Release Assay Analytical Pyrolysis 100 mi 200 km Existing collections (N = 500; 1-12 trees/site) New collections (N = 580; trees/site) Create Genetic Marker Map to identify allelic variation Identify Marker Trait Association Cell Wall Biosynthesis Database Establish common gardens for association and activation tag populations with 1000s of plants

11 HTP Characterization Pipeline for the Recalcitrance Phenotype Screening of 1000 s of samples Composition analytical pyrolysis, IR, confirmed by wet chemistry Pre-treatment new method with dilute acid and steam Enzyme digestibility sugar release with enzyme cocktail Detailed chemical and structural analyses of specific samples

su ugar yield [g sugar / g biomass] sugar yiel d [g sugar / g biomass s] 0.8 0.6 0.4 0.2 0.0 15 0.8 0.6 0.4 0.2 0.0 10 1.0 20 25 lignin content [%] 20 2.0 lignin S/G ratio [-] 30 3.

12 su ugar yield [g sugar / g biomass] sugar yiel d [g sugar / g biomass s] lignin content [%] lignin S/G ratio [-] Populus Association Study Tested for enhanced sugar release characteristics through pretreatment and enzymatic hydrolysis Hot water pretreatments at 160 and 180 o C HTP pretreatment and co-hydrolysis in 96 well-plates Preliminary observations: Sugar yield increases with S/G ratio Lignin content has minimal effect Some outlier poplar samples exhibit very high sugar release Characterization pipeline pp works Pretreatment conditions: Standard BESC poplar Theoretical sugar yield Studer, Wyman et al. 180 o C, 18Min 160 o C, 68Min

13 BESC: Alfalfa Cell Wall Chemistry Alfalfa (Medicago sativa L.) Initial Model Study Sugar release for alfalfa after enzymatic hydrolysis without acid pretreatment Isolate Cellulose 13 C CP/MAS NMR Alfalfa CrI % Wild CTR1 53 Anti sense gene down regulation of alfalfa lignin HCT and C3H lines had prominent effects on improving saccharification Transgenic alfalfas showed less recalcitrance for enzymatic hd hydrolysis Chen F. & Dixon R. A. Nature Biotechnology, 2007, 25(7) Wild CTR49A 51 C3H9A 54 C3H4A 54 HCT3A 54 HCT30A 54 13

BESC: Characterization Alfalfa Lignin Structure

Structure NMR HCT -30A H 2/6 H 3/5 H 14 C3H-4A H H

3/5 6 1 6 H 2 2 5 3 4 G lignin CTR Wild -1 CTR1 6 5 1

CH 3 3 S 3/5, G 3/4 S1/4,G1 G5 C? Cβ C C?

; Ziebell, A.; Davison, B.; Ragauskas, A.J.

14 BESC: Characterization Alfalfa Lignin Structure Differs from Transgenics Isolated Lignin Analyze Structure NMR HCT -30A H 2/6 H 3/5 H 14 C3H-4A H H 2/6 3/5 H lignin H 14 S lignin i C3H-9A H 14 H 2/6 H 3/ H G lignin CTR Wild -1 CTR G 3 4 CH S S 3 3 H 3 HC 3 C 4 4 CH CH 3 3 S 3/5, G 3/4 S1/4,G1 G5 C? Cβ C C? Cα C Me CR G 6 G 2 S 2/6 14 See Pu, Y.; Chen, F.; Ziebell, A.; Davison, B.; Ragauskas, A.J. BioEnergy Research (2009) 2, Quantitative 13 C NMR spectra of isolated alfalfa lignins

15 Quantitative 13 C NMR Characterization G unit 100% S unit H unit 80% 60% 40% 20% 15 0% CTR-1 C3H-9A C3H-4A HCT-30A H:S:G units composition of isolated alfalfa lignins H units increase from ~1% to 36~68% for C3H mutant, to ~75% for HCT transgenics. G units decrease by 36~75% for C3H mutant, ~77% for HCT transgenics S units decrease by 27~54% for C3H mutant, ~70% for HCT transgenics

16 Quantitative 13 C NMR Characterization 1.2 atom pe er Ar C CTR-1 C3H-9A C3H-4A HCT-30A 16 Methoxy group contents of isolated alfalfa lignins

17 Quantitative 13 C NMR Characterization 0.9 C at tom per Ar CTR-1 C3H-9A C3H-4A HCT-30A β--4 linkage decrease ~by 61% for C3H, ~40% for HCT transgenics 17

18 BESC: HSQC NMR Characterization Conformation of H:G:S Alfalfa Lignin Partial 13 C 1 H HSQC 2D correlation NMR (aromatic region) of alfalfa lignins 18 Changes in G S H clearly shown

BESC: HSQC NMR Characterization Alternations in Lignin

(aliphatic region) of alfalfa lignins 19 Relative content of

Relative content of phenylcoumaran (B) increases by 85% and

19 BESC: HSQC NMR Characterization Alternations in Lignin Subunit Linkages Partial 13 C 1 H HSQC 2D correlation NMR (aliphatic region) of alfalfa lignins 19 Relative content of 4 decreases for C3H and HCT transgenics. Relative content of phenylcoumaran (B) increases by 85% and 220% for C3H & HCT. Resinol (C) increases by 53% and 174% for C3H and HCT transgenics.

20 BESC: Lignin 31 P NMR Studies Increased Free Phenolics for Transgenic Alfalfa Lignin CH 3 CH 3 CH 3 P Cl + Lignin-H CH 3 CH 3 C H 3 CH 3 CH 3 P Lignin P R P Me P Me P R Guaiacol Condensed/ Syringyl 31 P NMR spectra of alfalfa lignins Terminal p hydroxyphenyl H increase by 890% and 945% for C3H and HCT transgenics 20 Terminal guaiacyl H decrease by 75% and 94% for C3H and HCT transgenics

21 BESC: Decrease in Transgenic Alfalfa Lignin MW and Impact on Recalcitrance GPC of isolated lignin confirms that there is a trend toward lower molecular weight lignin in the transgenic lines versus control. These results follow the trend that lignin with a higher H lignin subunit content are: 21 See Ziebell, A. et al. Journal of Biological Chemistry 2010, 285(50),

BESC: Low Recalcitrance Switchgrass Agrobacterium

cinnamate 22 The Samuel Roberts NBLE Foundation C4H HC H

4-coumaraldehyde HH 2C CAD H 4-coumaroyl alcohol H lignin

H H caffeoyl shikimic acid or quinic acid CoAS HCT CCoAMT

coniferaldehyde CAD F5H G lignini CH 3 HH 2C H coniferyl

HH 2C H H 5-hydroxyconiferyl alcohol l CH 3 H H CH 3

22 BESC: Low Recalcitrance Switchgrass Agrobacterium mediated transformation of switchgrass Lignin pathway HC cinnamate 22 The Samuel Roberts NBLE Foundation C4H HC H 4-coumaric acid 4CL CoAS H H 4-coumaroyl CoA CCR H 4-coumaraldehyde HH 2C CAD H 4-coumaroyl alcohol H lignin HCT R- H 4-coumaroyl shikimic acid or quinic acid C3H R- H H caffeoyl shikimic acid or quinic acid CoAS HCT CCoAMT CoAS H H caffeoyl CoA feruloyl CoA CCR H CH 3 H CH 3 H coniferaldehyde CAD F5H G lignini CH 3 HH 2C H coniferyl alcohol H CH 3 H H F5H CMT 5-hydroxyconiferaldehyde CH 3 HH 2C H H 5-hydroxyconiferyl alcohol l CH 3 H H CH 3 sinapaldehyde CAD Down Regulated CMT CH 3 HH 2C H CH 3 sinapyl alcohol S lignin

control and transgenic switchgrass Switchgrass

Klason lignin Control I 1 1.6 0.5 43.1 20.8 24.

23 BESC: Determining Structural Changes of Transgenic Switchgrass Composition % analysis of control and transgenic switchgrass Switchgrass Arab. Gal. Gluc. Xyl. Klason lignin Control I Transg I Control I Transg I Control Tiller Transg Tiller

24 BESC: Low Recalcitrance Switchgrass Evaluated by Saccharification/Fermentation Composition analysis of control and transgenic switchgrass Saccharification efficiency of T1 transgenic switchgrass stems with and without pretreatment Fermentation of control, T0 and T1 transgenic switchgrass without pretreatment 24 See 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, DI;

BESC: Low Recalcitrance Switchgrass Cellulose

25 BESC: Low Recalcitrance Switchgrass Cellulose Structure Does Not Contribute Cellulose isolation Analysis Crystallinity by NMR Molecular weights by GPC Molecular Weight Results 13 C CP/MAS NMR Results 25 Low recalcitrance of transgenic switchgrass is a lignin phenomenon > Understudy Currently

26 BESC: Control vs. Transgenic Switchgrass Conclusions Down regulated lignin in Alfalfa and Switchgrass are beneficial for reduced recalcitrance, enhanced pretreatment/deconstruction and ethanol production Down regulationof lignini incurs a host of changes in thestructuret t In Alfalfa HCT and C3H mutants the resulting lignin has more C C bondsanda shorter MW As anticipated lignin down regulation has no impact on the ultra structure of cellulose, solely a lignin phenomena Comparable approaches are being employed with HW energy crops Promising Pathway for Low Recalcitrance Agro Energy Crops for Biofuels 26

27 Thank You! SCIENCE RETREAT JUNE 2010 BESC is a U.S. Department of Energy Bioenergy Research Center supported by the ffice of Biological and Environmental Research in the DE ffice of Science

Understanding Gene Function and Control in Lignin Formation In Wood

Understanding Gene Function and Control in Lignin Formation In Wood Vincent L. Chiang North Carolina State University Raleigh, NC Tremendous effort has been devoted to developing genetically engineered